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Programme & Abstract Book CHROMATIN STRUCTURE & FUNCTION Nassau, Bahamas 15th - 18th November 2005 Organised By: Tony Kouzarides and Abcam

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Page 1: Programme & Abstract Book CHROMATIN - Abcam · Programme & Abstract Book CHROMATIN STRUCTURE & FUNCTION Nassau, ... androgen receptor dependent transcription ... 10:00 Benoit Guillemette

Programme & Abstract Book

CHROMATINSTRUCTURE & FUNCTIONNassau, Bahamas15th - 18th November 2005

Organised By:Tony Kouzarides and Abcam

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Programme & Abstract Book

The second

CHROMATINSTRUCTURE & FUNCTION

Nassau, Bahamas 15th - 18th November 2005

Organizers:Tony Kouzarides

(University of Cambridge)and

Abcam

Table of contents

Timetable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 2

Conference Programme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 3

Poster Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 8

Abstracts - Oral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 18

Abstracts - Poster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 57

Resort Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 173

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Disclaimer: Material contained within this booklet should be citied only with permission from the author(s).No live recording or photography is permitted during the oral or poster sessions.

Copyright © 2005 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.All information / detail is correct at time of going to print.

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Tuesday 15th November18:00 Keynote Speaker19:00 Poolside dinner reception

Wednesday 16th November09:00 - 10:30 Oral presentations

Drinks break11:00 - 12:30 Oral presentations

Lunch and free time16:00 - 17:30 Oral presentations

Drinks break18:00 - 21:00 Poster session and evening buffet

Thursday 17th November09:00 - 10:30 Oral presentations

Drinks break11:00 - 12:30 Oral presentations

Lunch and free time16:00 - 17:30 Oral presentations

Drinks break18:00 - 19:30 Oral presentations20:00 Beach Barbeque

Friday 18th November09:00 - 10:30 Oral presentations

Break11:00 - 12:15 Oral presentations

Lunch and departures

Keynote Speaker Sponsors:

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Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Timetable

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Conference Programme

Conference ProgrammeTuesday 15th NovemberChair: Tony Kouzarides

Keynote Speaker18:00 - 19:00 Danny Reinberg . . . . . . . . . . . . . . . . . . . . . . .Page 18

Histone marks and chromatin states

Wednesday 16th NovemberChair: Ali Shilatifard

09:00 - 09:30 Shelley Berger . . . . . . . . . . . . . . . . . . . . . . . .Page 19Factor and histone covalent modifications in genome regulation

09:30 - 09:45 Brad Bernstein . . . . . . . . . . . . . . . . . . . . . . . .Page 20Genomic studies of chromatin modifications in normal andmalignant cells

09:45 - 10:00 Hendrik Stunnenberg . . . . . . . . . . . . . . . . . . .Page 21Histone modification patterns associated with X-inactivation and escape

10:00 - 10:30 Sandra Hake . . . . . . . . . . . . . . . . . . . . . . . . . .Page 22Beyond the double helix: Understanding the functional importance of histone H3 and its variants

Break

11:00 - 11:30 Yang Shi . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 23A new kid on the chromatin block: identification of the first histone demethylase LSD1 and its regulation

11:30 - 11:45 Roland Schuele . . . . . . . . . . . . . . . . . . . . . . .Page 24LSD1 demethylates repressive histone marks to promoteandrogen receptor dependent transcription

11:45 - 12:15 Tony Kouzarides . . . . . . . . . . . . . . . . . . . . . . .Page 25Proline isomerisation of histone H3 regulates gene expression

12:15 - 12:30 Victoria Lunyak . . . . . . . . . . . . . . . . . . . . . . . .Page 26ESET dependent H3-K9 methylation upon restructuring of chromosomal domains during development.

Lunch and free time

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Wednesday 16th November (continued)Chair: Craig Peterson

16:00 - 16:30 Yi Zhang . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 27Histone modifications in polycomb silencing and cancer

16:30 - 16:45 Patrick Grant . . . . . . . . . . . . . . . . . . . . . . . . . .Page 28Generation and recognition of histone modifications by the SAGA and SLIK HAT complexes in health and disease

16:45 - 17:00 Vincent Geli . . . . . . . . . . . . . . . . . . . . . . . . . .Page 29Structure and function of the Set1 RNA recognition motif

17:00 - 17:30 Ali Shilatifard . . . . . . . . . . . . . . . . . . . . . . . . . .Page 30Yeast COMPASS points the way to human MLL and its role in pathogenesis of leukemia

Break

18:00 - 21:00 Posters

Thursday 17th NovemberChair: Shelley Berger

09:00 - 09:30 Kevin Strule . . . . . . . . . . . . . . . . . . . . . . . . . .Page 31Abstract title to be confirmed

09:30 - 09:45 Oliver Rando . . . . . . . . . . . . . . . . . . . . . . . . . .Page 32Genome-scale characterization of chromatin structure

09:45 - 10:00 Rolf Sternglanz . . . . . . . . . . . . . . . . . . . . . . . .Page 33Histone mutations that suppress the silencing defects of a yeast sir3 Ala2Gly mutation

10:00 - 10:15 Antonin Morillon . . . . . . . . . . . . . . . . . . . . . . .Page 34Histone variant HT2A-Z promotes promoter-proximal nucleosomedissociation upon transcription activation in S. cerevisiae.

10:15 - 10:30 Sharon Dent . . . . . . . . . . . . . . . . . . . . . . . . . .Page 35New functions for histone modifying enzymes

Break

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Thursday 17th November (continued)Chair: Shelley Berger

11:00 - 11:30 Jerry Workman . . . . . . . . . . . . . . . . . . . . . . . .Page 36Set2 methylates histone H3 K36 providing transcriptionalmemory that signals Rpd3S to deacetylate histones in transcribedregions to suppress spurious intragenic transcription

11:30 - 11:45 Nevan Krogan . . . . . . . . . . . . . . . . . . . . . . . . .Page 37Protein complexes and pathways involved in chromatin function

11:45 - 12:00 Bruno Amati . . . . . . . . . . . . . . . . . . . . . . . . . .Page 38Epigenetic determinants of Myc binding to the human genome

12:00 - 12:15 Francois Fuks . . . . . . . . . . . . . . . . . . . . . . . . .Page 39A direct mechanistic link between the Polycomb Group protein EZH2 and DNA methyltransferases

12:15 - 12:30 Julie Ahringer . . . . . . . . . . . . . . . . . . . . . . . . .Page 40Chromatin regulation and sumoylation in the inhibition of Ras induced vulval development in C. elegans

Lunch and free time

Chair: Sharon Dent

16:00 - 16:30 Peter Becker . . . . . . . . . . . . . . . . . . . . . . . . . .Page 41Site-specific acetylation defines an embryonic form of ISWI that associates with mitotic chromatin

16:30 - 16:45 Stephen Rea . . . . . . . . . . . . . . . . . . . . . . . . . .Page 42hMOF complex is required for histone H4 lysine 16 acetylation in mammalian cells

16:45 - 17:00 Susana Gonzalo . . . . . . . . . . . . . . . . . . . . . . .Page 43DNA methyltransferases control telomere length in mammalian cells

17:00 - 17:30 Craig Peterson . . . . . . . . . . . . . . . . . . . . . . . .Page 44Chromatin remodeling: Regulation of chromatin higher-order folding and DNA repair

Break

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Conference Programme

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Thursday 17th November (continued)Chair: Sharon Dent

18:00 - 18:30 Bob Kingston . . . . . . . . . . . . . . . . . . . . . . . . .Page 45Remodeling chromatin without covalent modification

18:30 - 18:45 Lianna Johnson . . . . . . . . . . . . . . . . . . . . . . .Page 46DNA and histone methylation in Arabidopsis

18:45 - 19:00 Eric Miska . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 47Short RNAs in C. elegans development and human cancer

19:00 - 19:30 Ramin Shiekhattar . . . . . . . . . . . . . . . . . . . . .Page 48Integrator, a multiprotein mediator of small nuclear RNAprocessing associates with the C-terminal repeat of RNApolymerase II

Friday 18th NovemberChair: Kevin Struhl

09:00 - 09:30 Kami Ahmad . . . . . . . . . . . . . . . . . . . . . . . . . .Page 49Abstract title to be confirmed

09:30 - 09:45 Jacques Cote . . . . . . . . . . . . . . . . . . . . . . . . .Page 50Dissection of the NuA4 HAT complex and crosstalk with other chromatin modifications and remodelers

09:45 - 10:00 Benoit Guillemette . . . . . . . . . . . . . . . . . . . . .Page 51H2A.Z is globally localized to the promoters of inactive genes and regulates nucleosome positioning

10:00 - 10:30 Karolin Luger . . . . . . . . . . . . . . . . . . . . . . . . .Page 52Structure and function of yeast nucleosome assembly protein 1

Break

11:00 - 11:30 Wolf Reik . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 53Regulation of imprinting and epigenetic reprogrammingin mammals

11:30 - 11:45 Aidan Doherty . . . . . . . . . . . . . . . . . . . . . . . . .Page 54Structure and function of the Jumonji C Domain

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Friday 18th November (continued)Chair: Kevin Struhl

11:45 - 12:00 Andreas Ladurner . . . . . . . . . . . . . . . . . . . . . .Page 55Human chromatin as a molecular target of cellular NAD metabolites

12:00 - 12:15 Jef Boeke . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 56Comprehensive mutagenesis of sites of histone modification

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Conference Programme

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Abstract 1 Ted Abel . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 57The role of CBP and histone acetylation in memory storage andsynaptic plasticity

Abstract 2 Melissa Adkins . . . . . . . . . . . . . . . . . . . . . . . .Page 58Spt6-mediated nucleosome reassembly is required fortranscriptional repression of the PHO5 gene

Abstract 3 Adriana Alejandro-Osorio . . . . . . . . . . . . . . . .Page 59The role of the histone deacetylase Rpd3p in coordinating theenvironmental stress response in yeast

Abstract 4 Erik Andersen . . . . . . . . . . . . . . . . . . . . . . . . .Page 60MET-1 and MET-2, two putative histone methyltransferases, areredundantly required for proper vulval cell fate specification

Abstract 5 Anthony Argentaro . . . . . . . . . . . . . . . . . . . . .Page 61Structure/function studies of the PHD-like domain of thechromatin-remodelling protein, ATRX

Abstract 6 Koh Meng Aw Yong . . . . . . . . . . . . . . . . . . . .Page 62The presence of EBV oriP within a transcription unit inhibitsreplication as well as transcription

Abstract 7 Slobodan Barbaric . . . . . . . . . . . . . . . . . . . . .Page 63Two nucleosomes at the yeast PHO84 promoter demonstratesdifferential requirement for chromatin remodelling activities

Abstract 8 Vivian Bardwell . . . . . . . . . . . . . . . . . . . . . . . .Page 64Polycomb group and SCF ubiquitin ligases are found in a BCL6corepressor complex

Abstract 9 Ralph Bash . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 65AFM recognition imaging: using antibodies to track proteinsduring in vitro remodeling

Abstract 10 Juraj Bies . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 66Pc2, the polycomb group protein recruits the c-Myb into PcGbodies and inhibits its activity

Abstract 11 Caroline Bouchard . . . . . . . . . . . . . . . . . . . . .Page 67c-Myc induces localized H2A.Z exchange in the promoters oftarget genes

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Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Poster index

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Abstract 12 Ray Camahort . . . . . . . . . . . . . . . . . . . . . . . .Page 68Genome-wide localization of the budding yeast histone variantCse4

Abstract 13 Kendra Cann . . . . . . . . . . . . . . . . . . . . . . . . .Page 69Multiple regions of the DNA-damage response protein TLS/FUScan regulate its relocalization to the nucleolei followingtranscriptional inhibition

Abstract 14 Rafael Casellas . . . . . . . . . . . . . . . . . . . . . . .Page 70Effect of DNA double-stranded break repair on gene expression

Abstract 15 Beverley Chilton . . . . . . . . . . . . . . . . . . . . . . .Page 71It Takes Two to Tango: autorepression by RUSH-1α/β

Abstract 16 Sung Hee Baek . . . . . . . . . . . . . . . . . . . . . . .Page 72A chromatin remodeling complex in regulation of a metastasissuppressor gene

Abstract 17 Pierre Close . . . . . . . . . . . . . . . . . . . . . . . . . .Page 73Elongator depletion causes defective transcript elongation ofgenes that regulate cell motility

Abstract 18 Megan Cole . . . . . . . . . . . . . . . . . . . . . . . . . .Page 74Genome-wide map of nucleosome acetylation and methylationin yeast

Abstract 19 Jean-Francois Couture . . . . . . . . . . . . . . . . . .Page 75Structural insights into lysine multiple methylation by SETdomain methyltransferases

Abstract 20 Qi Dai . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 76Snail-mediated repression in Drosophila requires Ebi andhistone deacetylation

Abstract 21 Ana D’Alessio . . . . . . . . . . . . . . . . . . . . . . . . .Page 77The kinetics of chromatin-driven active DNA demethylation inliving cells

Abstract 22 Jan-Hermen Dannenberg . . . . . . . . . . . . . . . .Page 78Role of mSin3A in development, regulating transcriptionalnetworks and tumorigenesis

9

Poster Index

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Abstract 23 Gregory David . . . . . . . . . . . . . . . . . . . . . . . .Page 79Functional specialization of the mSin3 complex in developmentand oncogenesis

Abstract 24 James Davie . . . . . . . . . . . . . . . . . . . . . . . . . .Page 80Recruitment of phosphorylated HDAC2 to promoters

Abstract 25 Cecile de la Cruz . . . . . . . . . . . . . . . . . . . . . .Page 81Developmental regulation of Suz12 localization

Abstract 26 Stephan Diekmann . . . . . . . . . . . . . . . . . . . . .Page 82Functional complementation of human centromere protein A(CENP-A) by Cse4p

Abstract 27 Ivana Djuretic . . . . . . . . . . . . . . . . . . . . . . . . .Page 83Silencing of a mammalian gene, Interleukin-4 in T helperlymphocytes

Abstract 28 Tom Donndelinger . . . . . . . . . . . . . . . . . . . . .Page 84Asymmetric eukaryotic cell division and cellular dimorphism

Abstract 29 Yannick Doyon . . . . . . . . . . . . . . . . . . . . . . . .Page 85ING tumor suppressor proteins are critical regulators ofchromatin acetylation required for genome expression andperpetuation

Abstract 30 Katherine Dunn . . . . . . . . . . . . . . . . . . . . . . . .Page 86Independent phosphorylation events on the amino-terminus ofhistone H3 associated with transcription

Abstract 31 Christine English . . . . . . . . . . . . . . . . . . . . . . .Page 87ASF1 binds to a heterodimer of histones H3-H4: a two stepmechanism for the assembly of H3-H4

Abstract 32 Alex Erkine . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 88Nucleosome displacement at promoters of yeast heat shockgenes is proportional to the degree of transient histone H3acetylation

Abstract 33 Laure Escoubet-Lozach . . . . . . . . . . . . . . . . .Page 89Application of genome-wide location analysis to studychromatin modifications in a model of signal-dependent generegulation

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Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

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Abstract 34 Yuhong Fan . . . . . . . . . . . . . . . . . . . . . . . . . .Page 90A new link between linker histone H1 and DNA methylation

Abstract 35 Barna Fodor . . . . . . . . . . . . . . . . . . . . . . . . . .Page 91The full complement of Su(var) gene function in Drosophila

Abstract 36 Alexey Fomenkov . . . . . . . . . . . . . . . . . . . . . .Page 92Role of p63 in development disorders

Abstract 37 Benjamin Freedman . . . . . . . . . . . . . . . . . . . .Page 93Linker histone dynamics, structure, and function in interphaseand mitotic egg extract chromosomes

Abstract 38 Jennifer Gallagher . . . . . . . . . . . . . . . . . . . . .Page 94Exploring establishment of Sir1-dependent silencing usingcomparative genomics

Abstract 39 Nicolas Gévry . . . . . . . . . . . . . . . . . . . . . . . . .Page 95Implication of histone variant H2A.Z in the regulation of thep53/p21CIP1/WAF1 pathway

Abstract 40 Anja Groth . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 96The role of human Asf1 in histone metabolism duringreplicational stress

Abstract 41 Matthew Guenther . . . . . . . . . . . . . . . . . . . . .Page 97The active and Polycomb-repressed genome in humanembryonic stem cells

Abstract 42 Shweta Hakre . . . . . . . . . . . . . . . . . . . . . . . . .Page 98Growth factor mediated gene regulation by TFII-I

Abstract 43 Melissa Harrison . . . . . . . . . . . . . . . . . . . . . . .Page 99The MBT repeat-containing protein LIN-61 regulates C. elegansvulval cell-fate specification

Abstract 44 Michael Hendzel . . . . . . . . . . . . . . . . . . . . . .Page 100The regulation of histone H1 binding in vivo

Abstract 45 Wilma A.Hofmann . . . . . . . . . . . . . . . . . . . . .Page 101Specific roles for nuclear actin and nuclear myosin I intranscription by RNA Polymerase II

11

Poster Index

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Abstract 46 Sui Huang . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 102Upstream binding factor association induces large scalechromatin decondensation

Abstract 47 Dana Huebert . . . . . . . . . . . . . . . . . . . . . . . .Page 103Global analysis of MLL and MLL-fusion mediated histonemodifications

Abstract 48 Christina Hughes . . . . . . . . . . . . . . . . . . . . .Page 104Linking Menin mutations to defects in histone methylation andaltered expression of target genes

Abstract 49 Irena Ivanovska . . . . . . . . . . . . . . . . . . . . . .Page 105Histone code in meiosis: histone kinase required forchromosomal architecture in Drosophila oocytes

Abstract 50 Danika Johnston . . . . . . . . . . . . . . . . . . . . . .Page 106Role of Drosophila ecdysone receptor and histonemethyltransferase TRR in gene regulation

Abstract 51 Sarah Johnstone . . . . . . . . . . . . . . . . . . . . .Page 107Embryonic stem cell transcription factors regulatedevelopmental transcription factors

Abstract 52 Paul Kaufman . . . . . . . . . . . . . . . . . . . . . . . .Page 108Replication-independent histone deposition by the HIRcomplex and Asf1

Abstract 53 Ik Soo Kim . . . . . . . . . . . . . . . . . . . . . . . . . .Page 109A chromatin remodeling complex in regulation of crosstalkbetween the Wnt and the NF-kappaB pathway

Abstract 54 Elena Kisseleva-Romanova . . . . . . . . . . . . .Page 110Identification of new mutants that lead to transcription internalentry in S. cerevisiae

Abstract 55 Christoph Koch . . . . . . . . . . . . . . . . . . . . . . .Page 111A microarray based approach to develop high resolution mapsof histone modifications in 1% of the human genome

Abstract 56 Hyockman Kwon . . . . . . . . . . . . . . . . . . . . . .Page 112BAF53 is essential for maintenance of chromosome territoriesand higher-order chromatin structure

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Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

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Abstract 57 Eric Lam . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 113Chromatin charting in living plants: a global study

Abstract 58 Robert Lane . . . . . . . . . . . . . . . . . . . . . . . . .Page 114Does chromatin inaccessibility contribute to mutually exclusiveolfactory receptor transcription?

Abstract 59 Martin Law . . . . . . . . . . . . . . . . . . . . . . . . . .Page 115Mutations in the ATRX chromatin remodelling protein causechanges in histone H3 lysine 9 methylation

Abstract 60 Kenneth Lee . . . . . . . . . . . . . . . . . . . . . . . . .Page 116Functional analysis of the ADA histone acetyltransferasecomplex

Abstract 61 Young Han Lee . . . . . . . . . . . . . . . . . . . . . . .Page 117Epigenetic regulation of the tumor suppressor Egr-1 gene byoncogenic Ras

Abstract 62 Lin Li . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 118SP1 and SP3 dynamic association with estrogen regulatedpromoters

Abstract 63 Karen Lower . . . . . . . . . . . . . . . . . . . . . . . . .Page 119ATRX acts in a localised manner to down regulate theexpression of alpha globin genes in ATR-X syndrome

Abstract 64 Malik Lutz . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 120Geminin binding to Cdt1 controls, but does not necessarilyblock, Cdt1 function during replication origin licensing

Abstract 65 Dale Mackay . . . . . . . . . . . . . . . . . . . . . . . . .Page 121Identification of an acetyltransferase of the DNA bindingprotein

Abstract 66 Asoke Mal . . . . . . . . . . . . . . . . . . . . . . . . . . .Page 122Linking HDAC1 and MyoD function in Rhabdomyosarcoma

Abstract 67 Roberto Mantovani . . . . . . . . . . . . . . . . . . . .Page 123Epigenetic changes to NF-Y controlled genes upon ERinduction

13

Poster Index

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Abstract 68 Jotin Marango . . . . . . . . . . . . . . . . . . . . . . . .Page 124The multiple myeloma SET domain (MMSET) protein is ahistone H3 and H4 methyltransferase with properties of atranscriptional co-repressor

Abstract 69 Philip Marsden . . . . . . . . . . . . . . . . . . . . . . .Page 125The cell-specific and hypoxia-regulated expression ofendothelial nitric oxide synthase (eNOS) is controlled bychromatin-based mechanisms

Abstract 70 Darin McDonald . . . . . . . . . . . . . . . . . . . . . .Page 126Polymeric nuclear actin in chromatin modification and double-strand break repair

Abstract 71 Dominik Mojzita . . . . . . . . . . . . . . . . . . . . . .Page 127Pdc2, the yeast homologue of CENP-B, is a part of thecentromere-kinetochore complex

Abstract 72 David Mosser . . . . . . . . . . . . . . . . . . . . . . . .Page 128Chromatin remodeling across the proximal promoter region ofthe interleukin-10 gene in macrophages

Abstract 73 Toshinori Nakayama . . . . . . . . . . . . . . . . . . .Page 129Long range histone modification of the Type2 cytokine geneloci in developing Th2/Tc2 cells

Abstract 74 James Nicholson . . . . . . . . . . . . . . . . . . . . .Page 130Structural studies of the oxidised histone octamer

Abstract 75 Barbara Nikolajczyk . . . . . . . . . . . . . . . . . . .Page 131The IL-1 beta gene is transcribed from a poised promoterarchitecture in monocytes

Abstract 76 Keisuke Nimura . . . . . . . . . . . . . . . . . . . . . .Page 132Preferential association of Dnmt3l with Dnmt3a2 on chromatinin ES cells

Abstract 77 Esperanza Nunez . . . . . . . . . . . . . . . . . . . . .Page 133Repetitive elements, transcriptional control and cell diversity

Abstract 78 Laura O’Neill . . . . . . . . . . . . . . . . . . . . . . . . .Page 134Distinctive histone modifications mark specific chromosomesin both human and mouse models

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Poster Index

Abstract 79 Hisanobu Oda . . . . . . . . . . . . . . . . . . . . . . . .Page 135Generation and characterization of Set9 and PR-Set7conditional gene knockout mice

Abstract 80 Søren Ottosen . . . . . . . . . . . . . . . . . . . . . . .Page 136Bromodomain protein 7 (Brd7) promotes nucleolar disruptionin response to DNA breaks

Abstract 81 Mahadeb Pal . . . . . . . . . . . . . . . . . . . . . . . .Page 137The role of TFIIB and the transcription bubble during theearliest stages of transcription by RNA polymerase II

Abstract 82 Maria V. Panchenko . . . . . . . . . . . . . . . . . . .Page 138pVHL partner and transcriptional co-activator Jade-1 is a novelsubstrate for HAT TIP60

Abstract 83 Tej Pandita . . . . . . . . . . . . . . . . . . . . . . . . . .Page 139Mammalian ortholog of Drosophila MOF is critical forembryogenesis and essential for DNA damage responses

Abstract 84 Biranchi Patra . . . . . . . . . . . . . . . . . . . . . . . .Page 140Genomewide localization of histone modifications duringmeiosis

Abstract 85 Rushad Pavri . . . . . . . . . . . . . . . . . . . . . . . .Page 141A fully reconstituted chromatin transcription system for thedetailed study of epigenetic mechanisms

Abstract 86 Inka Pawlitzky . . . . . . . . . . . . . . . . . . . . . . . .Page 142A novel regulatory region 5’ of the mouse IgH locus

Abstract 87 Laura Perez-Burgos . . . . . . . . . . . . . . . . . . .Page 143Domain organization at the chicken β-globin locus

Abstract 88 Yuri Postnikov . . . . . . . . . . . . . . . . . . . . . . . .Page 144Modulation of histone modifications by chromatin-bindingarchitectural proteins

Abstract 89 Brendan Price . . . . . . . . . . . . . . . . . . . . . . . .Page 145The Tip60 histone acetyltransferase is essential for theacetylation and activation of the ATM protein kinase

Abstract 90 Michael Rehli . . . . . . . . . . . . . . . . . . . . . . . .Page 146Genome-wide profiling of CpG-methylation by methyl-CpG iim-munoprecipitation

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Abstract 91 Kristen Riley . . . . . . . . . . . . . . . . . . . . . . . . .Page 147Involvement of the TAC1 complex in transcription elongation

Abstract 92 Flavio Rizzolio . . . . . . . . . . . . . . . . . . . . . . .Page 148Chromatin alteration on POF associated X/A balancedtranslocation

Abstract 93 Orit Rozenblatt-Rosen . . . . . . . . . . . . . . . . .Page 149Paf1 complex is associated with mRNA processing machinery.

Abstract 94 Ingemar Rundquist . . . . . . . . . . . . . . . . . . . .Page 150Analyses of linker histone - chromatin interactions in situ indifferent cell systems

Abstract 95 Michael Scher . . . . . . . . . . . . . . . . . . . . . . . .Page 151SirT3 is A nuclear histone deacetylase that translocates tomitochondria upon stress

Abstract 96 Gunnar Schotta . . . . . . . . . . . . . . . . . . . . . .Page 152H4-K20 methylation: a mark important for mouse development?

Abstract 97 Bernd Schuttengruber . . . . . . . . . . . . . . . . .Page 153Effect of long-range chromosomal interactions mediated by theFab-7 element on its target chromatin

Abstract 98 Alexandra Schulmeister . . . . . . . . . . . . . . . .Page 154Histone H3.3 phosphorylation during mitosis and meiosis inthe urochordate Oikopleura dioica

Abstract 99 Jill Schumacher . . . . . . . . . . . . . . . . . . . . . .Page 155The Tousled kinase functions in mitosis as a substrate andactivator of the Aurora B kinase

Abstract 100 David Schrump . . . . . . . . . . . . . . . . . . . . . . .Page 156Gene expression profiling of primary lung cancers exposed to5 aza 2’ deoxycytidine (DAC), depsipeptide FK228 (DP), orsequential DAC/DP

Abstract 101 Brian E. Schwartz . . . . . . . . . . . . . . . . . . . . .Page 157Nucleosome assembly pathways discriminate between sites inthe H3 and H3.3 histone tails

Abstract 102 Kristin Scott . . . . . . . . . . . . . . . . . . . . . . . . .Page 158A heterochromatin barrier partitions the fission yeastcentromere into discrete chromatin domains

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Abstract 103 Judith Sharp . . . . . . . . . . . . . . . . . . . . . . . . .Page 159XIST functions independently of BRCA1 in X chromosomeinactivation

Abstract 104 Shivendra D. Shukla . . . . . . . . . . . . . . . . . . .Page 160Histone modifications by ethanol in liver

Abstract 105 Robert Sims . . . . . . . . . . . . . . . . . . . . . . . . .Page 161Regulation of mRNA biogenesis by the coordinator complex,which specifically recognizes methyl H3K4

Abstract 106 Jeffrey Smith . . . . . . . . . . . . . . . . . . . . . . . . .Page 162Genetic identification of chromatin-related regulators ofribosomal RNA synthesis in Saccharomyces cerevisiae

Abstract 107 Yee Sun Tan . . . . . . . . . . . . . . . . . . . . . . . . .Page 163Skp1 regulates transcription via mono-ubiquitylation

Abstract 108 Hannah Tims . . . . . . . . . . . . . . . . . . . . . . . .Page 164Spontaneous and catalyzed nucleosome conformationalchanges

Abstract 109 Patrick Trojer . . . . . . . . . . . . . . . . . . . . . . . .Page 165A functional interplay between MLL and LSD1

Abstract 110 Patrick Trojer . . . . . . . . . . . . . . . . . . . . . . . .Page 166L3MBTL1 - the chromatin lock?

Abstract 111 María Isabel Tussié Luna . . . . . . . . . . . . . . .Page 167Pro-proliferative function of the long isoform of PML-RARαinvolved in acute promyelocytic leukaemia

Abstract 112 Wim Vanden Berghe . . . . . . . . . . . . . . . . . . .Page 168Differential interleukin-6 gene expression dynamics in benignand metastatic breast cancer cells reflects distinct chromatinsignatures at the promoter region

Abstract 113 Adam Wood . . . . . . . . . . . . . . . . . . . . . . . . .Page 169The Bur1/Bur2 complex is required for histone H2Bmonoubiquitination by Rad6/Bre1

Abstract 114 Dag H. Yasui . . . . . . . . . . . . . . . . . . . . . . . . .Page 170MeCP2 recruits SNF2h to the 15q11-13 imprinting controlregion

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Poster Index

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Danny ReinbergHistone marks and chromatin states

Danny Reinberg

Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department ofBiochemistry, UMDNJ/RWJMS, RWJMS Research Building, 683 Hoes Lane, Piscataway,Middlesex 08854, U.S.A.

Dynamic regulation of chromatin structure is essential for cellular processes like replication,transcription, differentiation, cell-cycle progression and DNA-repair. Posttranslationalmodifications of histones turned out to be important regulators of these biological processes.However, the histone modifications are not sufficient to carry the biological message. Rather,the histone “marks” serve as recognition modules for proteins which are able to distinguishbetween different modified residues and can even distinguish between different degrees ofmodifications on the same residue in the case of histone lysine methylation. For example thechromodomain of HP1 and PC bind histone H3 methylated at lysine 9 and lysine 27,respectively. It is of great interest to find novel chromatin binding factors to understand howchanges of the chromatin structure are regulated.

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Abstracts – Oral

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Shelley BergerFactor and histone covalent modifications in genome regulationShelley Berger

Gene Expressia and Regulation Program, The Wistar Institute, 3601 Spruce Street,Philadelphia, PA, 19104, U.S.A.

Genomic structure and function is regulated in part through covalent post-translationalmodifications (PTMs) of factors and histones, including acetylation (ac), methylation (me),phosphorylation (ph), ubiquitylation (ub), and sumoylation (su). Our research focuses onpatterns and temporal sequences of PTMs and the interrelationship of factor PTMs andhistone PTMs in S. cerevisiae and mammalian cells. In yeast current projects focus ondynamic PTMs during transcriptional regulation and sporulation. We are working to unravelthe molecular outcomes of these PTMs. In transcriptional regulation our recent studies focus on the roles of histone H3 Ser10ph,H2B K123ub, and H2B/H4su. We find that, on one hand, H3 Ser10ph prevents the bindingof certain corepressor complexes from binding to histones, and on the other hand, itpromotes the binding of several novel transcriptional coactivator molecules. Thus, Ser10phhas an interesting dual role in transcriptional regulation. Secondly, we find that ub/deub ofH2B K123 regulates transcriptional elongation by directly regulating histone binding of aRNA polymerase II kinase, Ctk1. Ctk1 binding, in turn, establishes a balance of H3 K4mevs. K36me in the ORF. Hence, K123ub has a unique attribute among histone PTMs: it mustbe both added and then removed for full transcriptional activation. Finally, we identifyH2B/H4su as the first known negative histone modification in S. cerevisiae. The sumo markappears to block certain positive marks such as acetylation and ubiquitylation.We are also studying H4 Ser1ph as a histone PTM that regulates gametogenesis. Duringboth S. cerevisiae sporulation and metazoan spermatogenesis, H4Ser1ph persists after thedecline of H3 Ser10ph, the well-known mitotic/meiotic modification involved in chromosomecondensation. Our results suggest that H4 Ser1ph is involved in chromatin compactionduring late stages of sporulation. We are currently analyzing whether Ser1ph directlycompacts nucleosomes, or whether it promotes association of other molecules that carryout the compaction. Each of these PTMs (and many others!) appears to be conserved through evolution. Thus,mechanistic studies in S. cerevisiae, a genetically and biochemically tractable modelorganism, help us to understand the molecular outcomes of these complex patterns andtemporal sequences of modifications, which can then be addressed in higher eukaryotes.

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Abstracts - Oral

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Brad BernsteinGenomic studies of chromatin modifications in normal andmalignant cells

Brad Bernstein1,2, Dana Huebert1,2, Mike Kamal2, Phil Kapranov3, TomGingeras3, Stuart Schreiber2,4 and Eric Lander2

1Department of Pathology, Massachusetts General Hospital and Harvard Medical School,Boston MA, U.S.A. 2Broad Institute of Harvard and MIT. 3Affymetrix, Inc. 4Howard HughesMedical Institute, Harvard Chemistry and Chemical Biology, Boston MA, U.S.A.

To gain insight into the role of chromatin in regulating genome structure and function, wemapped the patterns of histone H3 Lys4 and Lys27 methylation across several dozengenomic loci in human and mouse. The majority of loci contain punctate methylated sitesthat coincide with gene starts or regulatory elements, and appear to reflect their activationstatus. A smaller number of loci, including the HOX clusters, contain much broader regionsthat are continuously associated with Lys4 or Lys27 methylated histones. These ‘chromatindomains’ are cell type specific and correlate with the expression of underlying genes.Insulator elements and highly conserved sequences define the boundaries of thesedomains, while associated trithorax and polycomb proteins appear to function in theirepigenetic maintenance. The domains are also affected by leukemogenic MLL fusionproteins, and we propose that this activity underlies the pathogenesis of MLLrearrangements.

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Hendrik StunnenbergHistone modification patterns associated with X-inactivationand escape

Arie B. Brinkman, Thijs Roelofsen, Sebastiaan W.C. Pennings, Joost H.A.Martens, Thomas Jenuwein, Hendrik G. Stunnenberg

Radboud University, NCMLS, Geert Grooteplein 28, Nijmegen, Gelderland 6525 GA, TheNetherlands.

X-inactivation is associated with chromosome-wide removal of euchromatic histonemodifications and deposition of heterochromatic histone modifications. While the resultingchromatin is classically viewed upon as facultative heterochromatin that is uniform in nature,the human Xi has been shown to be microscopically segregated into two distinct types ofheterochromatin, characterized either by tri-methylated H3K27 or H3K9. However, thefunctional consequences of such segregation of these modifications at the single-gene levelare unclear. Here we have analyzed the allelic distribution of histone modifications at twonon-contiguous X-chromosomal regions in human somatic cells. Segregation of H3K27me3and H3K9me3 is also evident at the single-gene level, and transitions between thesechromatin types are marked with local H3K4 tri-methylation and H3/H4 acetylation around 5ends of active genes. While these modifications are well-documented euchromatic marks,they do not necessarily mark ongoing transcription. Surprisingly, H3K27me3 is not exclusivefor inactive genes: although it marks X-linked intergenic regions and inactive gene codingregions, it similarly marks some escape genes. At the latter genes, H3K27me3 appears as adominant silencing mark over active modifications. Most remarkably, we find that H3K9me3is not a hallmark of heterochromatin per se, since it is predominant in coding regions ofactive genes, a phenomenon that is not restricted to the X-chromosome. These resultsargue against the exclusiveness of individual marks for heterochromatin or euchromatin, butrather suggest that composite patterns of multiple interdependent or mutually exclusivemodifications together signal gene expression status.

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Abstracts - Oral

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Sandra HakeBeyond the double helix: Understanding the functionalimportance of histone H3 and its variants

Sandra B. Hake1, Benjamin A. Garcia2, Monika Kauer1, Elizabeth M.Duncan1, Rob Diaz1, Judith Recht1, Graham Dellaire3, Stephanie Morris4,Jeffrey Shabanowitz2, David P. Bazett-Jones3, Brian D. Strahl4, Donald F.Hunt2 and C. David Allis1.

1The Rockefeller University, New York, USA, 2University of Virginia, Charlottesville, USA,3The Hospital for Sick Children, Toronto, Canada, 4University of North Carolina School ofMedicine, Chapel Hill, USA

Chromatin, the repeating polymer of DNA and associated histone proteins, is thephysiological template of our genome. As such, elaborate mechanisms have evolved tointroduce meaningful variation into chromatin for purposes of altering gene expression andother important biological processes, including the repair of damaged DNA andchromosomal dynamics. Introduction of covalent histone modifications, chromatinremodeling by ATP-dependent complexes, and utilization of histone variants are three majormechanisms by which variation can be introduced into the chromatin fiber. Together, thisvariation might form a “histone code” that remains poorly understood. We favor the generalview that histone proteins are major carriers of epigenetic information. The fundamentalstructure of chromatin suggests that all DNA-templated processes, including a wide range ofepigenetic phenomena, are influenced by chromatin alterations with far-reachingimplications for human biology and disease. We are particularly interested in the biologicalsignificance and function of histone H3 variants and their post-translational modifications.

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Yang ShiA new kid on the chromatin block: identification of the firsthistone demethylase LSD1 and its regulation

Yu-Jiang Shi, Fei Lan and Yang Shi

Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA02115, U.S.A.

Posttranslational modifications of histone N-terminal tails impact chromatin structure andgene transcription. While the extent of histone acetylation is determined both byacetyltransferases and deacetylases, it has been unclear whether histone methylation isalso regulated by enzymes with opposing activities. Here, we provide evidence that LSD1(alias KIAA0601/BHC110/p110/nPAO), a homolog of nuclear amine oxidases, functions as ahistone demethylase and transcriptional co-repressor. LSD1 specifically demethylateshistone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs viaan oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1causes an increase in H3 lysine 4 methylation and concomitant de-repression of targetgenes, suggesting that LSD1 represses transcription via histone demethylation. Furtheranalyses reveal that LSD1 stability, chromatin accessibility and demethylase activity areregulated by multiple factors associated with LSD1, suggesting that LSD1-mediated histonedemethylation is regulated dynamically in vivo. Taken together, these results thus identify ahistone demethylase conserved from S. pombe to human and reveal dynamic regulation ofhistone methylation by both histone methylases and demethylases.

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Abstracts - Oral

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Roland SchueleLSD1 demethylates repressive histone marks to promoteandrogen receptor dependent transcription

Eric Metzger, Melanie Wissmann, Na Yin, Judith M. Mller, RobertSchneider, Antoine H. F. M. Peters, Thomas Gnther, Reinhard Buettner,and Roland Schuele

Universitt Freiburg, 66 Zentrale Klinische Forschung, Breisacherstrasse, Freiburg, Baden-Wrttemberg79106,Germany

Gene regulation in eukaryotes requires the co-ordinate interplay of chromatin modulatingproteins with specific transcription factors such as the androgen receptor (AR)1. Geneactivation and repression is specifically regulated by histone methylation status at distinctlysine residues2. Here we show that the lysine specific demethylase 1 (LSD1)3 co-localiseswith AR in normal human prostate and prostate tumour. LSD1 interacts with AR in vitro andin vivo and stimulates AR-dependent transcription. Conversely, LSD1 knockdown abrogatesandrogen-induced transcriptional activation and cell proliferation. Chromatinimmunoprecipitation (ChIP) analyses demonstrate that AR and LSD1 form chromatin-associated complexes in a ligand-dependent manner. LSD1 relieves repressive histonemarks by demethylation of histone H3 at lysine 9 (H3-K9), thereby leading to de-repressionof AR target genes. Importantly, we identify pargyline as an inhibitor of LSD1. Pargylineblocks demethylation of H3-K9 by LSD1 and consequently, AR-dependent transcription.Thus, modulation of LSD1 activity offers a novel strategy to regulate AR functions. Here, welink for the first time demethylation of a repressive histone mark with AR-dependent geneactivation, thus providing a mechanism by which demethylases control specific geneexpression.

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Tony KouzaridesProline isomerisation of histone H3 regulates geneexpression

Chris Nelson and Tony Kouzarides

The Gurdon Institute, Tennis Court Road, Cambridge, Cambridgeshire CB2 1QN, U.K.

Prolines can exist in a –cis or -trans conformation. Enzymes exist, (peptidyl prolylisomerases) which can convert prolines from one conformation to the other. In an effort toestablish if histones are modified in this way, we have analysed peptidyl prolyl isomerasesin S. cerevisiae We find that FPR4 is an enzyme that can isomerise specific prolines inhistone H3 and that this isomerisation affects lysine methylation of H3. The cross-talkbetween proline isomerisation and lysine methylation has a consequence in the regulationof transcription of certain genes in yeast. These data define a novel enzymatic activity thatmodifies histones and regulates gene expression.

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Abstracts - Oral

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Chromatin Structure & Function Nassau, Bahamas, 15th - 18th November 2005

Victoria Lunyak ESET dependent H3-K9 methylation upon restructuring ofchromosomal domains during development.

V.V. Lunyak1, Cramer T.2, Zhu X.1, Nunez E. 1, Hutt K.1, Roy R.3, García-Díaz A.3, Sze S.H.4, Montoliu L.3, and Rosenfeld M.G.1

1School of Medicine, USCD, San Diego, USA. 2Charite University, Virchow Klinikum, Berlin,Germany. 3Centro Nacional de Biotecnologia (CNB-CSIC), Madrid, Spain. 4Departments ofComputer Science and Biochemistry and Biophysics, Texas A and M University, U.S.A.

A central question in development is the spatial and temporal order of molecular events by whichgenes silenced in precursor cells are ultimately activated in the mature organ in the correct cell-restricted fashion. Our study of the dynamics of chromatin modifications within the growthhormone (GH) gene locus in the developing pituitary gland demonstrate that di- or tri- methylationof K9-H3 is a property of silenced GH gene, however the changes in the degree of H3-K9methylation are tightly linked to the development of the gland. Our results suggest that di- and tri-H3-K9 methylation partitioning serves unique roles in the structural and functional organization ofchromosomal domains. We will present data suggesting that chromatin remodeling could be oneof the essential components of the molecular events controlling restructuring of the chromosomaldomains within the cells traversing from the precursor to differentiated entity. Cumulative dataobtained by analysis of nuclear positioning of the GH gene locus within developing cells point tothe existence of HMTase activity capable of providing differential degrees of H3-K9 methylation,as well as to a developmental window during which the enzymatic activity could be expressed orassembled into the functional complex(es). Our data demonstrate a new in vivo function of ESETin the restructuring of chromosomal domains during development. Transgenic over-expression ofthe ESET protein earlier in development (in contrast to its normal temporal pattern of appearance)results in premature relocation of the developmentally regulated gene from condensed DAPI-stained chromatin to euchromatic compartments, observed by Immuno-FISH analysis. Therelocation of the GH gene to euchromatic compartment coincides with the exchange of tri-H3-K9for di-H3-K9 methylation. This event is not only targeted to nucleosomes in the immediate localeof the GH promoter, but rather responsible for the establishment of a specialized chromatindomain in the GH-gene locus. Furthermore, we have mapped the developmentally regulatedboundary/insulator element involved in establishing the differential domains of tri-H3-K9 and di-H3-K9 methylated chromatin. We speculate that the boundary /insulator function can be, in part,provided by transcriptional activation of SINE B2 repeat embedded in the DNA segment, which issufficient to provide boundary activity in an enhancer-blocking assay. By using a bioinformaticapproach, we have designed an algorithm which predicts putative boundary/insulators within themouse genome and will present some data on a functional assessment of their action. Our data support the hypothesis that the boundary/insulators elements are unlikely to be as staticas previously thought, but rather act as a dynamic infrastructure adapted to the transcriptionaland/or developmental state of the cell, providing the plasticity required to respond todevelopmental and environmental cues.

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Yi ZhangHistone modifications in polycomb silencing and cancer

Yi Zhang

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill,Chapel Hill, NC 27599, U.S.A.

Increasing evidence suggest epigenetic modifications, particularly covalent histonemodifications play an important role in regulating gene activity. Malfunction of histonemodifications can cause serious problems including cancer. Previously, we havedemonstrated that methylation of H3-K27 by the EED-EZH2 Polycomb group complex playsan important role in Polycomb silencing. Using a biochemical approach, we have purified aPRC1-like Polycomb complex that mediates histone H2A ubiquitination linking Polycombsilencing to histone H2A ubiquitination. Although both H3-K27 methylation and H2Aubiquitination are involved in PcG silencing, the relationship between these twomodifications is not known. We are setting-up assay systems to address this issue. Of thehistone methyltransferases characterized so far, the yeast Dot1 and its human counterpart,hDOT1L, are unique due to their lack of a SET domain, and that they methylate a lysineresidue (K79) located in the globular domain of histone H3. To understand the biologicalfunction of hDOT1L, we set out to look for its functional partners. We found that hDOT1L iscapable of interacting with a number of MLL (mix lineage leukemia) fusion partners involvedin acute myeloid leukemia. The domains of the fusion partners involved in the interactionare sufficient for mediating leukemogenesis when fused to MLL. Myeloid transformationassay demonstrated that the enzymatic activity of hDOT1L is required for MLL-hDOT1Lfusion to transform bone marrow cells in vitro. These observations in combination with therecent demonstration that MLL is a histone H3-K4 methyltransferase suggest that changefrom H3-K4 methylation to H3-K79 methylation of key MLL target genes may be a majormechanism in leukemogenesis. Current work is focused on identification of these key targetgenes and examination of their expression in normal and leukemia states.

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Patrick GrantGeneration and recognition of histone modifications by theSAGA and SLIK HAT complexes in health and disease.

Marilyn G. Pray-Grant, Stacey J. McMahon, Michael S. Torok, Jeremy A.Daniel, Stephen P. Baker, Sharon R. Dent1 and Patrick A. Grant.

Department of Biochemistry and Molecular Genetics, University of Virginia School ofMedicine, Charlottesville, Virginia 22908, U.S.A., 1Department of Biochemistry andMolecular Biology, M.D. Anderson Cancer Center, University of Texas, Houston, Texas77030, U.S.A.

In eukaryotes, the condensation of DNA into chromatin provides a considerable obstacle tothe nuclear machinery driving processes such as DNA replication, transcription or repair.Importantly the structure of chromatin is a dynamic one, which permits the localizeddecondensation and remodeling that facilitates the progress of these processes. Over thepast few years considerable progress has been made into how chromatin remodelingoccurs and in the identification of the enzymatic machines that mediate these processes.An emerging theme in the field of chromatin research has been the significant role thatposttranslational modifications of histones play in regulating nuclear function. Theconserved SAGA and SLIK transcriptional coactivator complexes modulate chromatinfunction via histone H3 and H2B acetylation and H2B deubiquitination. These complexesboth generate specific epigenetic marks, but also respond to others, thus orchestratinghistone modification patterns that dictate expression of protein coding genes. Thechromodomains of Chd1 and bromodomain of Gcn5 regulate the enzymatic activity andrecruitment of SAGA/SLIK to chromatin. We have also identified other novel proteinmodules as components of these complexes, which confer additional capability in therecognition of histone modifications. Furthermore, we have recently reported the yeast homologue of the humanataxin-7 as a component of SAGA and SLIK, which in its polyglutamine expanded form isresponsible for the neurodegenerative disease spinocerebellar ataxia type7 (SCA7). Herewe identify the mechanism by which this polyQ protein abrogates SAGA-dependentnucleosome acetylation and present evidence that Gcn5 inactivation causes SCA7phenotypes.

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Vincent GeliStructure and function of the Set1 RNA recognition motif

Lionel Trsaugues, Pierre-Marie Deh, Alfonso Rodriguez-Gil, RaphalGurois, Isabelle Varlet, Sebastian Chavez, Herman van Tilbeurgh andVincent Geli

CNRS,UPR9027, IBSM,CNRS, 31 chemin Joseph Aiguier, Marseille 13402, France

The yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains in addition to itscatalytic SET domain, a conserved RNA Recognition Motif (RRM1). We present here thecrystal structure and the secondary structure assignment in solution of the Set1 RRM1.Although RRM1 has the expected RRM-fold, it lacks the typical RNA binding features ofthese modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combiningRRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4methylation is not affected by a point mutation in RRM2 that preserves Set1 stability butaffects RNA binding in vitro. In contrast destabilizing RRM1 reduces the total amount ofSet1 within the cell. The destabilization of RRM1 leads to a concomitant increase of di-methylation of H3K4 at the 5’-coding region of active genes at the expense of tri-methylation, whereas both, di- and tri-methylation, decrease at the 3 coding region. Takentogether, our results suggest that Set1 RRMs bind RNA, but Set1 RNA binding activity is notlinked to H3K4 methylation. We will bring new insights about the function of Set1 RRMs.

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Ali ShilatifardYeast COMPASS points the way to human MLL and its rolein pathogenesis of leukemia

Ali Shilatifard

Saint Louis University Cancer Center, Saint Louis University School of Medicine, SaintLouis, MO 63104, U.S.A.

Chromosomal rearrangements and translocations play a major role in the pathogenesis ofhematological malignancies. The trithorax related mixed lineage leukemia (MLL) genelocated on chromosome 11q23 is rearranged in a variety of aggressive human B and Tlymphoid tumors as well as acute myeloid leukemia (AML) in both children and adults. Wedemonstrated previously that MLL translocation into the ELL gene result in the developmentof leukemia and that the C-terminal domain of ELL is required for the leukemic function ofthe chimera. In order to better define the role of MLL in pathogenesis of leukemia, we havebeen studying the biochemical properties of MLL and MLL-related proteins. We havedemonstrated that the yeast MLL homologue, Set1 exist in a complex we call COMPASS,which is a histone methyltransferases capable of mono- di and trimethylating the fourthlysine of histone H3. We now know that the Drosophila and the human homologues ofCOMPASS are also histone methyltransferases. The posttranslational modifications ofhistones by methylation have emerged as a key regulatory mechanism for both repressionand activation of gene expression. Studies from our laboratory and others during the pastfew years have brought about a watershed of information defining the molecular machineryand factors involved in the recognition and modification of nucleosomal histones bymethylation. I will discuss our recent findings regarding the molecular mechanism andconsequences of histone modification by COMPASS and its homolog MLL, in the regulationof gene expression, development and pathogenesis of cancer.

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Kevin Struhl

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,240 Longwood Avenue, CI-204, Boston, MA 02115, U.S.A.

Abstract unavailable at time of print

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Oliver RandoGenome-scale characterization of chromatin structure

Chih Long Liu, Guo-Cheng Yuan, Tommy Kaplan, Michael Dion, StuartSchreiber, Steve Altschuler, Nir Friedman and Oliver Rando

Bauer Center, Harvard University, 7 Divinity Ave., Cambridge, MA 02138, U.S.A.

We have designed a tiled oligonucleotide microarray to sequence the primary structure ofhalf a megabase of yeast chromatin. We have found that yeast promoters exhibit aremarkably stereotyped chromatin architecture, with upstream regulatory DNA sequencefound in a long nucleosome-free region surrounded by two well-positioned nucleosomeswith a characteristic modification pattern. These modifications associated with promoternucleosomes comprise one of two groups of histone modifications, with the other groupconsisting of histone modifications that occur over coding regions and whose levelscorrelate with transcription levels. In yeast, histone modifications occur in fewcombinations, suggesting that much less information is carried in histone modifications thanis theoretically possible.

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Rolf SternglanzHistone mutations that suppress the silencing defects of ayeast sir3 Ala2Gly mutation

Xiaorong Wang, Vinaya Sampath and Rolf Sternglanz.

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY11794, U.S.A.

Transcriptional silencing in S. cerevisiae is mediated by the binding of a Sir3-Sir4 complexto the N-terminal regions of histones H4 and H3 after their deacetylation by Sir2. Theknown histone binding domain of Sir3 occurs in the C-terminal third of the protein. The N-terminus of Sir3 is also important for its silencing function but the binding partner(s) of thisregion of Sir3 are not known. In order to learn more about the function of the Sir3 N-terminus, we have carried out a screen to identify suppressors of the silencing defect of asir3 Ala2Gly mutation. One of the suppressors identified was a dominant mutation thatchanged residue 77 of H3 from Asp to Asn. H3 Asp77 is in a region of the nucleosomewhere several groups have identified mutations that affect silencing both positively andnegatively. It is also very near Lys79, the residue methylated by Dot1. Two recessivesuppressor mutations, not in the genes for H3 and H4, were also identified in this screen,and are currently being characterized.

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Antonin MorillonHistone variant HT2A-Z promotes promoter-proximalnucleosome dissociation upon transcription activation in S.cerevisiae.

A. Morillon2, E. Kisseleva-Romanova1, N. Karabetsou1, A. Nair1, J. Mellor1

1University of Oxford, Biochemistry department, Oxford, UK, 2CNRS, CGM, Gif/Yvette, France

In addition to transcription factors, chromatin modifications play a crucial role in regulatingtranscription. It has been shown that chromatin structures can be remodeled by shiftingnucleosomes along the DNA, by covalently modifying histones and by changing histonecomposition of nucleosomes. However, control of histone variants exchange and theirfunctional relationships with others histone modifications remain poorly understood. Here weshow that H2A-Z histone variant encoded by HTZ1 in S. cerevisiae is preferentiallyincorporated onto promoter region during transcriptional repression and dissociated whentranscription is activated. We present evidence that Bdf1 and Swr1 are required for therapid incorporation of H2A-Z during nucleosome re-assembly when GAL1 gene is switchedoff. During transcription activation H2A-Z dissociation is concomitant with histone H3depletion on GAL1 and MET16 promoter-proximal regions and is dependent on the histonechaperone Asf1. Finally H2A-Z is required to control the timing of histone H3 depletionsuggesting that H2A-Z defines a specialized promoter nucleosome.

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Sharon DentNew functions for histone modifying enzymes

Sharon R. Dent, Yvonne A. Evrard, Ping Bu, Wenchu Lin, Ke Zhang andJohn Latham

Department of Biochemistry and Molecular Biology, U.T. M.D. Anderson Cancer, Houston,Texas 77401, U.S.A.

Our lab uses genetic approaches to define functions of histone modifying enzymes in vivo.We reported previously GCN5 deletion in the mouse leads to embryonic lethality due toincreased apoptosis. New data show that this apoptosis is p53 dependent and may betriggered by telomere defects. Telomeres are shortened in cells from GCN5 mutantembryos, and these cells display increased numbers of chromosomal end associations andfusions. In the absence of both p53 and GCN5, an even greater incidence of telomerefusion is observed. These double mutant embryos also display defects in neural tubeclosure and brain development. Very similar defects in neural development are observed inmice homozygous for GCN5 HAT catalytic site mutations and in mice that carry ahypomorphic allele of GCN5. These data provide the first evidence that GCN5 may berequired both for telomere maintenance and proper expression of genes required for neuraldevelopment in the mouse. In another project, we discovered that deletion of SET1suppresses defects in chromosome segregation caused by mutations in IPL1 in yeast.Mutations in other components of COMPASS also suppress the ipl1-2 mutation, butmutations in PAF1 or H2B K123 do not. These results indicate that Set1 and the COMPASScomplex normally oppose functions of Ipl1, and that these effects are independent of thefunctions of Set1 in transcription initiation and elongation that are mediated by Paf1 andH2B ubiquitylation. Moreover, we determined that Set1 is required for methylation of akinetochore protein, Dam1. Methylation of Dam1 by Set1 inhibits phosphorylation of flankingserines by Ipl1. Our findings demonstrate that Set1 has important functions in mitosis, andthey suggest that antagonism between lysine methylation and serine phosphorylation is afundamental mechanism for controlling protein function.

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Jerry WorkmanSet2 methylates histone H3 K36 providing transcriptionalmemory that signals Rpd3S to deacetylate histones intranscribed regions to suppress spurious intragenictranscriptionMichael J. Carrozza1, Bing Li1, Laurence Florens1, Selene K. Swanson1,Kenneth K. Lee1,Wei-Jong Shia1, Scott Anderson2, John R. Yates2,Michael P. Washburn1 and Jerry L. Workman1

1Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110,2The Scripps Research Institute LaJolla, CA 92037

Yeast Rpd3 histone deacetylase plays a surprisingly important role at actively transcribedgenes. We characterized two distinct Rpd3 complexes, Rpd3L and Rpd3S, by MudPITanalysis. Both complexes shared a three subunit core and Rpd3L contains unique subunitsconsistent with a promoter targeted corepressor. Rco1 and Eaf3 were subunits specific toRpd3S. Mutants of RCO1 and EAF3 exhibited increased acetylation in the FLO8 and STE11open reading frames (ORFs) and the appearance of aberrant transcripts initiating within thebody of these ORFs. Mutants in the RNA polymerase II-associated SET2 histonemethyltransferase also displayed these defects. Set2 functioned upstream of Rpd3S andthe Eaf3 methyl-histone binding chromodomain was important for recruitment of Rpd3S andfor deacetylation within the STE11 ORF. These data indicate that Pol II-associated Set2methylates H3 providing a transcriptional memory which signals for deacetylation of ORFsby Rpd3S. This erases transcription elongation-associated acetylation to suppressintragenic transcription initiation.

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Nevan Krogan Protein complexes and pathways involved in chromatinfunction

Nevan J. Krogan1,2, Michael-Christopher Keogh3, Minkyu Kim3, SeanCollins4, Andrew Emili1,2, Jonathan Weissman4, Timothy Hughes1,2,Stephen Buratowski3 and Jack Greenblatt1,2

1Banting and Best Dept. of Medical Research and 2Dept. of Molecular and MedicalGenetics, Univ. of Toronto, Toronto, Ontario, Canada M5G 1L6; 3Harvard Medical School,Boston, MA 02115; 4Howard Hughes Medical Institute, Univ. of California, San Francisco,CA 94143, U.S.A.

Proteins are being systematically TAP-tagged in the budding yeast Saccharomycescerevisiae, then purified and identified by mass spectrometry in order to define the stablyassociated components of protein complexes and weak interactions among proteincomplexes. In order to initiate a systems biology approach to chromatin metabolism andthe transcription apparatus for RNA polymerase II, including initiation factors, elongation andtermination factors, RNA processing proteins, and chromatin modifying proteins, we arecombining the clustered protein purification data with synthetic genetic array (SGA) analysisand microarray analysis of gene deletion and Tet-promoter mutants. SGA analysis wasused to define proteins that are genetically linked to known components of the RNApolymerase II transcriptional apparatus and chromatin modifiers, and all the resultingproteins were subsequently tagged and purified. Deletion mutants for all the non-essentialproteins in this genetic and biochemical network were then assembled into a 384-strainminiarray, and SGA was used to systematically construct double mutants for almost all~150,000 possible pairwise combinations of deletion mutations. The growth rates of thedouble mutants were then assessed by an automated vision system, following which thegenes, and therefore the protein complexes, were organized into putative pathwaysaccording to the similarity of their genetic interaction profiles. Proteins and proteincomplexes were also organized into putative pathways by clustering the gene expressionprofiles of a large number of gene deletion and Tet-promoter mutants. Experimental tests ofthe organization of pathways containing both well-known and newly discovered chromatinmodifying complexes and other proteins will be discussed. Protein purification and SGAanalysis are also being used to define protein complexes and functional pathways in otheryeast biological systems, including protein trafficking and DNA repair.

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Bruno Amati Epigenetic determinants of Myc binding to the human genome

Ernesto Guccione, Francesca Martinato, Lucilla Luzi, GiacomoFinocchiaro, Andrea Cocito and Bruno Amati

European Institute of Oncology, IFOM-IEO Campus, Milan, Italy

The determinants of transcription factor (TF) binding to eukaryotic genomes remain largelyelusive. Using quantitative chromatin Immunoprecipitation (q-ChIP), we estimated that over11% of all human loci bear high-affinity Myc-binding sites in their 5’ regulatory regions thatconform to the consensus CACGTG (or E-box) [1]. Not all E-box-containing promoters whereefficiently bound by Myc, however, suggesting that epigenetic determinants are paramountfor selectivity. High-throughput ChIP-on-chip studies suggested that Myc associates witheven larger numbers of genomic sites, of which roughly 20% contain an E-box [2, 3]. Startingfrom the available datasets [1-4], we have used a combination of bioinformatics and large-scale q-ChIP to dissect the correlations between histone marks and Myc binding. Our data show that stretches of H3K4 di- and tri-methylation previously identified by ChIP-on-chip [4] are generally confirmed by q-ChIP and are significantly associated with other marks,including di-methyl H3K79, and acetyl H3K9, H3K18, H4K5 and H4K12. Remarkably, themajority of these H3K4-methylated regions are also associated with CpG islands. Wetherefore term them “euchromatic islands”. Remarkably, Myc-binding sites are almost exclusively found within euchromatic islands. Thepresence of tri-methyl H3K4, in particular, shows a very high level of correlation with Myc,consistent with the observation that high-affinity E-boxes are preferentially found within CpGislands [1]. Importantly, our data also show that the E-box is a major specificity determinant,being present in over 60% of all Myc target sites, far above the estimates reached by ChIP-on-chip. In fact, sites previously classified as Myc targets by ChIP-on-chip [2, 3] but lackingtri-methyl H3K4 and E-boxes generally fail to score as targets by q-PCR. Finally, the Myc dimerization partner Max shows a near-perfect correlation with Myc binding,while the E-box binding factors USF1 and USF2 show an overlapping, but distinct pattern.We hypothesize that preferential accessibility of euchromatic islands and/or selectiverecognition of histone marks are rate-limiting steps for sequence-specific binding of TFs, anddetermine their selectivity for only a minority of all potential DNA sites in the human genome.

1.Fernandez, P., et al., Genomic targets of the human c-Myc protein. Genes Dev, 2003. 17: p. 1115-1129.2.Cawley, S., et al., Unbiased Mapping of Transcription Factor Binding Sites along Human Chromosomes21 and 22 Points to Widespread Regulation of Noncoding RNAs. Cell, 2004. 116(4): p. 499-509.3.Li, Z., et al., A global transcriptional regulatory role for c-Myc in Burkitt’s lymphoma cells. Proc Natl AcadSci U S A, 2003. 100(14): p. 8164-8169.4.Bernstein, B.E., et al., Genomic maps and comparative analysis of histone modifications in human andmouse. Cell, 2005. 120(2): p. 169-181.

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Francois FuksA direct mechanistic link between the Polycomb Groupprotein EZH2 and DNA methyltransferases

E. Vir, C. Brenner, R. Deplus, L. Di Croce, Y. de Launoit and F. Fuks

Free University of Brussels, GE, CP614, Campus Erasme, 808 route de Lennik, Brussels,Belgium 1070

The establishment and maintenance of epigenetic gene silencing is fundamental to celldetermination and function. Essential epigenetic systems involved in heritable repression ofgene activity are Polycomb Group (PcG) proteins and DNA methylation. Here we show that these two silencing pathways are mechanistically linked. We find thatthe PcG protein EZH2 interacts with DNA methyltransferases (DNMTs) and associates withDNA methyltransferase activity in vivo. Chromatin immunoprecipitations indicate that bindingof DNMTs to the EZH2-repressed MYT1 promoter requires the presence of EZH2 and thiscorrelates with silencing of the MYT1 gene. Furthermore, EZH2 binding to the MYT1promoter coincides with CpG methylation of MYT1. Our results suggest that EZH2 serves as a molecular beacon for DNA methyltransferasesand highlight a previously unrecognised direct connection between two key epigeneticrepression systems.

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Julie AhringerChromatin regulation and sumoylation in the inhibition of Rasinduced vulval development in C. elegans

Gino Poulin, Yan Dong, Andrew G. Fraser, Neil A. Hopper and JulieAhringer

The Gurdon Institute, Tennis Court Road, Cambridge, Cambridgeshire CB2 1QN, U.K.

Regulation of chromatin plays an important role in transcriptional control. Although a largenumber of chromatin associated proteins have been identified, their cellular functions andassociations are not well understood. In C. elegans, a number of synMuv genes encodechromatin associated proteins involved in transcriptional repression, including an ortholog ofRb and components of the NuRD histone deacetylase complex; these are involved inantagonizing Ras signalling in the vulva. Altered regulation of Ras signalling and of thesechromatin proteins has been linked with metastasis and/or cancer. To identify othercomponents of this regulatory mechanism, we carried out a genome-wide RNAi screen.After RNAi of 16,757 genes, we found 19 synMuv genes, 9 of which are new. Based on thesequence of these genes and genetic epistasis experiments, we propose that at least fourpost-translational modifications converge to inhibit Ras-stimulated vulval development:sumoylation, histone tail deacetylation, methylation, and acetylation. We further show thatmany of the synMuv genes also cooperate in gene regulation outside the vulva, negativelyregulating the expression of the Delta homologue lag-2. As most of the genes identified inthis screen are conserved in humans, we suggest that similar interactions may be relevantin mammals for control of Ras signalling, cross-talk between these pathways, and cellproliferation.

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Peter BeckerSite-specific acetylation defines an embryonic form of ISWIthat associates with mitotic chromatin

Roger Ferreira, Mariacristina Chioda, Anton Eberharter, Tiziana Bonaldi,Axel Imhof and Peter B. Becker

Adolf-Butenandt-Institut-Molekularbiologie, University of Munchen, 44 Schillerstr, MunchenD-80336, Germany

Nucleosome remodeling enzymes are involved in regulating all aspects of chromatinmetabolism, but little is known how their activity is regulated. The nucleosome remodelingATPase ISWI, which is part of several remodeling complexes in Drosophila, catalyses thesliding of histone octamers on DNA. We now found that ISWI is acetylated at lysine (K) 753by the acetyltransferase GCN5 in vivo and in vitro. The sequence surrounding K753resembles the N-terminus of histone H3, where the corresponding lysine, H3K14 is also aprominent substrate of GCN5. This finding provokes the exciting possibility that properties ofnucleosomes and remodeling enzymes may be regulated in concert by acetylation. We visualised ISWIK753ac during Drosophila development with a specific antibody.ISWIK753ac is restricted to embryonic development and particularly abundant in the earliestdevelopmental stages. Remarkably, ISWIK753ac was found concentrated on mitoticchromosomes of synchronously dividing preblastoderm nuclei, in contrast to bulk ISWI,which is excluded from condensed metaphase chromatin. Thus, the K753 acetylation marksan ISWI form with novel, unprecedented properties. We hypothesize that the association ofthis acetylated ISWI form with metaphase chromosomes may contribute to the extremeplasticity of preblastoderm chromatin and to the rapidity of nuclear division cycles duringearly embryonic development.

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Stephen ReahMOF complex is required for histone H4 lysine 16acetylation in mammalian cells

Stephen Rea1, Stefan Pfister2, Mikko Taipale1, Peter Lichter2 and AsifaAkhtar1

1Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg,Germany, 2Department of Molecular Genetics, German Cancer Research Centre,Heidelberg, Germany

Reversible histone acetylation plays an important role in regulation of chromatin structureand function. Here, we report that the human orthologue of Drosophila MOF, hMOF, is ahistone H4 lysine K16 specific acetyltransferase. Knockdown of hMOF in HeLa and HepG2cells causes a dramatic reduction of histone H4K16 acetylation as detected by western blotanalysis and mass spectrometric analysis of endogenous histones. We also provideevidence that similar to the Drosophila dosage compensation system, hMOF and hMSL3form a complex in mammalian cells. hMOF and hMSL3 siRNA treated cells also showdramatic nuclear morphological deformations, depicted by a polylobulated nuclearphenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leadsto accumulation of cells in G2 and M phases of the cell cycle. Treatment with specificinhibitors of the DNA damage response pathway reverts the cell cycle arrest caused byreduction in hMOF protein levels. Furthermore, hMOF depleted cells show increasednumber of phospho-ATM and gH2AX foci, and have an impaired repair response to ionizingradiation. Taken together, our data show that hMOF is required for histone H4 lysine 16acetylation in mammalian cells and suggest that hMOF has a role in DNA damage responseduring cell cycle progression. In addition, we have recently purified the hMOF complex andalso identified target genes of this complex. These novel findings will also be discussed.

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Susana GonzaloDNA methyltransferases control telomere length inmammalian cells

Susana Gonzalo1, Isabel Jaco1 , Mario F. Fraga2, Taiping Chen3, En Li3,Manel Esteller2 and Mara A. Blasco1

1Telomeres and Telomerase Group. Molecular Oncology Program. Spanish National CancerCentre (CNIO), Madrid, SPAIN, 2Epigenetics Group. Molecular Pathology Program. SpanishNational Cancer Centre (CNIO). Madrid, SPAIN, 3Epigenetics Program. Novartis Institutesfor Biomedical Research. Cambridge, Massachussetts, USA

Mammalian telomeres present heterochromatic features, which include trimethylation ofH3K9, trimethylation of H4K20, and binding of HP1. Proper assembly of telomericchromatin is critical for telomere homeostasis. Thus, defects in activities that participate inthe assembly of heterochromatin (Suv39h HMTases, Rb family members) result in telomerelength deregulation. We demonstrate an unprecedented role for mammalian DNA methyltransferases in telomerelength control. Mouse ES cells deficient for Dnmt1, or both Dnmt3a and Dnmt3b showdramatically elongated telomeres compared to wildtype cells. Mammalian telomere repeats(TTAGGG) lack the canonical CpG methylation site, however, we found that mousesubtelomeric regions are highly methylated as indicated by bisulfite sequencing, and thatthis epigenetic modification is dramatically lost in the absence of the Dnmts. Chromatinimmunoprecipitation showed that histone modifications that mark telomeric chromatin, suchas H3K9 and H4K20 tri-methylation, remain unaltered at subtelomeric and telomericdomains in the absence of DNA methyltransferase activities. Importantly, lack of Dnmtsresulted in increased telomeric recombination as indicated by sister chromatid exchangesinvolving telomeric sequences. Our results demonstrate that DNA methylation controls thelength of TTAGGG repeats at mammalian telomeres independently of histone methylationby repressing recombination between telomeric sequences.

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Craig PetersonChromatin remodeling: Regulation of chromatin higher-orderfolding and DNA repair

Craig L. Peterson1, Manolis Papamichos-Chronakis1, Michael Shogren-Knaak1,2, Haruhiko Ishii1, Michael J. Pazin3 and Jocelyn Krebs4.

1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester,MA 01605; 2Department of Biochemistry, Biophysics, and Molecular Biology, Iowa StateUniversity, Ames, IA 50011; 3Laboratory of Cellular and Molecular Biology, National Instituteof Aging, NIH, Baltimore, MD 21224; 4Department of Biological Sciences, University ofAlaska, Anchorage, AK 99508, U.S.A.

Chromosome structure plays a key role in the regulation of all DNA-mediated processes,including transcription and DNA repair. Dynamic changes in chromosome structure arecatalyzed by ATP-dependent chromatin remodeling enzymes and by histone modifyingenzymes, such as histone acetyltransferases. We will present two stories that illustratedistinct roles for both types of chromatin-based enzymes. First, we will discuss our recentwork on Ino80 and Swr1 which are ATP-dependent chromatin remodeling enzymes thathave been implicated in DNA repair. We have found that Ino80 is required for cell cyclecheckpoint adaptation in response to a persistent DNA double strand break (DSB). Thefailure of cells lacking Ino80 to escape checkpoint arrest correlates with an inability tomaintain high levels of histone H2AX phosphorylation and an increased incorporation of theHtz1p histone variant into chromatin surrounding the DSB. Inactivation of Swr1 eliminatesthis DNA damage-induced Htz1p incorporation and restores H2AX phosphorylation andcheckpoint adaptation. We propose that Ino80 and Swr1 function antagonistically atchromatin surrounding a DSB, and that they regulate the incorporation of different histoneH2A variants that can either promote or block cell cycle checkpoint adaptation.The second story will focus on histone H4 lysine 16 acetylation which is a prevalent andreversible post-translational modification in eukaryotic organisms. To characterize thestructural and functional role of this mark in the context of chromatin, we have used a nativechemical ligation strategy to generate histone H4 homogeneously acetylated at K16. Whenthis histone is incorporated into nucleosomal arrays, we find that acetylation of this singlelysine residue inhibits the formation of compact 30 nm-like fibers and impedes the ability ofchromatin to form cross-fiber interactions. Histone H4 K16Ac also inhibits the ability of theACF chromatin remodeling enzyme to mobilize a mononucleosome. This is the firstexample of a single histone modification that is sufficient to modulate higher-orderchromatin structure as well as perturb functional interactions between a nonhistone proteinand the chromatin fiber.

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Bob KingstonRemodeling chromatin without covalent modification

Jonathan Dennis, Rebecca Dunn, Nicole Francis, Ian King, *ChrisWoodcock and Bob Kingston

Massachusetts General Hospital, Boston, MA, U.S.A. and *University of Massachusetts,Amherst, MA, U.S.A.

Master regulatory genes, such as Hox genes, must be maintained in a heritably ‘on’ state insome cell lineages and in a heritably ‘off’ state in other cell lineages. Genetic studies haveidentified numerous genes that are required for this epigenetic level of regulation to workproperly. Many of these genes are classified as Polycomb-Group (PcG) or trithorax-Group(trxG) based on the screen that led to their initial isolation. PcG genes are required tomaintain repression. We have identified and characterized a complex called PRC1 thatcontains four PcG genes. This complex interacts with nucleosomal templates to blocktranscription and remodeling of those templates. Repressed templates have aredifferentially sensitive to various enzymatic probes and appear compacted when visualizedby microscopy. The Esc/E(z) complex (also called PRC2) has been proposed to targetPRC1 action via methylation of lysine 27 of histone H3. We are using recombinantcomplexes to address functional interactions between the PRC complexes and nucleosomaltemplates. To further characterize the functions of these complexes, we are developingtechnologies to analyze chromatin structure in living cells. Our goal is to develop anautomatable protocol to allow mapping of chromatin structure across entire Hox clusters incell lines under different stages of differentiation.

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Lianna JohnsonDNA and histone methylation in Arabidopsis

Lianna Johnson, Simon Chan, Xiaoyu Zhang, Yana Bernatavichute andSteven Jacobsen

UCLA, Life Sciences, Box 951606, Los Angeles, CA 90095-1606, U.S.A.

Three different classes of DNA methyltransferases are found in Arabidopsis. Each type ofmethyltransferase is targeted by a different mechanism. siRNAs are involved in targetingDRM2 to various repeated DNAs and also in targeting DNA for de novo methylation.Histone H3 methylation is critical for targeting CMT3 to specific regions of the chromatin.MET1 is specific for methylation of CG residues, and assumed to work like its homologs inmammals (Dnmt1), where hemimethylated DNA resulting from replication is the preferredsubstrate. Using as a model a SINE element found in euchromatin in Arabidopsis, mutantswhich block these pathways at various points will be examined for DNA methylation, histonemethylation and transcription to arrive at a simple overall model for targeting of DNA andhistone methylation.

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Eric MiskaShort RNAs in C. elegans development and human cancer

Eric Miska

Gurdon Institute, University of Cambridge, The Henry Wellcome Building, Tennis Court Rd.,Cambridge CB2 1QN, U.K.

In the last five years microRNAs (miRNAs) have emerged from the obscurity of the C.elegans heterochronic pathway to a new paradigm of gene regulation in plants andanimals. Currently, microRNAs represent 2% of all known human genes. Very little isknown about their biological function. We have taken a functional genomics approach to study the roles of microRNAs in C.elegans development. We have generated knockout strains corresponding to 96microRNAs, covering the majority of known microRNA genes. We will present anoverview of the classes of mutantphenotypes we have observed. One focus will be the issue of redundancy withinfamilies of microRNA genes. This study represents the first comprehensive analysis ofmicroRNA function.We are also interested in the roles of short RNAs in the control of gene expression atthe transcriptional level. We will present our work on how these short RNAs worktogether with a set of argonaute proteins to control germline development in C.elegans.

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Ramin ShiekhattarIntegrator, a multiprotein mediator of small nuclear RNAprocessing associates with the C-terminal repeat of RNApolymerase II

David Baillat1, Mohamed-Ali Hakimi2, Anders Näär3, Ali Shilatifard4, NeilCooch1, and Ramin Shiekhattar1,5

1The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, 2CNRS, UMR5163,Université Joseph Fourier, La Tronche, 38700, France, 3Harvard Medical School andMassachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA02129, 4Department of Biochemistry and Molecular Biology, Saint Louis University HealthSciences Center, Saint Louis, MI 63104

The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is an essential component oftranscriptional regulation and RNA processing of protein coding genes. A large body of dataalso implicates the CTD in the transcription and processing of RNAPII-mediated smallnuclear RNA (snRNA) genes. However, the identity of the complex(es) that associate withthe CTD and mediate the processing of snRNA genes have remained elusive. Here, wedescribe an RNA polymerase II complex that contains at least twelve novel subunits, termedthe Integrator, in addition to core RNAPII subunits. Two of the Integrator subunits displaysimilarities to the subunits of the cleavage and polyadenylation specificity factor (CPSF)complex. We show that Integrator is recruited to the U1 and U2 snRNA genes and mediatestheir 3’-end processing. The Integrator complex is evolutionarily conserved in metazoansand directly interacts with the C-terminal domain of the RNA polymerase II large subunit.

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Kami Ahmad

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,240 Longwood Avenue, CI-204, Boston, MA 02115, U.S.A.

Abstract unavailable at time of print

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Jacques CoteDissection of the NuA4 HAT complex and crosstalk with otherchromatin modifications and remodelers

Andranne Auger, Nicolas Lacoste, Marilyn Pray-Grant, Joelle Brodeur,Rhea T. Utley, Yannick Doyon, Olivier Jobin-Robitaille, Stphane Allard,Luc Gaudreau, Patrick Grant and Jacques Cotes

Laval University Cancer Research Center, Hotel-Dieu de Quebec (CHUQ),9 McMahonStreet, Quebec City, G1R 2J6 Canada

Acetylation of histone H4 N-termini is a major chromatin modification important for geneactivation. The NuA4 histone acetyltransferase complex is the only essential HAT in yeastand is responsible for H4 and H2A hyperacetylation in vivo. Thorough genetic andbiochemical analysis of the 13-subunit assembly identified 4 different subcomplexes, eachone linked to specific functions in the cell. Protein domains present in the complex regulateits association with chromatin substrates, in part through recognition of specific histonemodifications like H4/H2A phosphorylation and H3 methylation. For example, methylation ofH3 on lysine 4 and 36 regulates NuA4 action on chromatin. This effect is mediated by thesubunit Eaf3 and its chromodomain, which directly associates with MeK4 and MeK36 invitro. These interactions regulate NuA4 function not only during the process of transcriptionbut also in DNA repair. In addition, findings in yeast, Drosophila and human systemssuggests a link between NuA4 activity and the incorporation of histone variant H2AZ inchromatin. We demonstrate new substrate specificity for NuA4 as it efficiently acetylatesH2AZ in chromatin substrates. Depletion of NuA4 also affects H2AZ incorporation atspecific loci in vivo, supporting the concept of functional cooperation between NuA4 andSwr1 complexes during transcription regulation of specific genes.

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Benoit GuillemetteH2A.Z is globally localized to the promoters of inactive genesand regulates nucleosome positioning

Benoit Guillemette1, Alain R. Bataille2, Nicolas Gévry1, Maryse Adam1,Mathieu Blanchette3, François Robert2 and Luc Gaudreau1

1Centre de Recherche sur les Mécanismes du Fonctionnement Cellulaire, Département deBiologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada2Laboratoire de Chromatine et Expression du Génome, Institut de Recherches Cliniques deMontréal, Montréal, Québec, Canada, 3McGill Center for Bioinformatics, Lyman Duff MedicalBuilding, Montréal, Québec, Canada

H2A.Z is a highly conserved variant of canonical histone H2A that plays many distinct roles.In Saccharomyces cerevisiae, H2A.Z acts as an anti-silencing factor by preventing thespread of heterochromatin. H2A.Z is also functionally redundant with components of SAGAand SWI/SNF, and it is essential for the full expression of certain genes such as PHO5 andGAL1. We have previously shown that H2A.Z co-precipitates with, and is essential forRNAPII recruitment at the GAL1 promoter. Therefore, H2A.Z may possess dual roles inregulation of transcription. In order to better understand the function(s) of H2A.Z in yeast, we used a genome-widebinding assay (ChIP on chip) to map H2A.Z with at ~300bp resolution. We find that H2A.Z isglobally located within small regions (1-2 nucleosomes wide) across the genome. These Z-loci are also mostly located in the promoters of yeast genes, and surprisingly, H2A.Z bindingis higher in low-, rather than in highly-transcribed genes. A total of 63% of yeast promoterscontain a Z-locus. We also show that nucleosomes in genes that contain a Z-locus in theirpromoter show a regular binding pattern, whereas nucleosomes in non-Z-locus genes areless well organized. Using GAL1 as model gene, we find that deletion of HTZ1 causes ashift in a specific nucleosome positioned over the transcriptional start site. Finally, Z-loci arewider in telomere proximal regions, where the H2A.Z anti-silencing function was shown tobe important. These results suggest that H2A.Z contributes gene regulation by poising inactive genes bymodifying nucleosome phasing around promoters. They also support a model in whichH2A.Z could possess distinct mechanisms in the regulation of telomere-proximal genes thanin other genes.

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Karolin LugerStructure and function of yeast nucleosome assembly protein 1

Young-Jun Park, Jayanth Chodaparambil and Karolin Luger.

Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology,Colorado State University, Fort Collins, Colorado, USA 80523-1870

Nucleosome assembly protein 1 (NAP-1) is a key component in the modulation of chromatinstructure. It shuttles histones into the nucleus, assembles nucleosomes, and promoteschromatin fluidity by removal and replacement of histone H2A-H2B dimers, therebypromoting nucleosome sliding. Biochemical and biophysical experiments further suggestadditional roles of NAP-1 in ‘scavenging’ misassembled chromatin. Together, theseactivities are likely to affect transcription of many genes. The 3.0 Å crystal structure ofyeast NAP-1 reveals a homodimer with a novel fold. A long a-helix is responsible fordimerization via a novel antiparallel non-coiled coil, and an α/β domain is implicated inprotein – protein interactions. The four-stranded anti-parallel β-sheet that characterizes theα/β domain is found in all histone chaperones, despite absence of homology in sequence,structural context, or quarternery structure. This is the first structure of a member of thelarge NAP family of proteins, and suggests a mechanism by which histones are bound, andby which the shuttling of histones to and from the nucleus is regulated.

Supported by NIH / GM67777

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Wolf Reik

Regulation of imprinting and epigenetic reprogramming inmammals

A Lewis, A Murrell, F Santos, H Morgan, W Dean and W Reik

Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, CambridgeCB2 4AT, U.K.

Imprinting in mammals has important roles in development, including in fetal growth,postnatal adaptions, and adult behaviour. Imprinted genes occur in clusters, shareregulatory elements, and can be epigenetically regulated by imprinting centres. Our recentwork reveals how imprinting centres may control long range expression and epigeneticprofiles in imprinting clusters. Imprinting centre 1 (the insulator region upstream of H19 ondistal chromosome 7 in the mouse) mediates physical contact, on the maternalchromosome, with a silencer sequence in the Igf2 gene, about 100kb away, and theintervening DNA is looped out. On the paternal chromosome, the same region is in contactwith an activator sequence in Igf2. This higher order structure results in the maternal Igf2gene being located in a silent chromosome loop, away from the enhancers, while thepaternal Igf2 gene is located outside the silent loop in close proximity to enhancers. Thusthe insulator (IC1), presumably through its differential methylation,mediates long distance gene regulation and epigenetic marking in the Igf2-H19 cluster. In the neighbouring IC 2 cluster, a paternally expressed non-coding RNA transcript(Kcnq1ot1) is flanked by several paternally repressed protein coding genes. Most of thesegenes are specifically imprinted in the placenta, and lack differential DNA methylation. Thenon-coding transcript, directly or indirectly, mediates the targeting of repressive histonemodifications to the flanking genes, and silencing of the flanking genes in the placenta ismaintained in the absence of DNA methylation. This mechanism has striking similarities withthat of imprinted X chromosome inactivation. We argue that there are at least two differentbuilding principles by which different imprinting clusters are constructed.

Murrell et al. (2004) Nature Genet. 36, 889-893,Osborne et al (2004) Nature Genet. 36, 1065-1071,Lewis et al. (2004) Nature Genet. 36, 1291-1295,Constancia et al. (2004) Nature 432, 53-57.Reik and Lewis (2005) Nature Rev. Genet. 6, 403-410.

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Aidan DohertyStructure and function of the Jumonji C domain

Andrew Green1, David Lando2, Nigel C. Brissett1, Tony Kouzarides2 andAidan J. Doherty1

1Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, U.K.2Gurdon Institute and Department of Pathology, University of Cambridge, Tennis CourtRoad, Cambridge, CB2 1QR, U.K.

Jumonji domains (JmjC) are present in a wide range of proteins that often contain chromatinbinding domains (1). There is significant sequence similarity between JmjC and the FIH-1family of metalloenzymes. Combined structural and genetic approaches have identified thatFIH is a member of the 2-oxoglutarate-dependent dioxygenase superfamily (2,3). Thesecondary structure of the JmjC domain also predicts enzymatic activity (1,2), and itsfrequent association with putative chromatin modification activities suggests a role for JmjCin regulating the integrity of chromatin structure (1). This link with chromatin remodellingfunctions has been supported by the finding that the S. pombe JmjC domain protein, Epe1,modulates heterochromatization in fission yeast (4). Many enzymes regulate chromatinstructure via covalent modifications and JmjC represents an excellent candidate for anadditional novel modifying activity, possibly a hydroxylase, that regulates the integrity ofchromatin structure. As JmJc proteins are similar to members of the FIH family of protein hydroxylases (2), JmjCwas assayed for hydroxylase activity. In the presence of histones and co-factors (2-oxoglutarate, oxygen and Fe2+) we observed that JmjC is an active protein hydroxylaseand we identified the specific residues modified on the histone substrate. To confirm thatthis activity is specific to the JmjC protein, active site mutations in the JmjC domain wereproduced. These mutants were inactive in the hydroxlase assay, confirming that the activityis specific to JmjC. This work establishes, for the first time, that JmjC domains areenzymes (dioxygenases) that can catalyse histone-specific hydroxylation events in vitro.The crystal structure of a Jumonji domain revealed that the enzyme has a beta-barrel ‘jelly-roll’ conformation, similar to FIH-1 (2), that contains an iron co-ordination motif at thecatalytic centre with co-substrate 2-oxoglutarate bound in the active site. Thus JmjCappears to represent a new family of histone modifying enzymes.

1. Clissold, P.M. and Ponting, C.P. (2001) Trends Biochem Sci. 1, 7-9.2. Elkins JM, Hewitson KS, et al. Ratcliffe PJ, Schofield CJ. (2003) J Biol Chem. 278, 1802-6.3. Lando D, Peet DJ, Whelan DA, Gorman JJ, Whitelaw ML. (2002) Science 295, 858-861. 4. Ayoub N, Noma K, Isaac S, Kahan T, Grewal SI, Cohen A. (2003) Mol Cell Biol. 23, 4356-70.

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Andreas LadurnerHuman chromatin as a molecular target of cellular NADmetabolites

Georg Kustatscher, Bjrn Fritz and Andreas G. Ladurner

EMBL, Gene Expression Unit, Meyerhofstrasse 1, Heidelberg 69117, Germany

The role of NAD metabolites in chromatin structure and regulation is receiving increasingbiochemical attention. We have recently shown that the SirT1-metabolite O-acetyl-ADP-ribose binds the human macroH2A1.1 histone variant through its C-terminal macro domain.Structural evidence, as well as engineered mutants reveal how the metabolite is selectivelyrecognized. The recognition of nucleotide metabolites by human chromatin suggest theexistence of a novel regulatory paradigm in biology.In addition, we find that mutually-exclusive exon use in the MACROH2A1 gene produces asecond macroH2A isoforms (known as macroH2A1.2) which cannot bind O-acetyl-ADP-ribose. Alternative splicing may thus regulate the sensitivity of human chromatin toward thepotential metabolite regulator. We will discuss our most recent evidence, which suggestsdistinct tissue distribution between the known macroH2A variants. Further, we will report onour efforts to understand the functional consequences of NAD metabolite binding bychromatin. The histone variant macroH2A is a hallmark of mammalian heterochromatin. Ourstudies are directed at dissecting the role of these interesting NAD metabolites inmammalian chromatin.

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Jef BoekeComprehensive mutagenesis of sites of histone modification

Edel M. Hyland, Michael S. Cosgrove, Henrik Molina, Dongxia Wang,Akhilesh Pandey, Robert J. Cotter and Jef D. Boeke

339 Broadway Research Building,733 North Broadway, Baltimore, MD 21205, U.S.A.

The biological significance of recently described modifiable residues in the globular core ofthe bovine nucleosome remains elusive. We have mapped these modification sites onto theSaccharomyces cerevisiae histones and used a genetic approach to probe their potentialroles both in heterochromatic regions of the genome, and in the DNA repair response. Bymutagenizing these residues to mimic their modified and unmodified states we havegenerated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably,despite the apparent evolutionary pressure to conserve these near-invariant histone aminoacid sequences, the vast majority of mutant alleles are viable. However, a subset of thesevariant proteins elicit an effect on transcriptional silencing both at the rDNA locus and attelomeres, suggesting that post-translational modification(s) at these sites regulatesformation and/or maintenance of heterochromatin. Furthermore, we provide direct massspectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We alsoshow that substitutions at histone H4 K91, K59, S47, R92 and histone H3 K56, K115 lead tohypersensitivity to DNA damaging agents, linking the significance of the chemical identity ofthese modifiable residues to DNA metabolism. Finally we allude to the possible molecularmechanisms underlying the effects of these modifications. The project is currently beingexpanded to histones H2A and H2B.

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Ted Abel Abstract 1The role of CBP and histone acetylation in memory storageand synaptic plasticity

T. Abel1, M.P. Kaplan1, C. Vecsey1, K. M. Lattal2, J.M. Stein1, S.A. Fabian1,M.A. Attner1, J.D. Hawk1 and M.A. Wood1

1Biology, University of Pennsylvania, Philadelphia, PA, U.S.A. 2Behavioural Neuroscience,Oregon Health and Science University, Portland, OR, U.S.A.

Transcriptional activation is thought to be a key process in long-lasting forms of memoryand synaptic plasticity. This activation is directed by transcription factors and theircoactivators, which regulate gene expression via chromatin remodeling, histone modificationand interactions with the basal transcription machinery. One type of histone modificationassociated with transcriptional activation is acetylation, which is regulated by histoneacetyltransferases (HATs) and histone deacetylases (HDACs) that add or remove acetylgroups from histones, respectively. Recently, we have demonstrated that the transcriptionalcoactivator CREB-binding protein (CBP), a potent HAT, is involved in specific forms of long-term memory and synaptic plasticity. We have examined mice in which CBP activity inneurons is reduced either by the transgenic expression of an inhibitory form of cbp lackingthe HAT domain or by knocking in a mutation of the CREB transcription factor-binding KIXdomain of cbp. This genetic approach enabled us to compare the role of CBP-associatedHAT activity in memory and synaptic plasticity with the role of the KIX transcription factor-binding domain of CBP. We found that mutant mice expressing an inhibitory form of cbpexhibit impairments in spatial and contextual memory and in long-lasting forms ofhippocampal synaptic plasticity. KIX knock-in mice were also observed to have significantimpairments in contextual memory. A complementary method to study the role of histoneacetylation in synaptic plasticity and memory is to examine the effects of HDAC inhibitors,which increase the level of histone acetylation that correlates with transcriptional activation.We found that increasing histone acetylation using the HDAC inhibitor TSA enhances long-term contextual memory and facilitates synaptic plasticity via the transcription factor CREB.In summary, these results support the idea that histone acetyltransferases and histoneacetylation are critical to mechanisms of long-term memory storage. Histone acetylationmay provide an epigenetic mechanism for establishing gene-specific modifications thatresult in the coordinate expression of genes required for long-term memory storage.

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Melissa Adkins Abstract 2Spt6-mediated nucleosome reassembly is required fortranscriptional repression of the PHO5 gene

Melissa W. Adkins and Jessica K. Tyler

Department of Biochemistry and Molecular Genetics, University of Colorado HealthSciences Center, PO Box 6511, Mail Stop 8101, Aurora, CO 80045 U.S.A.

The packaging of the eukaryotic genome into chromatin is likely to have profound influenceon transcription from the underlying genes. We have previously shown that thedisassembly of promoter nucleosomes is obligatory for activation of the yeast PHO5 andPHO8 genes. Here we show that the PHO5 promoter nucleosomes are reassembledconcomitant with transcriptional repression and displacement of the TATA-binding protein(TBP) and RNA polymerase II. We identify the histone H3-H4 chaperone Spt6 as the factorthat mediates replication-independent nucleosome reassembly onto the PHO5 promoter aswell as the PHO8 promoter during transcriptional repression. Furthermore, we demonstratethat nucleosome reassembly is essential for transcriptional repression of the PHO5 andPHO8 genes. Finally, we show that in the absence of Spt6-mediated nucleosomereassembly, the activators Pho4 and Pho2 are displaced from the promoter in repressingconditions, yet transcription is sustained. As such, these studies demonstrate that the Pho4and Pho2 activators are not required for transcription in the absence of competingchromatin reassembly.

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Adriana Alejandro-Osorio Abstract 3The role of the histone deacetylase Rpd3p in coordinatingthe environmental stress response in yeast.

Adriana L. Alejandro-Osorio1 and Audrey Gasch2

1Department of Biomolecular Chemistry., 2Laboratory of Genetics and Genome Center ofWisconsin, University of Wisconsin-Madison, 3452 Genetics-Biotech Center, 435-g HenryMall, Madison WI 53706, U.S.A.

Unicellular organisms must be able to withstand frequent and stressful changes in theirenvironment. The budding yeast Saccharomyces cerevisiae initiates a large geneexpression program in response to diverse types of environmental stress (Gasch et al.2000, Causton et al. 2001). This response, called the environmental stress response(ESR), consists of ~300 genes whose expression is induced and ~600 genes whoseexpression is repressed in response to stressful conditions. A number of transcriptionfactors have been implicated in ESR regulation, however how these expression changesare coordinated is not understood. We have investigated the role of chromatin remodelingin ESR regulation and found that the histone deacetylase, Rpd3p, is required for initiation ofthe ESR. Cells lacking rpd3 fail to properly regulate expression of the ESR genes and aresensitive to a panel of stresses. This suggests that chromatin remodeling plays animportant role in coordinating the expression of this extensive gene expression response.

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Erik Andersen Abstract 4MET-1 and MET-2, two putative histone methyltransferases,are redundantly required for proper vulval cell fate specification

Erik Andersen and Bob Horvitz

MIT Biology 68, Room 417, 77 Massachusetts Ave., Cambridge, MA 02139, U.S.A.

Genetic screens for mutations that affect the vulval cell fate of Caenorhabditis elegans wasfirst to identify many homologs important in the regulation of human cancer. An increase ordecrease in the number of cells that adopt vulval cell fates is easily observed as either aMultivulva (Muv) or Vulvaless (Vul) phenotype respectively. These two types of mutants ledto the identification of components of the RTK/Ras, Notch, and Wnt pathways. The Muvphenotype of some mutants is due to loss-of-function mutations in two functionallyredundant genes, called the synthetic Multivulva (synMuv) genes. The synMuv genes aregrouped into three classes, A, B, and C. Animals with mutations in one or more geneswithin the same class are not Muv, whereas mutations in genes within any two classes areMuv. Several synMuv genes encode counterparts of a transcriptional repression complex,including HDA-1 HDAC, LET-418 Mi2, and LIN-53 RbAp48. Based upon this focus onchromatin remodeling, we wanted to determine if any histone methyltransferases (HMTases)play a role in vulval cell fate specification.We inactivated each of the 33 genes predicted to encode SET domain containing proteinsand assayed for vulval defects. Two putative HMTase genes - met-1 and met-2 - conferreda synMuv phenotype in combination with class A mutations. MET-1 is a homolog of yeastSET2p and predicted to methylate H3K36. MET-2 is the homolog of human SETDB1 andpredicted to methylate H3K9. Using quantitative western blots of wild-type, met-1, and met-2 extracts, we found that met-1 and met-2 are required for H3K36 and H3K9 trimethylation,respectively. Interestingly, met-1 and met-2 act redundantly with each other in vulval cellfate specification. Through further genetic studies, we hope to characterize how theseputative HMTases function in vivo to specify a developmental cell fate.

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Anthony Argentaro Abstract 5Structure/function studies of the PHD-like domain of thechromatin-remodelling protein, ATRX.

Argentaro A.1, Chapman L.2, Rhodes D.2, Yang J.C.2, Neuhaus D.2,Kowalczyk M.1, Higgs D.1 and Gibbons R.J.1

1MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, JohnRadcliffe Hospital, Oxford, OX39DS, U.K., 2Medical Research Council Laboratories,Cambridge CB22QH, U.K

The chromatin associated protein ATR-X was first identified because mutations in the ATRXgene cause a severe form of syndromal X-linked mental retardation associated with a-thalassemia. The ATRX protein contains a highly conserved cysteine-rich N-terminal region,part of which resembles a Plant homeodomain (PHD). NMR studies have shown that PHDdomains form a looped structure which binds zinc in a crossed braced topology betweenanti-parallel beta strands. Such domains are frequently found in chromatin-associatedproteins and are thought to mediate protein-protein interactions. The N-terminal region ofATRX is clearly different from the majority PHD domains but similar sequences are found inde novo methyltransferases (DNMT3b) and the related DNMT3L protein, hence this newsequence has been referred to as the ADD domain. At present neither the structure norfunction of the ADD domain is known, however since >60% of disease-causing mutationsoccur within this region of the ATRX protein it is likely to be of considerable functionalimportance.Based on the previously solved structure of the PHD domain we have made predictionsabout the fold of the variant PHD domain in ATRX using several structural predictionprogrammes although preliminary data suggest that the extended PHD-like, ADD domainmay form a new fold distinct from that of the PHD domain. Analysis of natural mutations ofthe ATRX protein may also test structural predictions. Most mutations show significantlyreduced levels of ATRX protein despite apparently normal or somewhat increased levels ofRNA, suggesting that they de-stabilise the ADD structure. In general, mutations, affectingresidues predicted to lie on the surface of the ADD domain have less severely reducedlevels of protein and these mutations do not appear to affect the structural integrity of thePHD-like fold. Such mutations may give rise to ATRX syndrome by affecting critical proteininteractions involved in the normal nuclear role of ATRX.

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Koh Meng Aw Yong Abstract 6The presence of EBV oriP within a transcription unit inhibitsreplication as well as transctiption

Koh Meng Aw Yong and Siu Chun Hung

Department of Microbiology, National University of Singapore, Block MD4 Level 5, 5Science Drive 2, Singapore 119260

The interplay between DNA replication and transcription has never been truly understood.While there has been some work done supporting claims that the two are antagonistic orsynergistic to each other, others claim that neither is affected by the other. In our study, wehope to cast more light on this contentious relationship between replication and transcriptionusing the latent origin or replication, oriP of the DNA virus Epstein-Barr virus (EBV). Bymaking use of the fact that EBV initiates replication by recruiting host cellular replicationmachinery to the oriP in the presence of EBV latent protein EBNA-1, we inserted oriPimmediately downstream of a SV40 promoter as part of the transcription unit. We thenassayed for DNA replication and transcription using Southern and Northern blots analysisrespectively. Our results indicate that DNA replication is inhibited by the presence of theSV40 promoter immediately upstream of the oriP. In addition, transcription was alsoinhibited such no full length transcripts were produced. This inhibition occurred even in theabsence of EBNA-1. Based on the current data observed, our hypothesis is that as thetranscriptional elongation complex reads through the transcriptional unit, it is physicallystalled at the oriP in a sequence dependent manner. This results in no transcript beinggenerated; and at the same time, it inhibits the initiation of replication by physicallypreventing the replication machinery complex from assembling on the oriP.

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Slobodan Barbaric Abstract 7Two nucleosomes at the yeast PHO84 promoter demonstratesdifferential requirement for chromatin remodelling activities

Bojana Sili1, Sabrina Stürzl2, Tim Luckenbach2, Philipp Korber2, SlobodanBarbaric1 and Wolfram Hörz2

1Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University ofZagreb, 10000 Zagreb, Croatia. 2Adolf-Butenandt-Institut Molekularbiologie, UniversitätMünchen, Schillerstr. 44, 80336 München, Germany.

We have investigated remodeling of the chromatin structure at the yeast PHO84 promoter,which is coregulated with the well studied PHO5 and PHO8 promoters in response tophosphate availability. The PHO84 promoter is the strongest promoter of the PHO family,containing five binding sites for the specific activator Pho4. Under repressive conditionsthere is a short hypersensitive region in the promoter, containing two closely positionedPho4 binding sites. Upon induction the promoter chromatin structure is altered, so that atleast one nucleosome upstream and one downstream from the hypersensitive region areremodeled. This way two additional Pho4 binding sites become accessible. Remodelingleads to histone depletion from the promoter region. We found that the rate of chromatinremodeling and subsequent promoter activation was strongly delayed in mutants deleted forSnf2, Ino80, Gcn5 or the histone chaperone Asf1. Nonetheless, after prolonged inductionfull remodeling and activation were achieved in the absence of Ino80, Gcn5 or Asf1.However, in the absence of Snf2 remodeling was only partial and resulted in the disruptionof the downstream but not of the upstream nucleosome, showing different requirements forremodeling of these two nucleosomes. Therefore, regarding the Snf2 requirement forchromatin remodeling, the PHO84 promoter appears to present a hybrid of the twocoregulated promoters: the PHO8 which is fully dependent on Snf2, and the PHO5 whereonly the rate of remodeling is affected.

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Vivian Bardwell Abstract 8Polycomb group and SCF ubiquitin ligases are found in aBCL6 corepressor complex

Gearhart, M.D., Corcoran, C.M., Wamstad, J.A. and Bardwell, V.J.

Department of Genetics, Cell Biology and Development, University of Minnesota,Minneapolis, MN 55455, U.S.A.

The corepressor BCOR potentiates transcriptional repression by the oncogene BCL6 andsuppresses the transcriptional activity of a common Mixed-Lineage Leukemia (MLL) fusionpartner, AF9. Mutations in human BCOR cause X-linked, male lethal Oculofaciocardiodentalsyndrome. Here we identify a BCOR complex containing several mammalian homologs ofPolycomb Group (PcG) proteins, including the chromatin modifier RNF2, an E3 ligase forthe mono-ubiquitylation of H2A (Ub-H2A). Thus the BCOR complex employs PcG proteinsto expand the repertoire of enzymatic activities that can be recruited by BCL6. RNF2 andUb-H2A are associated with PcG repression of Hox genes in Drosophila and the inactive Xchromosome (Xi) in mammals. We find that BCOR complex components and Ub-H2Alocalize to a BCL6 target in B cells but BCOR does not localize to Xi. The BCOR complexalso contains SKP1 and FBXL10, components of a second E3 ubiquitin ligase. In additionits F box and leucine rich repeats, FBXL10 contains chromatin binding motifs and a JmjCdomain suggesting the BCOR complex may have enzymatic activities for poly-ubiquitylationand trimethyl histone demethylation. In addition, we are examining the role of BCOR inhematopoietic development using BCOR deficient ES cells and in other aspects ofdevelopment using a mouse model system.

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Ralph Bash Abstract 9AFM recognition imaging: using antibodies to track proteinsduring in vitro remodeling

Ralph Bash1,3, Hongda Wang1, Chris Anderson1, Dennis Lohr3 and StuartLindsay1,2,3

1Biodesign Institute, 2Department of Physics and Astronomy and 3Department of Chemistryand Biochemistry, Arizona State University, Tempe, AZ 85287, U.S.A.

We are using recognition imaging to study remodeling of promoter chromatin, a key event inthe initiation of transcription. This technique uses antibodies during AFM imaging torecognize specific proteins. Antibodies used were tested against all proteins that might beencountered in the experiments. Some antibodies cross-react badly and others do not. Wecorrelated these results with ELISA assays, finding the two techniques in agreement, so thecross reactions represent limitations of the antibodies and not the recognition technique.We have also begun to establish quantitative criteria for analyzing recognition images, anddescribe software we have written for this purpose. This technique has been applied tochromatin remodeling reactions with hSwi-Snf, demonstrating removal of histone H2a fromnucleosome cores.

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Juraj Bies Abstract 10Pc2, the polycomb group protein recruits the c-Myb into PcGbodies and inhibits its activity.

Marek Sramko, Jan Markus, Linda Wolff and Juraj Bies

Center of Molecular Medicine, Cancer Research Institute, Slovak Academy of Sciences,Bratislava, Slovakia and Laboratory Cellular Oncology, National Cancer Institute, NIH,Bethesda, MD U.S.A.

Post-translational modifications of c-Myb oncoprotein play an important role in regulation ofits activity. We have shown that conjugation of SUMO-1 affects the transactivation capacityand the proteolytic turnover of c-Myb. Importance of this modification is underscored by thefact that both SUMO-1-modified lysines (K499 and 523) are located in the conserved regionof the negative regulatory domain, frequently lost during oncogenic activation of c-Mybprotein (Bies et al. 2002). c-Myb specific SUMO-ligase PIASy was identified to increase anextent of SUMOylation to the negative regulatory domain of c-Myb (Dahle et al., 2003).However, recent experiments showed, that SUMOylation of c-Myb was unaffected in PIASydeficient mice when compared to wild-type animal (Wong et al., 2004). These resultssuggest, that in addition to PIASy, there must be another c-Myb-specific SUMO-1 E3 ligasethat catalyzes SUMOylation of c-Myb. The polycomb protein Pc2, which has SUMO E3-ligase activity for the corepressors CtBPand CtBP2, interacts with c-Myb and increases conjugation of SUMO-1 to its negativeregulatory domain. Pc2 changes subnuclear localization of SUMOylated c-Myb transcriptionfactor. It recruits Myb from PML nuclear bodies into PcG bodies-like structures, where c-Myb colocalizes with another member of the polycomb repressive complex, Bmi.Coexpression of Pc2 strongly decreases transactivation activity of c-Myb. Interestingly,covalent conjugation of SUMO-1 protein to the negative regulatory domain of c-Myb wasdispensable for downregulation of c-Myb activity, as similar inhibition of both wild type, andSUMOylation-deficient mutant of c-Myb was detected.

Bies J et al. (2002). J Biol Chem. 277(11): 8999-90Dahle O et al. (2003) Eur J Biochem.; 270(6):1338-48.Wong et al. (2004). Mol Cell Biol. 24(12):5577-86.

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Caroline Bouchard Abstract 11c-Myc induces localized H2A.Z exchange in the promoters oftarget genes

Caroline Bouchard, Till Kortüm and Martin Eilers

Institute for Molecular Biology and Tumour Research (IMT), Marburg, Germany

The c-myc proto-oncogene encodes a transcription factor which can both activate andrepress gene expression. Post-translational covalent modifications (acetylation, methylation)of the histone tails and replacement of core histones by variant forms play a crucial role incontrolling transcription. We have previously found that binding of Myc to its target genecyclin D2 induces histone H3 and H4 acetylation at a single nucleosome (1). However, thecomplete and ordered cascade of events leading to the transcription of cyclin D2 is stillunclear. The ATPase Domino (the Drosophila melanogaster homolog of the mammalianp400 protein), as part of the Tip60 chromatin remodeling complex, has been shown tocatalyze the exchange of the Drosophila histone variant H2Av in its phosphorylated formafter acetylation by Tip60 at DNA double strand breaks against unphosphorylated H2Av (2).In mammals, two histone variants (H2A.Z and the DNA-damage-associated H2A.X) havefunctional overlap with H2Av. The highly conserved H2A.Z is important for the maintenanceof constitutive heterochromatin, for setting boundaries between euchromatin andheterochomatin and has also been implicated in both activation and repression oftranscription (3).Here, we used chromatin immunoprecipitation (ChIP) to evaluate the Myc-dependentchanges in histone methylation and histone replacement at the level of individual Myc targetpromoters. We now show in different cellular model systems that Myc binding is associatedwith the local displacement of the histone variant H2A.Z within the promoter region. Ourdata suggest a universal role of H2A.Z in the regulation by Myc of its target genes. The co-factors recruited by H2A.Z in order to regulate transcription are being identified. Additionally,the molecular mechanism(s) which underlie(s) the displacement of H2A.Z upon Myc bindingare currently under investigation.

1. Bouchard C et al., Genes Dev. 2001 Aug 15;15(16):2042-7.2. Kusch T et al., Science 2004 Dec 17;306(5704):2084-7. 3. Kamakaka RT and Biggins S, Genes Dev. 2005 Feb 1;19(3):295-310.

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Ray Camahort Abstract 12Genome-wide localization of the budding yeast histonevariant Cse4

R. Camahort, K. Collins, S. Biggins, C. Seidel and J. Gerton

Stowers Institute for Medical Research, Gerton Lab., 1000 E. 50th St., Kansas City, Jackson64110 , U.S.A.

The kinetochore is a complex, multi-protein structure required for proper chromosomesegregation in all eukaryotes. The Saccharomyces cerevisiae kinetochore consists of over65 known proteins which work in concert to facilitate equal distribution of the replicatedgenome. The budding yeast CenH3 histone variant Cse4 is a histone H3-like innerkinetochore protein that is vital to kinetochore function. Cse4 homologs are found in allhigher eukaryotes, and include Drosophila melanogaster Cid protein, Caenorhabditiselegans HCP-3, and the functional vertebrate ortholog CENP-A. Cse4 is thought to localizespecifically to centromeric nucleosomes, and function exclusively in the recruitment ofadditional kinetochore proteins and proper chromosome segregation. Using whole genomeDNA microarrays, we have looked at genome wide localization of Cse4 in S. cerevisiae.This data indicates that Cse4 may be found at more places along the budding yeastgenome than previously thought. Additionally, post-translational modifications and protein-protein interactions have been investigated in the hopes of elucidating the mechanism ofCse4 localization and function.

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Kendra Cann Abstract 13Multiple regions of the DNA-damage response proteinTLS/FUS can regulate its relocalization to the nucleoleifollowing transcriptional inhibition.

K. L. Cann, M. Arntfield, L. DeLange and G. G. Hicks

Manitoba Institute of Cell Biology and the University of Manitoba, Winnipeg, MB, Canada

The heterogeneous nuclear ribonucleoprotein TLS (Translocated in LipoSarcoma; FUS) wasinitially identified as a fusion partner of CHOP in over 90% of human myxoid liposarcomas, and ofERG in human myeloid leukemia.In these fusions, the N-terminus of TLS provides a potent transcriptional activation domain tocomplement the DNA-binding domain of CHOP or ERG. We have previously shown the TLS-/-mice have widespread genomic instability.We have also found that TLS is required for a proper cellular response to both gamma-irradiationand mitomycin C, which cause DNA double-strand breaks(DSBs) and interstrand cross-links (ICLs), respectively. TLS has been implicated in transcription,splicing, and RNA shuttling, but its mechanisms of action in genomic stability and the DNAdamage response have yet to be identified. To better understand TLS function, we chose toinvestigate TLS localization and its known association with the nucleolus, a subnuclear structureinvolved in rRNA transcription, ribosome assembly and cell cycle regulation. Endogenous TLSand EGFP (enhanced green fluorescent protein)-tagged TLS is restricted to the nucleus, but isexcluded from the nucleoli. After transcriptional inhibition, both endogenous TLS and EGFP-TLSrelocalize to the nucleoli. Ultraviolet-irradiation and the topoisomerase inhibitor camptothecin canalso induce this relocalization.There are two major functional domains in TLS: an N-terminal SYGQQS-repeat region and a C-terminal ribonucleotide-binding domain, containing three RGG repeat regions, a Zn-finger motif,and an RNA-recognition motif (RRM). An EGFP-tagged N-terminal fragment of TLS (amino acids1-216) still relocalizes to the nucleoli following actinomycin D treatment, identifying this region ascontaining the necessary response signal. This N-terminal fragment encompasses two regions ofSYGQQS repeats, and a deletion that removed the first of these SYGQQS-repeat regions (delta1-91) retained the ability to relocalize following treatment with actinomycin D. A deletion constructthat had both regions of SYGQQS repeat regions removed (delta1-193) did not relocalize to thenucleoli, identifying a critical signalling or interaction domain at amino acids 92-193. An internaldeletion of this sequence, the second region of SYGQQS repeats (delta92-193), yielded amodified version of TLS that was still capable of relocalizing following transcriptional inhibition.Because the TLS(delta92-193) construct contained the first region of SYGQQS repeats, theseresults suggest that there is a redundancy of function between the two SYGQQS repeat regions.Therefore, both SYGQQS repeat regions can function independently to mediate the relocalizationof TLS following transcriptional inhibition. This avenue of investigation is identifying andcharacterizing critical domains of TLS, which will help elucidate its wild-type function in the cellularresponse to stress.Ultimately, it will also help lead to potential mechanisms of action in genomic stability and the DNAdamage response.

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Rafael Casellas Abstract 14Effect of DNA double-stranded break repair on geneexpression

Marika Orlov1, Elizabeth Crouch1, Stanley Gorsky2, Andre Nussenzweig3,Tom Misteli2, Robert D. Phair4, Michael Kruhlak3 and Rafael Casellas1

1Genomic Integrity, NIAMS, National Institutes of Health, Bethesda, U.S.A., 2Cell Biology ofGenomes, NCI, National Institutes of Health, Bethesda, U.S.A., 3Experimental ImmunologyBranch, NCI, National Institutes of Health, Bethesda, U.S.A., 4Bioinformatics Services,Rockville, U.S.A.

DNA double-stranded breaks (DSBs) result in H2AX phosphorylation and recruitment ofDNA repair factors into nuclear micro-domains (foci) at the sites of lesion. Interestingly, bothfoci formation and damage-induced chromatin remodeling are massive and extendmegabases away from the actual DSB. The physiological significant of these higher orderchromatin structures is unclear. We have investigated the effects of DSB repair and fociformation on RNA polymerase assembly and elongation. By means of photobleachingtechniques and molecular scissors we show that DNA lesions initiate a time dependantinhibition of transcriptional elongation in the vicinity of the DSB. With the use ofcomputational modeling of imaging data, we show that recruitment and incorporationfrequencies of polymerase components are affected by DNA damage. Both transcriptionalsuppression and defects in polymerase assembly are not the direct result of DNA damageper se but they are instead orchestrated by ATM activity. We propose that micro-domainsand chromatin remodeling shut down transcriptional activity adjacent to lesion sites toensure proper repair of DNA ends.

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Beverley Chilton Abstract 15It Takes Two to Tango: autorepression by RUSH-1α/β

Aveline Hewetson and Beverly S. Chilton

Department of Cell Biology and Biochemistry, Texas Tech University Health SciencesCenter, 3601 4th Street - MS 6540, Lubbock, TX 79430, U.S.A.

RUSH/SMARCA3 is regulated by a steroid-dependent alternative splicing mechanism.RUSH-1α is the progesterone-dependent isoform. RUSH-1β is the truncated, estrogen-dependent isoform. Although the proteins have identical DNA-binding domains, little isknown about their structure-function relationships. Progesterone-dependent transcriptionalactivation of the RUSH/SMARCA3 gene is mediated by a bipartite PRE half-site/overlappingY-box combination in the proximal promoter (-162/+90). Estrogen represses the samepromoter construct via two Sp3/Sp1 sites. Gel supershift assays confirmed that Sp3/Sp1proteins bind both sites at a15:1 ratio. ERα/Sp1 interactions were eliminated with gelshift/supershift assays. In transient transfection assays, progesterone (R5020) inducedtranscriptional activation of the promoter in the presence (p < 0.05) and absence (p < 0.001)of the Sp3/Sp1 sites. Dual ablation of these sites resulted in a dramatic increase (p < 0.001)in promoter activity confirming Sp3 repression of progesterone-dependent transcription.Estrogen-treatment produced a dramatic decrease in the amount of RUSH message with aconcomitant shift to RUSH-1β isoform expression. In gel shift assays with nuclear extractfrom estrous animals, only Sp3 binds the distal site, and neither Sp3 nor Sp1 binds theproximal site.ever, repression also involves an authentic (ChIP) RUSH/SMARCA3 bindingsite (-616/-611) in the 5’-UTR. Gel shift/supershift assays with nuclear extract fromprogesterone-treated animals and a-specific antibodies confirmed exclusive, site-specificbinding of RUSH-1α when the gene is transcriptionally active. Transient transfection assayswith mutant constructs showed that elimination of the binding site increased (p < 0.001)transcriptional activation.m estrous animals and b-specific antibodies confirmed exclusivebinding of RUSH-1β to the same site when the promoter is transcriptionallyquiescent. alternative splicing and isoform specific autorepression of the RUSH/SMARCA3gene.

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Sung Hee Baek Abstract 16A chromatin remodeling complex in regulation of ametastasis suppressor gene

Hee June Choi, Jung Hwa Kim, Bogyou Kim, Ji Min Lee, Ik Soo Kim andSung Hee Baek

Seoul National University 216, 20 Sillim-Dong, Kwanak-Ku, Seoul 151-742, South Korea

Defining the molecular strategies that integrate diverse signaling pathways in expression ofspecific gene programs critical in homeostasis and disease remains a central issue inbiology. In cancer biology, this is particularly pertinent, because down-regulation of tumormetastasis suppressor genes is a common occurrence, and the underlying molecularmechanisms are not well established. Recently, we reported that the down-regulation of ametastasis suppressor gene, KAI1, in prostate cancer cells involves the inhibitory actions ofbeta-catenin, along with a reptin chromatin remodeling complex (Kim et al., Nature 434,921-926, 2005). This inhibitory function of beta-catenin/reptin chromatin remodelingcomplex requires both increased levels of beta-catenin expression and recruitment ofhistone deacetylase activity. The coordinated actions of beta-catenin/reptin componentsthat mediate the repressive state serve to antagonize a Tip60 coactivator complex, requiredfor activation, with the balance of these opposing complexes controlling the expression ofKAI1 and metastatic potential. Here we found an intriguing signal recognition code of whichsignaling factors cause reptin chromatin remodeling complex to confer repressive functionto control expression of KAI1 and its metastatic potential. Biochemical purification of areptin-containing complex revealed that it unexpectedly contains SUMO processingenzymes. Reptin is a direct target of SUMOylation and lack of SUMOylation exerts toantagonize the repressive function of reptin. This work provides a novel insight for linkingSUMO modification of chromatin remodeling complex to cancer metastasis.Mutations in the IKBKAP gene, encoding a subunit of Elongator, cause familialdysautonomia (FD), a severe neurodevelopmental disease with complex clinicalcharacteristics. Elongator was previously linked with transcriptional elongation and histoneacetylation. Here, we used RNA interference (RNAi) to identify Elongator target genes.Strikingly, several of these genes encode proteins implicated in cell motility. Indeed,characterization of IKAP RNAi cells and fibroblasts derived from FD patients uncovereddefects in this cellular function. Whereas Elongator is recruited to both target and non-targetgenes, only target genes display histone H3 hypo-acetylation and progressively lowerRNAPII density through the coding region. Decreased target gene transcription in FDfibroblasts can be overcome by treatment with histone deacetylase inhibitor. Taken together,our results indicate that defects in Elongator function result in reduced transcriptionalelongation of several genes. The resulting cell motility deficiencies may underlie theneuropathology of FD patients. Our data indicate that FD should be classified as atranscription disease.

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Pierre Close Abstract 17Elongator depletion causes defective transcript elongation ofgenes that regulate cell motility

Pierre Close1,2, Nicola Hawkes2, Isabelle Cornez1, Catherine Creppe1,Charles A. Lambert3, Bernard Rogister4, Ulrich Siebenlist5, Marie-PauleMerville1, SU.S.A.n A. Slaugenhaupt6, Vincent Bours1, Jesper Q.Svejstrup2 and Alain Chariot1.

1Laboratory of Medical Chemistry and Human Genetics, Center for Biomedical IntegrativeGenoproteomics, University of Liege, Liege, Belgium., 2Cancer Research UK LondonResearch Institute, Clare Hall Laboratories, South Mimms, United Kingdom., 3Laboratory ofConnective Tissues Biology, Center for Biomedical Integrative Genoproteomics, Universityof Liege, Liege, Belgium., 4Center for Cellular and Molecular Neurobiology, University ofLiege, Liege, Belgium., 5Laboratory of Immunoregulation, NIAID, NIH, Bethesda, MD,U.S.A.., 6Center for Human Genetics Research, Massachusetts General Hospital andHarvard Medical School, Boston, MA, U.S.A.

Mutations in the IKBKAP gene, encoding a subunit of Elongator, cause familialdysautonomia (FD), a severe neurodevelopmental disease with complex clinicalcharacteristics. Elongator was previously linked with transcriptional elongation and histoneacetylation. Here, we used RNA interference (RNAi) to identify Elongator target genes.Strikingly, several of these genes encode proteins implicated in cell motility. Indeed,characterization of IKAP RNAi cells and fibroblasts derived from FD patients uncovereddefects in this cellular function. Whereas Elongator is recruited to both target and non-targetgenes, only target genes display histone H3 hypo-acetylation and progressively lowerRNAPII density through the coding region. Decreased target gene transcription in FDfibroblasts can be overcome by treatment with histone deacetylase inhibitor. Taken together,our results indicate that defects in Elongator function result in reduced transcriptionalelongation of several genes. The resulting cell motility deficiencies may underlie theneuropathology of FD patients. Our data indicate that FD should be classified as atranscription disease.

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Megan Cole Abstract 18Genome-wide map of nucleosome acetylation andmethylation in yeast

Dmitry K. Pokholok1, Christopher T. Harbison1, Stuart Levine1, MeganCole1,2, Nancy M. Hannett1, Tong Ihn Lee1, George W. Bell1, KimberlyWalker1, P. Alex Rolfe3, Elizabeth Herbolsheimer1, Julia Zeitlinger1, FranLewitter1, David K. Gifford1,3 and Richard A. Young1,2

1Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge,Massachusetts 02142, U.S.A., 2Department of Biology, Massachusetts Institute ofTechnology, Cambridge, Massachusetts 02139, U.S.A., 3MIT Computer Science andArtificial Intelligence, Laboratory,Cambridge, Massachusetts 02139, U.S.A.

Eukaryotic genomes are packaged into nucleosomes whose position and chemicalmodification state can profoundly influence regulation of gene expression. We profilednucleosome modifications across the yeast genome using chromatin immunoprecipitationcoupled with DNA microarrays to produce high-resolution genome-wide maps of histoneacetylation and methylation. These maps take into account changes in nucleosomeoccupancy at actively transcribed genes and, in doing so, revise previous assessments ofthe modifications associated with gene expression. Both acetylation and methylation ofhistones are associated with transcriptional activity, but the former occurs predominantly atthe beginning of genes, whereas the latter can occur throughout transcribed regions. Mostnotably, specific methylation events are associated with the beginning, middle, and end ofactively transcribed genes. These maps provide the foundation for further understanding theroles of chromatin in gene expression and genome maintenance.

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Jean-Francois Couture Abstract 19Structural insights into lysine multiple methylation by SETdomain methyltransferases

Jean-Francois Couture1, Lynnette M. A. Dirk2, Joseph S. Brunzelle3,Robert L. Houtz2 and Raymond C. Trievel1

1University of Michigan, 4412 Medical Science I, 1301 Catherine Street, Ann Arbor, Michigan48109, U.S.A, 2Department of Horticulture, University of Kentucky, 401D Plant ScienceBuilding, Lexington KY 40546-0312, 3Life Sciences CAT, Dept of Mol. Pharm. and Biol.Chem., Feinberg School of Medicine, Northwestern University, Boston MA, U.S.A.

Site-specific protein lysine methylation has emerged as an important covalent modificationthat regulates numerous genomic functions, including transcription, mitosis, and DNAdamage checkpoints in the cell cycle. This modification is catalyzed by a family of proteinlysine methyltransferases (PKMTs), the majority of which possess a conserved catalyticSET domain. Sequence and structure variations within the SET domain enable PKMTs torecognize and methylate distinct substrates, including histones, transcription factors, andother nuclear proteins. In addition to their lysine selectivity, these enzymes can catalyzemono-, di-, or trimethylation of the lysine epsilon-amino group. This property is referred toas their product specificity and is biologically important because many methyllysine-bindingfactors can discriminate among different degrees of methylation. Despite its significance, themechanism by which SET domain PKMTs catalyze lysine multiple methylation has remainedpoorly understood.To elucidate this mechanism, we have determined the crystal structures of the Y334Fmutant of human SET8 (hSET8), a histone H4 Lys-20-specific methyltransferase, incomplex with histone H4 peptides bearing unmodified, monomethylated, and dimethylatedLys-20. The Y334F active site mutation alters the product specificity of hSET8 from a mono-to a dimethylase, but does not otherwise affect histone H4 binding or methylation comparedwith the native enzyme. Collectively, the structures reveal that the Y334F substitution alterswater-mediated hydrogen bonding to the Lys-20 epsilon-amino group, permitting it to adoptalternative conformations that facilitate dimethylation. Moreover, biochemicalcharacterization of the mutant reveals that it binds to the unmodified and monomethylatedLys-20 histone H4 peptides with equivalent affinity, but cannot methylate themonomethylated peptide. These observations suggest that the hSET8 Y334F mutantdimethylates Lys-20 via a processive mechanism in which histone H4 remains bound withinthe active during the catalytic cycle. Taken together, these studies furnish insights into thestructural determinants of lysine multiple methylation by SET domain PKMTs.

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Qi Dai Abstract 20Snail-mediated repression in Drosophila requires Ebi andhistone deacetylation

Qi Dai, Mattias Bergman and Mattias Mannervik

Department of Developmental Biology, Wenner-Gren Institute, Arrhenius Laboratories E3,Stockholm University, Stockholm, Sweden

Drosophila Ebi and its mamalian homolog TBL1 are WD40 proteins with a divergent F-boxdomain. Previous studies have linked Ebi and TBL1 to two different pathways; ubiquitinconjugation through SCF-type ligases, and N-CoR/SMRT/HDAC3-mediated transcriptionalrepression. We found that Ebi specifically affects the function of the Snail repressor inDrosophila embryos, since in ebi germline clone mutants Snail target genes are de-repressedin the mesoderm. De-repression of a Snail-dependent reporter gene in ebi mutant embryos,and genetic interactions with snail support a requirement for Ebi in Snail function. Snail-mediated repression was previously shown to depend on the CtBP co-repressor. We foundthat both CtBP and Ebi can interact with Snail protein in vitro, but through different interactiondomains. A minimal Ebi-interaction domain that fails to bind CtBP constitutes a potentrepression domain in both S2 cells and in transgenic embryos. This suggests that Snail usesEbi as co-repressor independently of CtBP. The repression activity of this domain can beattenuated either by knockdown of HDAC3 or by TSA treatment, indicating an involvement ofhistone deacetylation. We suggest that Ebi as part of a SMRTER/HDAC3 co-repressorcomplex is required for Snail function in Drosophila, and that histone deacetylation is part ofthe mechanism by which Snail represses transcription.

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Ana D’Alessio Abstract 21The kinetics of chromatin-driven active DNA demethylation inliving cells.

Ana C. D’Alessio, Ian C.G. Weaver and Moshe Szyf.

Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec,Canada.

Gene expression profiles of vertebrate cells are regulated through the epigenome, whichconsists of chromatin, and its modifications, and the pattern of DNA methylation at CpGdinucleotides. Active genes are associated with acetylated histones and hypomethylatedDNA, whereas inactive genes are associated with hypoacetylated histones andhypermethylated DNA. However, the relationship between these two components of theepigenome is still unclear. The objective of this study is to define the temporal relationshipbetween chromatin and transcriptional machinery events leading to active DNAdemethylation.DNA demethylation of an endogenous gene in a replicating cell could either be an active ora passive replication-dependent process. We took advantage of a previously describedtransient transfection, replication-independent, active demethylation assay in living cells. Inthis system, pharmacological induction of histone acetylation by inhibition of histonedeacetylases with trichostatin A (TSA) triggers active DNA demethylation. We used ChIPassays and bisulfite mapping to study the temporal and caU.S.A.l relationship betweenchromatin and active DNA demethylation. We show that histone acetylation is followed by RNA Pol II (Pol II) binding to the methylatedpromoter. Following interaction of Pol II, the promoter becomes demethylated. Activedemethylation of this promoter is dependent on transcription, as demethylation is blockedthrough the addition of Actinomycin D, an inhibitor of Pol II. DNA demethylation is laterfollowed by appearance of trimethylated lysine 4-H3, which is characteristic of elongatingforms of Pol II. In agreement with this model, only demethylated DNA is associated withtrimethylated lysine 4-H3.To determine whether the same mechanism applies to endogenous genes, we performedan expression micro-array analysis on TSA treated cells to identify endogenous genes thatwere induced by histone acetylation. We found that G antigen family (GAGE) could bedemethylated in response to TSA. More importantly, the same order of events as found withthe exogenous gene, were associated with GAGE. This work provides a mechanism for howgenes silenced through histone hypoacetylation and DNA methylation, such as oncogenes,may become activated in tumourigenesis

Supported by grant from the National Institute of Cancer of Canada 2005 to MS.

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Jan-Hermen Dannenberg Abstract 22Role of mSin3A in development, regulating transcriptionalnetworks and tumorigenesis.

Jan-Hermen Dannenberg, Gregory David2, Sheng Zhong1, Jaco van derTorre, Wing H. Wong1 and Ronald A. DePinho

Department of Medical Oncology, Dana Farber Cancer Institute; Departments of Medicineand Genetics, Harvard Medical School; Boston, MA, U.S.A. 1Department of Statistics,Stanford University, Stanford, CA, U.S.A., 2Present address: Department of Pharmacology,New York University, School of Medicine, New York, NY, U.S.A.

mSin3A is a core component of a large multi-protein co-repressor complex (mSin3/HDAC)with associated histone deacetylase enzymatic activity. Physical interactions of mSin3Awith many sequence-specific transcription factors have implicated a role for the mSin3A co-repressor complex in diverse signaling pathways and linked biological processes. Todissect the complex nature of mSin3A’s actions, we monitored the impact of conditionalmSin3A deletion on the developmental, cell biological and transcriptional levels. mSin3Awas shown to play an essential role in early embryonic development and in the proliferationand survival of primary, immortalized and transformed cells. Although we established a rolefor mSin3A/HDAC in p53 deacetylation and activation, genetic deletion of p53 was notsufficient to attenuate the mSin3A null cell lethal phenotype thereby pointing to mSin3A’sbroad biological activities beyond regulation of the p53 pathway. Correspondingly, time-course gene expression profiling following mSin3A deletion revealed deregulation of genesinvolved in cell cycle regulation, DNA replication, DNA repair, apoptosis, chromatinmodifications and mitochondrial metabolism. In silico promoter analysis of the mSin3Atranscriptome revealed significant enrichment for Myc-Mad, E2F and p53 cis-regulatoryelements in promoters of up-regulated genes following mSin3A depletion. Significantly,using bioinformatics combined with ChIP analyses on verified mSin3A target genes weidentified specific cis-regulatory elements binding the transcriptional activator Stat and thenucleosome remodeling factor Falz, thereby expanding further the mSin3A network ofregulatory factors. Together, these integrated genetic, biochemical and computationalstudies demonstrate the involvement of mSin3A in the regulation of diverse pathwaysgoverning many aspects of normal and neoplastic growth and survival. Studies addressing a role for mSin3A in tumorigenesis showed that mSin3A-heterozygosityresults in an increased sensitivity towards DNA-damaging agents and a pronouncedresistance to DMBA-induced lymphomagenesis. Mechanisms underlying this novel role formSin3A in lymphomagenesis will be discussed.

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Gregory David Abstract 23Functional specialization of the mSin3 complex indevelopment and oncogenesis

Gregory David1,2, Natalie Simpson1, Mei Finnerty1, Jan-HermenDannenberg2 and Ronald A. DePinho2

1Department of Pharmacology, NYU School of Medicine, New York, 2Departments of AdultOncology, Dana Farber Cancer Institute, Boston

The highly conserved Sin3-histone deacetylase complex, a prominent co-repressorcomplex, is characterized in mammalian cells by the presence of the mSin3A protein andthe closely related mSin3B protein. Aberrant recruitment of the mSin3 complex alterschromatin structure and is involved in the pathogenesis of several human tumors. We usedgenetic inactivation in the mouse to elucidate the molecular pathways by which the mSin3complex affects development and oncogenesis. Specifically, we have generated conditionalknock-out mouse strains for mSin3A, mSin3B and the essential component of the mSin3complex, mSds3, and we have previously shown that an integral mSin3 complex is requiredfor pericentric heterochromatin structure and proper chromosomal segregation. In addition,we have observed that mSin3A and mSin3B are both essential during embryogenesis buttheir inactivation results in dramatically different developmental and cellular phenotypes. Weused biochemical and cellular approaches to dissect the molecular pathways engaged byeach component of the mSin3 complex and uncovered specific roles for each of the mSin3proteins. Although mSin3A engages numerous transcriptional pathways necessary for cellsurvival and proliferation, mSin3B is dispensable for cell viability. However, organismal andcellular phenotypes resulting from mSin3B inactivation strongly suggest a specificcontribution to the E2F repressors pathway. Finally, the role of the mSin3 complex in tumorsuppression has been investigated in vivo and will be discussed. Altogether, these studiesunderline the role of the mSin3 complex in cellular proliferation and differentiation andreveal an unsuspected functional specialization for each one of the mSin3 proteins in highereukaryotes.

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James Davie Abstract 24Recruitment of phosphorylated HDAC2 to promoters

Jian-Min Sun, Hou Yu Chen and James R. Davie

University of Manitoba, NA, CancerCare Manitoba, 675 McDermot Avenue, Winnipeg,Manitoba (Province), R3E 0W3, Canada

Histone deacetylase 2 (HDAC2) is one of the histone-modifying enzymes that regulate geneexpression by remodeling chromatin structure. Along with HDAC1, it is found in the Sin3and NuRD multi-protein complexes, which are recruited to promoters by DNA bindingproteins. In this study, we show that HDAC2 in human breast cancer cells is mostly notphosphorylated. However, a minor population of HDAC2, preferentially cross-linked to DNAby cisplatin and formaldehyde, is mono-, di- or tri-phosphorylated. Moreover, the formationof Sin3 and NuRD complexes, as well as their recruitment to promoters by factors like p53,Rb, YY1, p50, p65, Sp1 and Sp3 are dependent on HDAC2 phosphorylation. The majorityof the HDAC2 population, in an unmodified form, is bound to chromatin but is not cross-linked to DNA by formaldehyde and therefore would not be detected in standard chromatinimmunoprecipitation (ChIP) assays. This observation raises a cautionary note in the use ofthe standard ChIP assay that the efficiency of formaldehyde cross-linking of chromosomalproteins to DNA can vary greatly.

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Cecile de la Cruz Abstract 25Developmental regulation of Suz12 localization

de la Cruz C.C., Fang J., Plath K., Worringer K.A., Nusinow D.A., ZhangY. and Panning B.

Department of Biochemistry and Biophysics, University of California, San Francisco, CA,94143, U.S.A.

Chromatin modifications are among the epigenetic alterations essential for geneticreprogramming during development. The Polycomb group (PcG) gene family mediateschromatin modifications that contribute to developmentally regulated transcriptionalsilencing. Trimethylation of histone H3 on lysine 27, mediated by a PcG protein complexconsisting of Eed, Ezh2, and Suz12, is integral in differentiation, stem cell self-renewal, andtumorigenesis. Eed and Ezh2 are also implicated in the developmentally regulated silencingof the inactive X chromosome, as they are transiently enriched on the inactive Xchromosome when X chromosome silencing is initiated. Here we analyze the dynamiclocalization of Suz12 during cellular differentiation and X-inactivation. Though Suz12 is arequisite member of the Eed/Ezh2 complexes, we found that Suz12 exhibits a notabledifference from Ezh2 and Eed: while Ezh2 and Eed levels decrease during stem celldifferentiation,Suz12 levels remain constant. Despite the differential regulation in abundance of Suz12 andEed/Ezh2, Suz12 is also transiently enriched on the Xi during early stages of X-inactivation,and this accumulation is Xist RNA dependent. These results suggest that Suz12 may havea function that is not mediated by its association with Eed and Ezh2, and that this additionalfunction is not involved in the regulation of X-inactivation.

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Stephan Diekmann Abstract 26Functional complementation of human centromere protein A(CENP-A) by Cse4p

Gerhard Wieland, Sandra Orthaus, Sabine Ohndorf, Stephan Diekmannand Peter Hemmerich

IMB1, Westfluegel, Beutenbergstr 11, Jena, Thueringen 07745, Germany

We have employed a novel in vivo approach to study the structure and function of theeukaryotic kinetochore multiprotein complex. RNA interference was used to block thesynthesis of proteins CENP-A and Clip-170 in human cells. By coexpression, homologouskinetochore proteins from Saccharomyces cerevisiae were then tested for their ability tocomplement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog,was specifically recruited to kinetochores and was able to complement the RNAi-inducedcell cycle arrest in CENP-A depleted human cells. Thus, Cse4p can structurally andfunctionally substitute CENP-A cross-species. Bik1p, the budding yeast homolog of humanCLIP-170, also specifically localized at kinetochores in mitosis but Bik1p did not rescue theCLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivocomplementation assay provides a powerful new tool to study the function and evolutionaryconservation of multiprotein complexes from yeast to man.

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Ivana Djuretic Abstract 27Silencing of a mammalian gene, Interleukin-4 in T helperlymphocytes

Ivana Djuretic1,2, K. Mark Ansel1,2 and Anjana Rao1,2

1Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, U.S.A. ,2CBR Institute for Biomedical Research, Boston, Massachusetts 02115, U.S.A.

T helper cell differentiation has been an excellent model for studying the regulation ofmammalian gene expression by changes in chromatin structure. The murine Interleukin-4/5/13 (T helper 2) locus, in particular, has been a subject of many studies which resulted inidentifying various elements that regulate its transcription including enhancers, silencers anda locus control region. In this study we focus on identifying the role of two regulatoryelements, HS IV and HSS3, in silencing the Il4 gene in T helper 1 cells. By targeted deletionwe determined that HS IV functions as a silencer for Il4 while the function of HSS3 is stillunclear. Deletion of both HSS3 and HS IV partially rescues the HS IV-deficient phenotype,suggesting a possible interaction between these two genetic elements. The predominanthistone modification at these two sites is H3K27 methylation, indicating that Polycombproteins might be involved in silencing of Il4. Interestingly, a peak of H3K4 dimethylation inaddition to H3K27 trimethylation is also found at HS IV. A combination of these twomodifications has not been previously observed. We have also identified two transcriptionfactors, T-bet (T box expressed in T cells) and Runx3, which bind to HS IV. In additionintergenic transcripts can be detected in T helper 2 cells, which express Il4, but not T helper1 cells, which normally silence it. The final goal of this study will be to determine therelationship between intergenic transcription at the Il4/5/13 locus, histone modifications atHS IV and HSS3, and transcription factors that bind to them, and how they cooperate toregulate the expression of Th2 cytokines.

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Tom Donndelinger Abstract 28Asymmetric eukaryotic cell division and cellular dimorphism

Tom Donndelinger and Elizabeth Oldenkamp

Mercy Medical Center, 1512 12th Ave Rd., Nampa 3686, U.S.A.

Chromatin asymmetry at cell division is strictly conserved in eukaryotic cells and persists inthe nuclei of daughter cells. With our greatly improved tissue detail, mitotic asymmetrybecomes unavoidably apparent. In over one hundred years of formalin-fixed paraffinembedding tissue processing there have been no significant improvements. Our re-engineered tissue processing method, which preserves structural and dedicated watermolecules while cutting at one micron, gives ten to one hundred times better detail thantraditional tissue processing methods. Images are referenced to published electronmicroscopic data at 1000X. Asymmetry in post-mitotic chromatin patterns in daughter cellshas been identified and subjected to an extensive phylogenetic trace. The results stronglysuggest that all eukaryotic cell division produces some degree of asymmetric structuralfeatures of chromatin in the daughter cells. These biologic features of the “0” and “1” cellsalways occur in cell division point out the possibility of distinct subsetting of overlapping butnon-identical proteomics. Cytoplasmic and chromatin differences have been traced in alltissues and all organs of a large number of metazoans and plants with no exceptionsobserved. The asymmetry continues in cancerous tissues and some virus infected cells.This new detail implies a greater degree of complexity of organisms than was previouslyunderstood. Cells as tissue components are not heterogeneous in their morphology; rather,they are dimorphic. Additional studies forcing cells into two-dimensional space from threedimensions, strongly suggests these post-mitotic pairs are preferentially physicallyentangled. The format of entangled, binucleated protozoans and diatoms has apparentlybeen conserved throughout evolution. We are progressing with proof of concept with highresolution flow cytometry with antibodies to phosphorus biased, strictly conserved histonefeatures in metaphase chromosome sets. Single cell systems biology and communicationmay be too simple to understand cell life. Basic understandings of cell division,communication, and disease may have to be altered in light of this new data.

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Yannick Doyon Abstract 29ING tumor suppressor proteins are critical regulators ofchromatin acetylation required for genome expression andperpetuation

Yannick Doyon1, Christelle Cayrou1, Mukta Ulla2, Anne-Julie Landry1,Valérie Côté1, William S. Lane3, Song Tan4, Xiang-Jiao Yang2 and JacquesCôté1

1Laval University Cancer Research Center, Hôtel-Dieu de Québec (CHUQ), Quebec CityQc, Canada G1R 2J6, 2Molecular Oncology Group, McGill University Health Center,Montreal Qc, Canada H3A 1A1, 3Harvard Microchemistry Facility, Harvard University,Cambridge MA 02138, 4Department of Biochemistry and Molecular Biology, PennsylvaniaState University, University Park PA 16802, U.S.A.

Members of the ING family of tumor suppressors regulate cell cycle progression, apoptosisand DNA repair as important cofactors of p53. In yeast, the ING paralogs are key to thecatalysis of chromatin modification by specific HAT or HDAC complexes. In human, ING1and ING3 are stable components of the mSin3A HDAC and Tip60/NuA4 HAT complexes,respectively. We now report the purification of the three remaining members of the humanING family. While ING2 is in a HDAC complex similar to ING1, ING4 associates with theHBO1 acetyltransferase required for normal progression through S phase and the majorityof histone H4 acetylation in vivo. ING5 fractionates with two distinct complexes containingHBO1 or nucleosomal H3-specific MOZ/MORF HATs and interacts with the MCM proteins,supporting a role for MYST-ING complexes in DNA replication.As the ING, HBO1 and MOZ/MORF proteins contribute to oncogenic transformation, themulti subunit assemblies characterized here underscore the critical role of epigeneticregulation in cancer development.

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Katherine Dunn Abstract 30Independent phosphorylation events on the amino-terminusof histone H3 associated with transcription

Katherine L. Dunn and James R. Davie

Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue Winnipeg,Manitoba R3E 0V9, Canada.

Approximately 30% of human cancers contain a mutation to one of the ras oncogenes.Stimulation of the ras-mitogen activated protein kinase (ras-MAPK) pathway leads to rapidphosphorylation of histone H3 at serines 10 and 28 and expression of immediate-earlygenes. H3 phosphoacetylated at serine 10 lysine 14 has been found associated withimmediate-early genes following stimulation of the Ras-MAPK pathway. Ras-transformedcells contain higher levels of H3 phosphorylated at serine 10 and display a less condensedchromatin structure. We hypothesize that phosphorylation of H3 at serine 28 contributes to chromatin remodelingand immediate early gene activity. Subsequent to ras- MAPK pathway stimulation H3phosphorylated on serine 10 or 28 was excluded from regions of highly condensedchromatin and was present in increased amounts in ras-transformed cells. H3phosphorylated at serine 10 or at serine 28 was dynamically acetylated, however H3 tailsphosphorylated at serine 28 had a higher steady state level of acetylation. When visualizedwith indirect immunofluorescence, most foci of H3 phosphorylated at serine 28 do notcolocalize with foci of H3 phosphorylated at serine 10 or foci of H3 phosphoacetylated atserine 10 lysine 14, indicating that these two phosphorylation events are targetedseparately. Furthermore, foci of H3 phosphorylated at serine 28 associate with foci ofphosphorylated RNA polymerase II, as do foci of H3 phosphorylated at serine 10,suggesting that these two modifications promote transcription independently.

This work was supported by a grant from the National Cancer Institute of Canada with fundsfrom the Canadian Cancer Society, a Canada Research Chair to J.R.D., and a K.M.Hunter/Canadian Institutes of Health Research doctoral research award to K.L.D.

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Christine English Abstract 31ASF1 binds to a heterodimer of histones H3-H4: a two stepmechanism for the assembly of H3-H4

Christine M. English1, Jessica K. Tyler1, Brian Tripet1, Nasib K. Maluf2 andMair E.A. Churchill3

1The Department of Biochemistry and Molecular Genetics, University of Colorado HealthSciences Center at Fitzsimons, Aurora CO 80045, 2Department of Pharmaceutical Sciences,University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver CO80262., 3Department of Pharmacology, University of Colorado Health Sciences Center atFitzsimons, Aurora CO 80045, U.S.A.

The first step in the formation of the nucleosome is commonly assumed to be the depositionof a histone H3-H4 heterotetramer onto DNA. Anti-silencing function 1 (ASF1) is a majorhistone H3-H4 chaperone that deposits histones H3 and H4 onto DNA. Towardsunderstanding the mechanism of deposition of histones H3 and H4 onto DNA, we havedetermined the stoichiometry of the Asf1-H3-H4 complex. We have established that a singlemolecule of Asf1 binds to a heterodimer of H3-H4 using gel filtration, amino acid, reversed-phase chromatography and analytical ultracentrifugation analyses. We propose that Asf1blocks H3-H4 heterotetramer formation by occluding the H3-H3 dimerization interface. Thiswork suggests that the formation of the H3-H4 heterotetramer on DNA is likely to occur bythe sequential or concerted deposition of two H3-H4 heterodimers.

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Alex Erkine Abstract 32Nucleosome displacement at promoters of yeast heat shockgenes is proportional to the degree of transient histone H3acetylation.

T. Y. Erkina and A. M. Erkine

USD School of Medicine, Lee Medical Bldg., 414 E. Clark Street, Vermillion, SD 57069,U.S.A.

It is often not clear if histones of the promoter nucleosomes are just post-translationallymodified or outright removed from promoters during induction of transcription. Using chromatin immuno-precipitation in combination with real-time PCR, we studied thekinetics of chromatin changes at promoters of HSP12, SSA4, HSP82 genes. Our resultsindicate that the extent of nucleosome displacement differs for individual heat shock genepromoters. Importantly, the HSP12 promoter with a higher degree of nucleosomedisplacement showed significantly less visible displacement of acetylated H3 than the SSA4promoter with lower degrees of nucleosome displacement. For HSP12 promoter, the level ofacetylated H3 temporarily increased prior to the nucleosome departure. For all promoterstested, the higher level of nucleosome displacement correlated with the transientlyincreased level of histone H3 acetylation. To test whether this rule is dictated by thepromoter elements or by the HSF as a general activator of these genes, we comparedindividual promoters in strains expressing different truncated versions of HSF. Deletion ofeither of the two activating regions in HSF led to diminished nucleosome displacement.Deletion of both regions simultaneously severely decreased nucleosome displacement forall the promoters tested. In accordance with the above rule, the histone acetylation level atindividual promoters followed the degree of nucleosome displacement showing thedependence of these processes on HSF as a general heat shock gene regulator, and noton individual promoter elements.

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Laure Escoubet-Lozach Abstract 33Application of genome-wide location analysis to studychromatin modifications in a model of signal-dependent generegulation

Laure Escoubet-Lozach1, Jean Lozach1, Christopher W. Benner1, RomanSasik1, Ivan Garcia-Bassets2, Rosa Luna1, Donna Reichardt1, JanaCollier1, Colleen Eckhardt3, Kristin Stubben3, Jennifer Lapira3, GaryHardiman3, B. Ren4, Michael G. Rosenfeld2 and Christopher K. Glass1

1Cellular and Molecular Medicine, School of Medicine, 2Howard Hughes Medical Institute,School of Medicine, 3BioMedical Genomics Microarray Facility, Department of Medicine,4Howard Hughes Medical Institute, Cellular and Molecular Medicine, University of CaliforniaSan Diego, La Jolla, CA 92093, U.S.A..

Macrophages are phagocytic cells that play essential roles in native and acquired immunity.Upon stimulation with an inflammatory mediator, such as LPS, macrophages regulateseveral hundred genes that contribute to an anti-microbial response. In this model of signal-dependent gene regulation, we investigate the chromatin modifications occurring inresponse to LPS and study to which extent these modifications are linked to generegulation. This study has been performed at a genome-wide level using the ‘ChIP-on-chip’technology associating Chromatin Immunoprecipitation (ChIP) and promoter microarray(chip). For this purpose, we developed a murine promoter array containing approximately34,000 features (Mu34K) and representing the proximal promoters of 26,921 genes andtiled regions of 48 genes.Here, we present a comprehensive set of data comparing the H3K9 and H3K18 acetylationmarks as well as the H3K4, H4R3, H3R17, H3K9 and H3K27 methylation marks inuntreated and LPS-treated macrophages, we analyse their association and theirinterdependence, their involvement in gene regulation and explore mechanisms by which asignal such as LPS leads to such chromatin modifications.

Grants : P50 HL56989, P30 DK063491, American Heart Association 0325103Y

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Yuhong Fan Abstract 34A new link between linker histone H1 and DNA methylation

Yuhong Fan1, Tatiana Nikitina2, Riddhi Bhattacharyya1, Jie Zhao1, EricBouhassira1, Chris Woodcock2 and Arthur Skoultchi1

1Department of Cell Biology, Albert Einstein College of Medicine, 402 Chanin, 1300 MorrisPark Ave., Bronx, NY 10461, U.S.A., 2Department of Biology, University of Massachusetts,Amherst, Massachusetts 01003, U.S.A.

H1 linker histones are thought to play a key role in the folding of chromatin into higher orderstructures. Mice contain eight H1 subtypes that differ in expression during development.Previous studies in ourlaboratory showed that mice develop normally when any one of six different H1 genes isinactivated homozygously, whereas mice lacking three H1 subtypes, H1c, H1d and H1e,generated by three rounds of gene inactivation in ES cells, die by mid-gestation with abroad range of defects. To further understand the role of H1 in chromatin structure and geneexpression, we derived ES cells (embryonic stem cells) from the H1cH1dH1e triple nullblastocysts and wild-type littermates. Using these cells, we showed that marked reductionin H1 amount leads to global changes in chromatin structure in vivo, including decreasednucleosome spacing and reduced chromatin compaction. To further study the mechanismsby which H1 affects gene expression, gene expression profiles were examined by Affymetrixmicroarray analysis. Despite the global changes in chromatin structure in these cells,expression of only a small number of genes is affected. Interestingly, a significantproportion of the affected genes are normally regulated by DNA methylation. Bisulfitesequencing analysis of DNA showed that the level of DNA methylation at certain loci isaffected in the triple H1 null ES cells. In contrast, the global DNA methylation status isunchanged. These results suggest that regulation of gene expression by H1 is mediated, atleast in part, through epigenetic regulation of DNA methylation.

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Barna Fodor Abstract 35The full complement of Su(var) gene function in Drosophila

Barna D. Fodor, Masato Yonezawa, Amit Sharma, Sandro Lein, GunterReuter and Thomas Jenuwein

Research Institute of Molecular Pathology (IMP), 7 Dr. Bohrgasse, Vienna, Landstrasse A-1030, Austria

The genome of eukaryotes is organized into distinct euchromatic and heterochromaticsubdomains. Heterochromatin has fundamental roles in the structural organization ofchromosomes, genome stability, and controlling epigenetic programs. Genetic screens in S.pombe and Drosophila have identified a number of genes called suppressors of variegation[Su(var)s] most of which encode components of heterochromatin. In this study we applied asingle nucleotide polymorphism (SNP) mapping approach to identify the previously isolated,unknown Su(var) mutations in Drosophila (~60) in collaboration with the lab of GunterReuter. First, Su(var) mutations were mapped between P-elements with molecularlycharacterized insertion sites by recombination mapping. Later the chromosomes from theserecombinants were analyzed by SNP markers. With this approach a genomic region of ~ 50kb that harbors the Su(var) locus can easily be designated.To date, we conducted linkage analysis on 24 Su(var)s and further mapped 14 by SNPmapping. We were able to identify novel Su(var)s indicating the involvement of thesumoylation pathway in heterochromatin formation. Other mutations have been narroweddown to less then hundred kb segments. In these regions several interesting candidateshave been identified. These results prove the utility of our mapping strategy. Once thecorresponding genes are identified, study of their mammalian homologs will follow. Theproject will contribute to the full definition of heterochromatin.

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Alexey Fomenkov Abstract 36Role of p63 in development disorders

Huang Y.P, Kim Y., Fomenkov A. and Ratovitski E.

Johns Hopkins University School of Medicine, Richard Ross Research Building, Room 733Rutland Avenue, Baltimore, MD 21205, U.S.A.

The stratified epithelia of the skin and oral mucosa are the most protective types of keratinized orcornified epithelia. To maintain this barrier, epithelial tissues undergo constant renewal and repair.A novel gene, p63, was found to be a key regulator of squamous epitheliadifferentiation/stratification and stem cell renewal during embryonic development. P63 nullmice showed crucial defects in skin development, including lack of stratification and structuresdependent upon epidermal-mesenchymal interactions, such as hair follicles, teeth, mammarygland, and prostate. However, the molecular mechanisms underlying a critical role for p63 instratification/development are still poorly understood.By using proteomic, genomic and cellular modeling approaches, our research team has madeimportant contributions into p63 field. p63 physically and functionally associated withmembers of RNA transcription/splicing machinery (e.g. ABBP1, SRA4, and C-terminal domainof RNA polymerase II). This novel finding supported the idea that p63 modulates specific RNAsplicing of the fibroblast growth factorreceptor-2 towards epithelial-specific isofom, FGFR2-K-SAM (KGFR) and thus determineepithelial cell fate. p63 function is regulated by a specific proteasome-dependent degradationmechanism through association with RACK1 and stratifin. This process is requiredphosphorylation and sumoylation of p63 and contributes into a molecular program leading tonormal skin and bone differentiation. The environmental stimuli (e.g. ultraviolet, chemicalagents, etc.) that induce DNA damage also enhanced p63 protein modifications leading toselective degradation of p63.As a transcriptional factor, p63 plays an important role in regulation of its downstream genetargets by both direct binding to the promoter area and by protein interaction with othercomponents of RNA processing complex. Our results demonstrated that p63 regulatestranscription of numerous genes specifically involved in cell-cell adhesion (e.g. K5, K14,plakoglobin, enviplakin, BPAG-1, and ¡V2, integrin alpha3beta1. These proteins were shown tobe critical components of cytoskeleton and hemi-desmosomes that define skin stratificationand mediate cell-cell junctions. These and other novel findings support the notion why p63 nullmice failed to develop proper skin structures.Mutations associated with the p63 DNA-binding domain found in patients with ectodermal

dysplasia, facial clefting, split hand/foot malformation, limb-mammary syndrome and acro-dermato-ungual-lacrimal-toothsyndrome and may directly affect the transcription properties of p63. While, a number ofmutations associated with AEC syndrome (ankyloblepharon, ectodermal dysplasia andclefting, AEC) and Rapp-Hodgkin syndrome has been mapped inside the p63 sterile Ą-motif.These mutations were suggested to inappropriately modulate the p63 protein-proteininteractions mediated by the sterile alpha-motif.

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Benjamin Freedman Abstract 37Linker histone dynamics, structure, and function in interphaseand mitotic egg extract chromosomes

Benjamin S. Freedman, Thomas J. Maresca and Rebecca Heald

University of California at Berkeley, Life Science Addition, Room #315, University ofCalifornia at Berkeley, Berkeley, CA 94720-0001, U.S.A.

Classic electron microscopic and nuclease digestion experiments have established thatlinker histones bind to nucleosomal arrays and compact them into a thicker chromatin fiber.It is unclear how these experiments, which were assayed at the scale of chromatin underprocessed conditions, relate to the in vivo organization of entire chromosomes. We recentlyaddressed this issue using physiologically active Xenopus egg extracts and sperm nucleithat progress through the cell cycle. We found that replicated, mitotic chromosomesassembled in extracts depleted of the embryonic linker histone H1M (or B4) had a distortedmorphology and were about twofold longer than normal (Maresca T.J. et al., 2005). Thus,H1M helps compact whole chromosomes, which is consistent with the classical role forlinker histones at the smaller scale of nucleosomal chromatin. Currently, we are using thisroughly physiological, vertebrate system to investigate several aspects of linker histonefunction: (1) To extend our findings from mitotic to interphase chromosomes, we are usingfluorescence microscopy to measure nuclear size, chromosome morphology, andchromosome length (by FISH) in nuclei assembled with or without H1M. (2) H1M must firstbe loaded onto DNA during interphase in order to compact mitotic chromosomes. Tounderstand the molecular mechanism underlying this loading step, we are performing H1Mimmunoprecipitations to identify binding partners. We are also comparing the bindingdynamics of H1M to chromosomes during interphase versus mitosis. (3) H1M has two majordomains, a globular head and a highly basic, unstructured carboxyl tail. We are substitutingthese domains for the full-length protein to evaluate their separate contributions tochromosome morphology. We are also engineering phosphorylation-state H1M mutants, toinvestigate the long-standing mystery of why many linker histones are phosphorylatedduring mitosis.

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Jennifer Gallagher Abstract 38Exploring establishment of Sir1-dependent silencing usingcomparative genomics

Jennifer Gallagher, Lenny Teytelman, Joshua Babiarz and Jasper Rine

University of California, Berkeley, 16 Barker Hall #3202, Berkeley, CA 94720, U.S.A.

Chromosomal regions are silenced by altering the chromatin structure in those regions toform heterochromatin. Silencing in yeast are most evident at the cryptic mating type loci(HM), telomeres, and at eukaryotic genes inserted into rDNA array. Silenced chromatin canbe stably inherited through many generations even in the absence of a protein, Sir1p, whichfunctions in its establishment. In the presence of Sir1p, silencing at the HM loci is efficientlyestablished to silence the cryptic mating loci. Recent studies have shown that silenced chromatin can rapidly evolve and the proteincomponents can vary greatly from species to species. Saccharomyces cerevisiae containsone SIR1 gene, while another Saccharomyces species, S. bayanus, contain four orthologsof the Saccharomyces SIR1 gene, all of which are diverged somewhat from each other. Wehave that least three of the proteins encoded by these orthologs are localized to the HMR-Esilencer in Saccharomyces bayanus and all are likely to function in silencing. Wehypothesize that multiple Sir1 proteins from S. bayanus may represent a division of laborthat is consolidated into one protein in S. cerevisiae. This hypothesis implies that thesilencers themselves may have evolved more elaborate structures in S. bayanus.Differences in the HML and HMR silencers may explain why different Sir1 proteins toestablish silencing at HML and HMR. In the recently identified Orc1 interaction domain ofSir1, six of eight amino acids essential for the Orc1 interaction are invariant in the closestortholog of S. bayanus and two are similar (Hou, PNAS 2005). In contrast, the S. bayanusparalogs of the closest Sir1 ortholog all contain from two to four of the eight invariantresidues in the Orc interaction domain. Based on the sequence alignments, the Sir1paralogs could be recruited to the chromatin via a different DNA binding proteins.

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Nicolas Gévry Abstract 39Implication of histone variant H2A.Z in the regulation of thep53/p21CIP1/WAF1 pathway.

Nicolas Gévry and Luc Gaudreau

Departement de Biologie, Faculte des Sciences, Centre de Recherche sur les Mecanismesdu Fonctionnement Cellulaire, Universite de Sherbrooke, Sherbrooke, Quebec, Canada.

In addition to post-translational modifications of histone tails and ATP-dependant chromatinremodelling, the incorporation of core histone variants into nucleosome plays a key role ingoverning gene expression. Histone variant H2A.Z has been shown to be important for theproper induction of several genes in yeast. In spite of the high homology between yeastand human, it remains to be determined if H2A.Z is important for mammalian genetranscription. Here, we show the implication of H2A.Z in the p53/p21CIP1/WAF1 pathwayboth regulating transcription of p21, and the senescence induction program. High-resolutionChIP analyses using quantitative PCR demonstrates preferential binding of H2A.Z in theregion overlapping the p53 binding sites of p21. Moreover, this localization of H2A.Zsurrounding the p53 binding sites is not present in p53-deficient cell lines. Down regulationof H2A.Z expression by shRNA technology led to premature senescence of untransformedhuman fibroblasts, including p21 induction, senescence-associated heterochromatic foci,and beta-gal staining. Collectively, our data suggests that the incorporation of the histonevariant H2A.Z into chromatin inhibits p53-p21 transcription and the development ofpremature senescence.

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Anja Groth Abstract 40The role of human Asf1 in histone metabolism duringreplicational stress

Groth A., Loyola A., Ray-Gallet D., Quivy J-P., Lukas J., Bartek J. andAlmouzni G.

Institute Curie, UMR218, Pavillon Pasteur, 26 Rue d’Ulm, Paris, 75248 France

Maintenance of chromosomal integrity requires tight coordination of histone biosynthesiswith DNA replication. We have recently shown that extracts from human cells exposed toreplication inhibitors display an increased capacity to support replication-coupled chromatinassembly (Groth et al., 2005). This enhanced activity is dependent on the histonechaperones Asf1a and Asf1b (hAsf1), which can synergize with the chromatin assemblyfactor CAF-1 in repair- and replication-coupled chromatin assembly (Tyler et al., 1999; Melloet al., 2002). In cytosolic extracts from unperturbed S-phase cells, hAsf1 exists inequilibrium between an active histone-containing multi-chaperone complex and an inactivehistone-free form. Interference with DNA replication results in accumulation of soluble S-phase histones and mobilization of the majority of hAsf1 into the active complex. Our datasuggest that hAsf1 provides the cells with a buffering system for histone excess generatedin response to stalled replication and explains how mammalian cells maintain a criticalactive histone pool available for deposition during recovery from replicational stresses.Moreover, our findings points to hAsf1 as a candidate sensor of histone availability. We arecurrently exploring such a potential regulatory function for hAsf1 by searching for novelinteractors potentially facilitating integration of histone availability with DNA replication,checkpoint signaling, and histone biosynthesis.

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Matthew Guenther Abstract 41The active and Polycomb-repressed genome in humanembryonic stem cells

Matthew G. Guenther1*, Tong Ihn Lee1*, Richard G. Jenner1*, Laurie A.Boyer1*, Brett Chevalier1*, Stuart S. Levine1*, Roshan M. Kumar1, SarahE. Johnstone1,2, Heather L. Murray1, Jacob P. Zucker3, Bingbing Yuan1,George W. Bell1, Elizabeth Herbolsheimer1, Nancy M. Hannett1, Megan F.Cole1,2, Kaiming Sun1, Duncan T. Odom1, Thomas L. Volkert1, David P.Bartel1,2, Douglas A. Melton3’5, David K. Gifford1,5, Rudolf Jaenisch1,2 andRichard A. Young1,2,5

1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge,Massachusetts 02142, U.S.A., 2Department of Biology, Massachusetts Insitute ofTechnology, Cambridge, Massachusetts 02139, U.S.A., 3Howard Hughes Medical Institute,Department of Molecular and Cellular Biology, Harvard University, Cambridge,Massachusetts 02138, U.S.A., 4MIT CSAIL, 32 Vassar Street, Cambridge, Massachusetts02139, U.S.A., 5Broad Institute of MIT and Harvard, One Kendall Square, Building 300,Cambridge, Massachusetts 02139, U.S.A.*These authors contributed equivalently to this work

The capacity of embryonic stem (ES) cells to self-renew and to give rise to multiple somaticlineages holds promise for human regenerative medicine. To gain insights into global generegulation in ES cells, we determined the locations of RNA polymerase II and the PolycombRepressive Complex-2 (PRC2) subunit Suz12 across the entire non-repeat portion of thehuman genome. This map of active and repressed portions of the genome identifies activepromoters for one-third of human genes and reveals the targeted repression of keydevelopmental regulators by PRC2. Our results produce a high-confidence transcriptomefor ES cells and provide insights into how PRC2 contributes to regulation of stem cellpluripotency and self-renewal.

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Shweta Hakre Abstract 42Growth factor mediated gene regulation by TFII-I

Shweta Hakre1,3 and Ananda L. Roy1,2,3,4

Departments of Pathology1 and Biochemistry2 and Programs in Immunology3 andGenetics4 Tufts University School of Medicine, Boston, MA 02111, U.S.A.

TFII-I is a signal induced multi-functional transcription factor that can regulate transcriptionalactivity of the growth-responsive c-fos gene. Although TFII-I has multiple spliced isoforms,in murine fibroblasts the delta (D) and beta (b) isoforms are predominantly expressed. Weshow here that these two isoforms have distinct subcellular localization and mutuallyexclusive transcription functions in the context of growth factor signaling in vivo. While TFII-ID is largely cytoplasmic in the resting state and translocates to the nucleus upon growthfactor signaling, TFII-Ib is nuclear in basal state and is exported out of the nucleus upongrowth factor stimulation. Employing quantitative chromatin immmunoprecipitation assaysand gene silencing experiments with RNAi, we demonstrate that the TFII-Ib is basallyrecruited to the c-fos promoter in vivo but the recruitment is abolished in the presence ofmitogenic stimulation. In contrast, upon growth factor signaling,TFII-ID undergoes induced tyrosine phosphorylation, translocates to the nucleus and isrecruited to the same site on the c-fos promoter. Importantly, we also demonstrate thatupon growth factor signaling, the TFII-I delta isoform interacts with Erk1/2 (MAPK) kinase inthe cell cytoplasm and imports the Erk1/2 to the nucleus, thereby transducing growth factorsignaling. Our results identify a novel growth factor signaling pathway controlled byopposing activities of two TFII-I spliced isoforms.

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Melissa Harrison Abstract 43The MBT repeat-containing protein LIN-61 regulates C.elegans vulval cell-fate specification

Melissa Harrison1, Bob Horvitz1 and Xiaowei Lu2

1Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute ofTechnology,Cambridge, MA 02139., 2Department of Cell Biology, University of VirginiaSchool of Medicine, Charlottesville, VA 22908, U.S.A.

Chromatin remodeling plays an important regulatory role in gene expression, a criticalcomponent in controlling cell-fate specification. However, there is limited knowledge of themeans by which individual chromatin-remodeling factors cause changes in developmental-fate decisions. Studies of vulval development in the nematode Caenorhabditis eleganshave proven to be useful in elucidating mechanisms involved in cell-fate specification. TheA dult vulva arises from three of six ectodermal blast cells, all of which are initiallycompetent to adopt a vulval cell fate. Vulval specification is negatively regulated by at leastthree redundant functions provided by the synthetic Multivulva (synMuv) class A, B, and Cgenes. Loss-of-function mutations in two or more gene classes result in a Multivulva (Muv)phenotype, characterized by more than the normal three blast cells adopting vulval cellfates. Many of the synMuv genes, including lin-35Rb, dpl-1DP, efl-1E2F, lin-53RbAp48,hda-1HDAC1, let-418Mi-2, trr1TRRAP, mys-1MYST, and epc-1E(Pc), have homologs inother species that function in chromatin remodeling and transcriptional regulation.lin-61, a gene identified in screens for class B synMuv mutants, encodes a proteincontaining four MBT (malignant brain tumor) repeats. MBT repeats are loosely-defined~100 amino acid sequences present in several nuclear proteins, such as the DrosophilaPolycomb group protein Sex Comb on Midleg. Structural analysis of MBT repeats fromother proteins shows that these domains have similarity to Tudor and chromodomains thathave been shown to bind modified histones. Antibody staining demonstrates that LIN-61 isa ubiquitously-expressed, DNA-localized nuclear protein. LIN-61 staining patterns aresimilar in most synMuv mutant backgrounds, suggesting expression is independent of thefunction of other synMuv proteins. Ten loss-of-function alleles have been identified throughgenetic screens. Analysis of missense mutants has identified substitutions in highly-conserved residues that may disrupt protein stability, whereas proteins mutant in a non-conserved region of the MBT repeat or outside the MBT repeats are stable, if functionallycompromised. Experiments to identify the proteins that interact with LIN-61 in vivo areongoing.

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Michael Hendzel Abstract 44The regulation of histone H1 binding in vivo

John Th’ng, Ming Ye and Michael J Hendzel

University of Alberta, Rm 3332, Cross Cancer Institute, 11560 University Ave., Edmonton,Alberta, T6G1Z2, Canada

Using fluorescence recovery after photobleaching (FRAP) to quantify the binding propertiesof histone H1 subtypes in living cells, we have defined the C-terminal domain (CTD) as theprimary determinant of binding affinity to chromatin. In this report, I will review experimentsthat demonstrate that the C-terminal domain of histone H1 becomes structured uponbinding to chromatin. Differences in the binding affinity of H1 histone subtypes correlatewith the length of the CTD but not with the overall net charge of the domain. Remarkablysingle amino acid substitutions within the CTD can destabilize binding as much as 90% andidentify specific segments of the histone H1 CTD that are particularly important in definingthe affinity of binding. By swapping CTDs between individual H1 subtypes, we were also able to define a smallcontribution mediated by the N-terminal domain. By positioning the fluorescent tag oneither the C-terminus or the N-terminus and by using fluorescent tags of substantiallydifferent size, we have been able to define the extent that fluorescent tagging disruptshistone H1 binding. These experiments reveal that green fluorescent protein and itsvariants reduce the binding affinity of H1 histones and raise concerns about conclusionsdrawn from studies employing histone proteins that use fluorescent protein tags. Usingsmall fluorescent tag of less than 15 amino acids, we find that although H1 subtypes aredynamically bound to chromatin in vivo, they bind more stably than previously appreciated.Histone H1.5, for example, can remain bound for up to 20 minutes before dissociating. Histone H1 subtypes showed different preferences for the regions of chromatin that theybind. Nonetheless, all H1 subtypes were regulated by phosphorylation and thisphosphorylation could be inhibited by the cyclin dependent kinase inhibitor, roscovitine.When binding of histone H1 was compared between cancer cell lines and normal cell lines,cancer cell lines were observed to destabilize histone H1 binding to different extents thatcorrelated with differences in steady-state histone H1 phosphorylation status. Interestingly,histone H1 binding affinity also correlated with radiosenstivity. Collectively, our results are beginning to unravel the unique properties of individual histoneH1 subtypes, the mechanisms of regulating histone H1 binding, and the existence of tertiarystructure in the CTD.

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Wilma A.Hofmann Abstract 45Specific roles for nuclear actin and nuclear myosin I intranscription by RNA Polymerase II

Wilma A.Hofmann1, James A. Goodrich2, Primal de Lanerolle1

1Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL,60612, U.S.A., 2Department of Chemistry and Biochemistry, University of Colorado atBoulder, Boulder, CO, 80309-0215, U.S.A.

It has been known for many years that actin is abundant in the nucleus. Nevertheless, thephysiological functions of actin in the nucleus are not well defined. The discovery of another“cytoplasmic protein” in the nucleus, namely nuclear myosin I, has piqued interest in thefunctional role of these proteins in the nucleus. We have found that nuclear actin andnuclear myosin I (NMI) are intimately involved in the basic process of transcription. ?-Actinas well as NMI are tightly bound to and co-purify with the RNA polymerase II core enzyme.The functional involvement of actin and NMI in transcription was demonstrated in vivo aswell as in vitro. Microinjecting antibodies to actin or NMI inhibited transcription andchromatin immunoprecipitation assays showed that both proteins are present at thepromoter region of transcribing genes. In vitro transcription assays using highly purifiedproteins confirmed that both actin and NMI are required for transcription initiation. However,a combination of pre-initiation complex (PIC) assembly assays and protein-binding studiesshowed that they are involved in subtly different ways. While actin is needed at the pre-initiation stage of transcription and necessary to integrate RNA polymerase II into thedeveloping PIC, NMI is not necessary for PIC-formation per se. In fact, NMI appears to benecessary for the formation of the first phosphodiester bond during transcription initiation.Taken together, our results demonstrate not only a crucial role for nuclear actin and nuclearmyosin I in the basic process of transcription but also present mechanistic evidence on howthose proteins are involved in transcription.

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Sui Huang Abstract 46Upstream binding factor association induces large scalechromatin decondensation

Danyang Chen, Andrew S. Belmont and Sui Huang

Northwestern University Medical School, Ward 11-240, 303 E. Chicago Ave., Chicago, IL60611, U.S.A.

The function of upstream binding factor (UBF), an essential component of the RNApolymerase I (pol I) preinitiation complex, is unclear. Recently, UBF was found distributedthroughout ribosomal gene repeats rather than being restricted to promoter regions. Thisobservation has led to the speculation that one role of UBF binding may be to inducechromatin remodeling. To directly evaluate the impact of UBF on chromatin structure, weused an in vivo assay in which UBF is targeted via a lac repressor fusion protein to aheterochromatic, amplified chromosome region containing lac operator repeats (Capenter,A. E. and Belmont, A. S. 2004, Methods Enzymol 375, 366-381). We show that theassociation of UBF with this locus induces large-scale chromatin decondensation. Thisprocess does not appear to involve common remodeling complexes, including SWI/SNFand histone acetyltransferases (HATs), and is independent of extensive histone acetylation.However, UBF recruits the pol I specific, TATA box binding protein (TBP) containing complexSL1 and pol I subunits. Our results suggest a working hypothesis in which the dynamicassociation of UBF with ribosomal DNA (rDNA) clusters recruits the pol I transcriptionmachinery and maintains these loci in a transcriptionally competent configuration. Thesestudies also provide an in vivo model simulating rDNA transactivation outside the nucleolus,allowing temporal and spatial analyses of chromatin remodeling and assembly of the pol Itranscription machinery.

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Dana Huebert Abstract 47Global analysis of MLL and MLL-fusion mediated histonemodifications

Dana J. Huebert1,2, Christina M. Hughes2,3, Matthew Meyerson2,3, Stuart L.Schreiber2,4, Bradley E. Bernstein1,2

1Massachusetts General Hospital, Dept of Pathology, Building 149 13th St, Charlestown,MA, 2Broad Institute of Harvard and MIT, 320 Bent Street, Cambridge, MA, 3Dana-FarberCancer Institute, Dept of Medical Oncology, 44 Binney St, Boston, MA, 4Howard HughesMedical Institute, Harvard University, Dept of Chemistry and Chemical Biology, 12 OxfordSt., Cambridge, MA, U.S.A.

Rearrangements of MLL (mixed lineage leukemia) are implicated in up to 40% of acutemyelogenous leukemias. The MLL protein product methylates lysine 4 of histone 3, amodification generally associated with transcriptional activation. Genomic analyses haveshown that lysine 4 methylation is typically punctate and found at gene starts. However,broad, cell-type specific domains span the active portions of the Hox clusters. Thesedomains may keep chromatin in a permissive state, allowing the cell to epigeneticallymaintain active transcription. MLL has recently been shown to co-localize with RNApolymerase at active genes, an unusual finding given its known role as an oncogene. Weinvestigated the function of an MLL fusion protein using chromatin immunoprecipitation,real-time PCR and microarrays. The data suggest that the fusion protein localizes toregions distinct from those bound by wild-type MLL. We speculate that, while wild-type MLLhas an important role in maintaining active chromatin at many genes, the fusion proteinfunctions at a subset of genes, including Hox, which mediate its pathogenesis.

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Christina Hughes Abstract 48Linking Menin mutations to defects in histone methylationand altered expression of target genes

Christina M. Hughes, Laura E. MacConaill, Andrew Goldstein, OritRozenblatt-Rosen and Matthew Meyerson

Dana Farber Cancer Institute, Mayer 434, 44 Binney St., Boston, Suffolk 02115, U.S.A.

Some of the mutations that cause cancer directly alter the expression of genes involved inthe regulation of cellular growth and differentiation. Gene expression, however, is regulatednot only by the sequence of the genes themselves, but by the structure of the chromatin,including modifications of histones. This suggested the possibility that proteins that regulatechromatin structure and histone modification could be involved in the genesis of humancancer. We and others have shown that the tumor suppressor menin, the protein product ofthe MEN1 gene, which is mutated in familial endocrine neoplasia type 1, associates with aSET1 (COMPASS)-like histone methyltransferase (HMTase) complex. Some, but not all, ofthe MEN1 mutations associated with familial cancers affected HMTase activity in vitro. Toelucidate the role(s) menin plays we are exploring the interaction(s) of menin with the boththe MLL and MLL2 proteins, which we believe are key protein components of similar butdistinct menin interacting histone methyltransferase complexes. Because we areparticularly interested in linking the defects in the HMTase complexes to the ability of themenin mutants to cause cancer, we are analysing the complexes to determine how thepatient-derived menin mutations, in particular the mutations that do not have an affect onHMTase activity, alter the complexes. We are also asking how the mutations affect theregulation of menin target genes, for example the homeobox gene Hoxc8, to study the linksbetween the defects gene expression with the formation of the MEN1 tumors. Finally, wehave shown that menin is specifically phosphorylated and are in the process of determiningwhether the phosphorylation of menin affects its association with, or the activity of, theHMTase complexes.

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Irena Ivanovska Abstract 49Histone code in meiosis: histone kinase required forchromosomal architecture in Drosophila oocytes

Irena Ivanovska, Tulasi Khandan and Terry L. Orr-Weaver

Whitehead Institute, 9 Cambridge Center, Cambridge, Massachetts 02142, U.S.A.

Chromosomes undergo dramatic morphological changes that facilitate their functions.During sexual reproduction, meiotic chromosomes assume specialized configurations thatensure accurate segregation of homologous chromosomes and promote faithful propagationof the genetic material. Although histone modifications have well-established functions inchromosomal structural changes, their roles in meiotic chromosomal structures are largelyunknown. We discovered a histone code in meiosis characterized by a specific pattern ofhistone modifications. Phosphorylation of Histone H2A by a recently-identified kinase, NHK-1, is an important component of this code. Lack of Histone H2A phosphorylation in theDrosophila nhk-1 mutant results in female sterility due to defects in the karyosome (theprophase I arrest state of the chromosomes) and subsequently in the metaphase I arrest.These defects are a result of failure to disassemble the synaptonemal complex and to loadcondensin onto the mutant chromosomes. The absence of Histone H2A phosphorylation inthe nhk-1 mutant leads to loss of specific histone acetylations, while other modificationsremain unaffected, consistent with the histone code hypothesis. As meiotic progression iseasily visualized in Drosophila ovaries, this system has enabled us to decipher the temporalrelationships between the relevant histone modifications. Embryos laid by nhk-1 mutantfemales arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis isaffected as well. These studies reveal a critical role for histone modifications inchromosome dynamics in meiosis and mitosis.

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Danika Johnston Abstract 50Role of Drosophila ecdysone receptor and histonemethyltransferase TRR in gene regulation

Danika Johnston, Yurii Sedkov, Svetlana Petruk, James B. Jaynes andAlexander Mazo

Thomas Jefferson University, 485 JAH, 1020 Locust Street, Philadelphia, PA 19107, U.S.A.

Type I steroid hormone receptors shuttle between the nucleus and cytoplasm, howevermost hormone receptors are of the type II category and predominantly nuclear. Thesenuclear receptors are present at their binding site irrespective of the transcriptional status oftheir target genes. In the presence or absence of ligand, Type II receptors can recruit co-activator or co-repressor proteins, respectively, to the target gene in order to alter itstranscriptional status usually through chromatin structure modifications. The current modelfor the Drosophila Ecdysone receptor (heterodimer of Usp and EcR) suggests that thisreceptor belongs to the Type II group. Our results indicate that EcR, and it co-activator,histone methyltransferase (HMTase) Trithorax Related (TRR), can form a protein complexoutside DNA and that they are not present in vivo at the promoters of repressed targetgenes in the absence of ecdysone. Confirming experiments suggest that both EcR andTRR are shuttling between the nucleus and cytoplasm in an ecdysone dependent fashion,but interestingly, these two proteins may be shuttling independently of each other. This dataquestions the current model of Ecdysone Receptor functioning as a Type II receptor andprovoked us to investigate the presence or absence of nuclear receptor components andtheir co-regulators at the promoters of certain ecdysone induced genes both before andafter induction. Preliminary results from chromatin immunoprecipitation experimentsindicate that, following induction with ecdysone, EcR and Usp, as well as their co-activatorTRR, are recruited to the promoters of both Hsp27 and hedgehog (hh), two ecdysoneinduced genes. Results also showed an increase in the presence of pCAF, a histoneacetyltransferase, and a decrease in SMRTER, a transcriptional co-repressor afterecdysone treatment. These results will aid in understanding the mechanism of nuclearreceptors and histone modifying complexes to alter the transcription of developmentalgenes.

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Sarah Johnstone Abstract 51Embryonic stem cell transcription factors regulatedevelopmental transcription factors

Sarah E.Johnstone1,2, Laurie A. Boyer1,6, Tong Ihn Lee1,6, Megan F.Cole1,2, Stuart S. Levine1, Jacob P. Zucker3, Matthew G. Guenther1,Roshan M. Kumar1, Heather L. Murray1, Richard G. Jenner1, David K.Gifford1,4,5, Douglas A. Melton3,5, Rudolf Jaenisch1,2, and Richard A.Young1,2,5*

1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge,Massachusetts 02142, U.S.A., 2Department of Biology, Massachusetts Institute ofTechnology, Cambridge, Massachusetts 02139, U.S.A., 3Howard Hughes Medical Institute,Department of Molecular and Cellular Biology, Harvard University, Cambridge,Massachusetts 02138, U.S.A., 4MIT Computer Science and Artificial Intelligence Laboratory(CSAIL), 32 Vassar Street, Cambridge, Massachusetts 02139, U.S.A ., 5Broad Institute ofMIT and Harvard, One Kendall Square, Building 300, Cambridge, Massachusetts 02139,U.S.A.

Embryonic stem (ES) cells have two important properties: the ability to self-renew and thecapacity to develop into any cell type. The transcription factors OCT4, SOX2, and NANOGhave essential roles in early development and are required for the propagation ofundifferentiated ES cells in culture. To gain insights into transcriptional regulation of humanES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scalelocation analysis. Interestingly, target genes for these factors frequently encode transcriptionfactors, many of which are developmentally important homeodomain proteins. Theseresults provide new insights into the transcriptional regulation of stem cells and reveal howOCT4, SOX2, and NANOG contribute to self-renewal and pluripotency.

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Paul Kaufman Abstract 52Replication-independent histone deposition by the HIRcomplex and Asf1

Erin M. Green1, Andrew J. Antczak1, Aaron O. Bailey2, Alexa A. Franco1,Kevin J. Wu1, John R. Yates 3rd2 and Paul D. Kaufman3

1Lawrence Berkeley National Laboratory and Department of Molecular and Cell Biology,University of California, Berkeley, Berkeley, CA 94720, U.S.A., 2Department of Cell Biology,The Scripps Research Institute, La Jolla, CA 92037, U.S.A., 3Program in Gene Function andExpression, University of Massachusetts Medical School, Worcester, MA 01605-2324, U.S.A.

The orderly deposition of histones onto DNA is mediated by chromatin assembly proteins.In budding yeast, products of four HIR genes (HIR1, HIR2, HIR3 and HPC2) regulatehistone gene expression, pericentric chromatin, and heterochromatin, but had beenuncharacterized biochemically. Mass spectroscopic analysis of tandem affinity-purified Hirproteins shows that these four proteins comprise the HIR complex. The HIR complex co-purifies with histone deposition protein Asf1 under mild ionic conditions. Each of the fourHir proteins contribute to the stability of the HIR complex and its localization to a histonegene promoter; in contrast, Asf1 is not required for HIR complex localization to a histonegene promoter.The HIR complex and Asf1 together deposit histones onto DNA in a replication-independentmanner. However, the HIR complex does not alter the replication-linked histone depositionactivity of Chromatin Assembly Factor-1. The HIR and Asf1 proteins thus represent aconserved eukaryotic pathway for histone replacement throughout the cell cycle.

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Ik Soo Kim Abstract 53A chromatin remodeling complex in regulation of crosstalkbetween the Wnt and the NF-kappaB pathway

Ik Soo Kim, Jung Hwa Kim, Hee June Choi, Bogyou Kim, Ji Min Lee andSung Hee Baek

Seoul National University 216, 20 Sillim-Dong, Kwanak-Ku, Seoul 151-742, South Korea

Deciphering the molecular strategies, by which specific signal pathways regulate biologicalprocesses by switching genes to the appropriate on or off state, remains an important issuein biology. Transcription of most genes in the nucleus is regulated by the coordinate actionof cofactors and chromatin remodeling complexes. BAF60c has been reported to becomponents of ATP-dependent chromatin remodeling complexes. Here we present cDNAmicroarray data revealing crosstalk between the NF-kappaB and the Wnt/beta-cateninpathway, and found that a subset of NF-kappaB target genes including BAF60c are down-regulated by activation of the Wnt/beta-catenin pathway. We will present a molecularmechanism of crosstalk between the NF-kappaB and the Wnt/beta-catenin signalingpathway.

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Elena Kisseleva-Romanova Abstract 54Identification of new mutants that lead to transcriptioninternal entry in S. cerevisiae

Kisseleva-Romanova E., Youdell M., Nair A., Karabetsou N., Morillon A.and Mellor J.

University of Oxford, C8, Dept of Biochemistry, Microbiology Unit, South Parks Road,Oxford, OX1 3QU, U.K.

During transcription, the histone chaperones Spt6 and Spt16 (FACT) assist RNApolymerase II (RNAPII) to disassemble and re-assemble histones on DNA. In S. cerevisiae,it has been shown that in absence of Spt6 or Spt16 activity, nucleosomal histone levels arereduced which result in RNAPII-dependent internal initiation inside FLO8, STE11 or VPS72genes. To provide a better understanding of this mechanism, we have started tosystematically screen for other components involved in internal initiation at STE11 gene.Interestingly, we identified factors interacting with elongating RNAPII and which are relatedto covalent histones modifications and chromatin remodelling processes. To discriminatebetween complexes controlling histone expression and histone assembly in these mutants,we compared levels of global histone H3 to the occupancy of histone H3 at several lociincluding STE11. We will discuss the possibility of a novel aspect of histones chaperonemetabolism and its consequence on RNAPII transcription.

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Christoph Koch Abstract 55A microarray based approach to develop high resolutionmaps of histone modifications in 1% of the human genome

Christoph M. Koch1, Shane C. Dillon1, Robert M. Andrews1, Gayle K.Clelland1, Sarah Wilcox1, Joanna M. Fowler1, Keith D. James1, AlexanderW. Bruce1, Phillippe Couttet1, Oliver M. Dovey1, Peter D. Ellis1, Andrew J.Mungall1, Pawandeep Dhami1, Bee Ling Ng1, Heike Fiegler1, Cordelia F.Langford1, Paul Flicek2, Ewan Birney2, Nigel P. Carter1, David Vetrie1, IanDunham1

1The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,Cambridge,CB10 1SA, U.K., 2EMBL – European Bioinformatics Institute, Wellcome TrustGenome Campus, Hinxton, Cambridge, CB10 1SD, UK

The NHGRI has established a pilot project (ENCODE) to explore computational andexperimental methods to develop an encyclopaedia of DNA elements in the humangenome. Initially the project targets 1% of the genome chosen according to the criteriaoutlined at http://www.genome.gov/10506161. We have constructed a microarrayrepresenting the 44 ENCODE regions consisting of 24156 PCR fragments with an averagesize of 1 kb. It covers ~80% of the targeted regions including repetitive elements wherepossible. We are using this microarray to assay DNA samples enriched for sequencesinvolved in specific biological processes and functions generated by chromatinimmunoprecipitation (ChIP). ChIP experiments are being performed with a variety ofantibodies for specific histone modifications in a lymphoblastoid cell line (GM06990), anerythroleukemia cell line (K562) and a foetal lung fibroblastoid cell line (HFL-1). Our aim isto investigate whether histone modifications can be used as a robust indicator of functionalelements in the genome. Therefore we have developed high resolution maps of histonemodifications and correlated these maps with a range of genomic DNA features includingC+G content, genes/exons, repeat elements, SNP density and regions of conserved DNAsequence identified by comparative sequencing across multiple species as well as theexpression profiles of the cell lines. Preliminary analysis reveals strong enrichments of acetylated histone H3 and di- and tri-methylated histone H3 (H3K4me2 and H3K4me3) at 5 ends of transcriptional start sites.Mono-methylated histone H3 (H3K4me1) was found to be widely distributed and notexclusive focussed to transcriptional start sites similar to acetylated histone H4 (H4ac).While comparing enrichments between different cell lines we found a correlation of theexpression status of genes and the absence or presence of H3K4me3 at the transcriptionalstart site/promoter. Active promoters in each cell line show a robust enrichment of H3K4me3while inactive promoter do not.

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Hyockman Kwon Abstract56BAF53 is essential for maintenance of chromosometerritories and higher-order chromatin structure

Ki Won Lee, Mi Jin Kang, Jae Hwan Shim, Su Jin Kwon, Yunhee KimKwon and Hyockman Kwon

Department of Bioscience and Biotechnology, 210 Natural Science, 89 Wangsan-riMohyeon-myon, Yongin, Kyongki-do, 449-791, Korea

In interphase mammalian cells, chromosomes are compartmentalized into discretechromosome territories. Chromosome territory has been viewed as a collection ofchromosomal subdomains, but the structure of chromosomal subdomain remains largelyelusive. Here we show that BAF53 knockdown by siRNA interference causes disintegrationof chromosome territories and opens up higher-order chromatin structure. These distinctstructural changes of chromosomes were accompanied with a cell cycle defect and theinduction of several unexpressed genes such as Prm 2. Relaxation of chromatin fibers frombackfolding in chromosomal subdomains explains well these results and can be simulatedby dissociation of chromatin loops from loop base spring according to the multi-loopsubcompartment model. In agreement with the role of BAF53/beta-actin dimer as anucleation center for actin oligomerization, we discover that BAF53 knockdowndisassembles nuclear actin array. Based on these observations we propose a tentativemodel for loop base spring.

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Eric Lam Abstract 57Chromatin charting in living plants: a global study.

Eric Lam

Biotech Center, Rutgers University, 216 Foran Hall, 59 Dudley Road, New Brunswick, NewJersey 08901, U.S.A.

In the phenomena known as position effects, integration loci affect the expression levels oftransgenes. For instance, a transgene that is highly expressed when integrated intoeuchromatic regions may be poorly expressed when inserted into heterochromatic regions.Recent studies suggest that chromosomes in interphase nuclei may be organized, and thatthe 3-D positions of genes in the interphase nuclei may influence their expression. Thegoals of our Chromatin Charting project are: 1) to produce a set of resources to betterquantify position effects in the Arabidopsis genome and 2) to track the three dimensional (3-D) positions and dynamic properties for specific regions of the genome in interphase nucleiof live plants. To these ends, we are presently generating over 5,000 Ds-tagged lines ofArabidopsis. The Ds-tagging cassette contains a CaMV35S::luciferase which allowsmonitoring of position effects, and a lac operator repeat (array) which allows the tagged locito be visualized by expressing fusions of fluorescent-proteins with lacI-NLS within the nucleiof live Arabidopsis plants. We are mapping the tagged loci with the goal of selecting 3,000lines with which tagged loci would occur on average once every 40 kbp. The selected lineswill be used to evaluate the position effects on gene activity at a global level. In addition, avisualization line expressing an HcRed-lacI-NLS fusion protein would be crossed to theseselected transposants for visual tracking of the inserted loci. Lastly, we are also trying toimprove our fluorescent protein tracking system by creating a multicolor system for morequantitative evaluation of the location and dynamics of the tagged loci. These materialsshould be useful for the characterization of mutations and corresponding genes thatinfluence gene expression through altering chromatin architecture in eukaryotes. Ourprogress with this project would be presented.

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Robert Lane Abstract 58Does chromatin inaccessibility contribute to mutuallyexclusive olfactory receptor transcription

Marijo Kambere and Robert P. Lane

Wesleyan University, Hall Atwater Laboratory, Lawn Avenue, Middletown, CT 06457, U.S.A.

The mammalian olfactory system is able to detect and distinguish among tens ofthoU.S.A.nds of odorants in the environment. This ability is dependent on an organizingprinciple in which each sensory neuron in the nose expresses only one olfactory receptor(OR) allele from among >1000 OR genes in the genome. We are interested inunderstanding the regulatory mechanisms that govern this mutually exclusive expression.Previous genomic and genetic studies suggest that OR genes reside in repressed regionsof the genome, and we are exploring the hypothesis that mutually exclusive transcription ofonly one OR allele is facilitated by limiting transcriptional access by chromatin modifications.We have used chromatin immunoprecipitation (ChIP) assays to generate preliminary resultson the histone modifications at an OR locus that we have previously shown becomes activein a differentiated olfactory sensory cell line. For all six modifications tested, histones aroundthe tested OR locus in undifferentiated cells show acetylation and methylation patternsconsistent with inactive euchromatin. This result suggests that the ground state for OR lociin premature sensory neurons is “closed”. We are now assaying histone acetylation andmethylation patterns at multiple OR loci to test whether all OR genes are similarly “closed”in undifferentiated cells, and investigating how chromatin patterns change during thedifferentiation process. We anticipate having preliminary results for these studies by thedate of this meeting.

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Martin Law Abstract 59Mutations in the ATRX chromatin remodelling protein causechanges in histone H3 lysine 9 methylation.

Martin Law, Richard Gibbons and Douglas Higgs

MRC Molecular Haematology Unit, WIMM, Oxford, Weatherall Institute of MolecularMedicine, John Radcliffe Hospital, Oxford, OX3 0J1, U.K.

Mutations in the chromatin remodelling protein, ATRX, cause a rare X-linked developmentaldisorder characterised by severe mental retardation, abnormalities of the face, skeleton andurogenital system with short stature and mild alpha thalassaemia - the ATR-X syndrome.The precise role of the ATRX protein is currently unclear although several lines of evidencesuggest that it may influence transcription by altering chromatin structure. In common withmembers of the SNF2 family of chromatin remodelling proteins ATRX possesses conservedATPase/helicase motifs and can disrupt histone-DNA contacts in vitro. ATRX alsopossesses an extended variant PHD domain, which is a cysteine rich zinc finger motif alsoseen in other chromatin-associated proteins. The PHD-like domain and the ATPase domainare the most common sites of mutations that cause the ATR-X syndrome, underlining theirfunctional importance. ATRX has been shown to interact with several proteins that caninfluence transcription (DAXX, HP1 and EZH2), although the significance of theseinteractions is not yet clear.One mechanistic link between ATRX and the alteration of gene expression is that ATR-Xpatients exhibit diverse changes in DNA methylation patterns. In ATR-X patient cells,ribosomal genes become substantially hypomethylated at the promoter and coding region.In light of the recently described links between chromatin remodelling, DNA methylation andhistone methylation, this study sought to further define the role of ATRX in theinterconnection of these epigenetic pathways. Here, chromatin immunoprecipitation wasused to show that in ATRX patient cell lines, the ribosomal genes show reduced histone H3lysine 9 tri- methylation which parallels the reduction in DNA methylation. These epigeneticchanges however produce no detectable alteration in the rate of ribosomal genetranscription. These data support the idea that, in human cells, chromatin remodelling,histone H3 lysine 9 methylation and DNA methylation are linked.

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Kenneth Lee Abstract 60Functional analysis of the ADA histone acetyltransferasecomplexKenneth K. Lee, Samantha Pattenden, Daeyoup Lee, Laurence Florens,Selene K. Swanson, Michael Washburn and Jerry L. Workman

Stowers Institute for Medical Research, Kansas City, MO, U.S.A.

The histone acetyltransferase, Gcn5 is present in three macromolecular complexes inSaccharomyces cerevisiae, SAGA, SLIK and ADA. The ADA complex is the leastunderstood of the three and is the only one that does not bind transcriptional activators. Ourgoal was to characterize the function of the ADA complex through both in vivo and in vivoapproaches. Using TAP tag purification in yeast, we purified and separated the three GCN5containing complexes. MudPIT mass spectrometry of the purified ADA complex revealedtwo novel subunits, Sgf29 and a previously uncharacterized protein we named Ahc2. Weshowed that deletion of AHC2 results in the loss of both Ahc2 and Ahc1; however this lossdoes not affect HAT activity of the remaining complex in vivo. In contrast, deletion ofSGF29, which results in the loss of Sgf29 only, does lead to a decrease in HAT activity invivo. Examination of the DNA and nucleosome binding activity of ADA found that thecomplex was able to bind both; however binding was reduced in the absence of Ahc1 andAhc2. Next, we determined the effects of deleting ADA specific components on transcriptionusing expression microarrays. We found a number of genes in the pheromone matingpathway that were downregulated greater than two fold. We are currently examining the rolethat the ADA complex may play in the regulation of the pheromone pathway. We are alsocurrently examining the binding of the ADA complex throughout the genome using ChIP/chipanalysis of both intergenic and open reading frames. Our comprehensive analysis of theADA complex allows us to understand how it functions independently of SAGA and SLIK.

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Young Han Lee Abstract 61Epigenetic regulation of the tumor suppressor Egr-1 gene byoncogenic RasSoon Young Shin, Tae Seop Shin and Young Han Lee

Hanyang University, 411, College of Science and Technology, 1271 Sa 1-dong, sangrok-gu/Ansan-si, Gyeonggi-do, 426-791 Korea

The early growth response-1 (Egr-1) gene (also known as NGFI-A, zif268, krox 24, or Tis8)is a transcription factor that contains three zinc-finger motifs in the DNA-binding domain andpreferentially binds to GC-rich regulatory elements. Egr-1 is induced rapidly by a variety ofgrowth factors, cytokines, and cellular stresses, suggesting a multifunctional role of Egr-1 incell growth, survival, differentiation, neuronal plasticity, and development. Egr-1 is down-regulated in a variety of human tumors including human breast carcinoma andfibrosarcoma, and decrease of Egr-1 level is strongly associated with the progression oftumorigenesis. Forced expression of the Egr-1 gene into tumor cells inhibits cell growth andtumorigenicity by induction of TGF-beta1, fibronectin, and plasminogen activator inhibitor-1,which are important in the growth control and stabilization of extracellular matrix proteins,implying that functional loss of Egr-1 may contribute to tumorigenic potential. Although agreat deal of progress in identifying and characterizing the signaling that positively regulatethe Egr-1 gene expression, much less is known about negative regulatory mechanismresponsible for loss of Egr-1 expression. Ras-MEK-ERK-Elk-1 is the major signalingpathway which induces the Egr-1 expression through serum response element (SRE)-mediated transcriptional activation in response to mitogenic signals. However, we havefound that chronic expression of oncogenic Ras suppresses the Egr-1 transcription which isabrogated by treatment with trichostatin A. In this presentation, well discuss how oncogenicH-Ras paradoxically down-regulates Egr-1 expression in a point of chromatin remodeling.

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Lin Li Abstract 62SP1 and SP3 dynamic association with estrogen regulatedpromotersLin Li and James R. Davie

Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0V9Canada

Transcription factors Sp1 and Sp3 are involved in the regulation of many genes involved incellular processes including regulation of estrogen responsive genes. Although Sp1 andSp3 are structurally similar and bind the same DNA sequence, these factors have severaldistinguishing features including locating to different subnuclear sites. In this study weapplied kinetic chromatin immunoprecipitation assays to investigate the temporalassociation of Sp1, Sp3 and ER on α-amanitin-synchronized estrogen responsivepromoters in MCF-7 cells cultured in the presence or absence of estradiol. The promoterschosen for study have one or more Sp binding sites positioned near an imperfect estrogenresponse element (ERE) (TFF1), half site ERE (cathepsin D, retinoic acid receptor α,transforming Growth Factor α) or no ERE (cyclin D1, c-myc). Regardless of the promotercontext, unliganded and liganded ER cycled on these promoters with different frequencies.Distinct temporal patterns of Sp1 and Sp3 association with these promoters were observed.In the presence of estradiol, Sp3 cycles on these promoters and typically the association ofSp3 lags behind that of liganded ER, suggesting that ER may facilitate the binding of Sp3 tothese promoters.

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Karen Lower Abstract 63ATRX acts in a localised manner to down regulate theexpression of alpha globin genes in ATR-X syndromeKaren M. Lower, Doug R. Higgs and Richard J. Gibbons

Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, JohnRadcliffe Hospital, Headington, Oxfordshire OX3 9DS, U.K.

ATRX is a member of the SNF2 family of chromatin remodelling proteins, however the exactfunction of this protein remains unknown. Mutations in the ATRX gene result in theintellectual disability disorder ATR-X syndrome which is associated with a mild form of alphathalassemia. The latter feature is due to down regulation of the alpha globin genes, whichare located at the telomeric end of the short arm of chromosome 16. It is not known howATRX regulates the expression of the alpha globin genes. One hypothesis is that ATRXacts to remodel the chromatin environment surrounding this gene cluster. If this is the case,it is reasonable to expect that the genes surrounding the alpha globin cluster might also bealtered in their expression patterns when ATRX is mutated. To address this hypothesis, wecarried out semi-quantitative real time PCR on 20 ATR-X and 20 control lymphoblast celllines to analyse the expression level of the genes surrounding the alpha globin cluster.Eight genes in the terminal 400 kb of 16p were analysed. Results show that of the genesanalysed, expression did not significantly vary between control and ATR-X cell lines.Therefore, this suggests that ATRX is somehow directly targeting the alpha globin locus,and that the down regulation of expression of these genes is not due to a global effect onthe chromatin structure of the surrounding region.

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Malik Lutzmann Abstract 64Geminin binding to Cdt1 controls, but does not necessarilyblock, Cdt1 function during replication origin licensingMalik Lutzmann, Olivier Cuvier, Domenico Maiorano and Marcel Méchali

Institute of Human Genetics, CNRS-UPR1142, 141 rue de la Cardonille, 34396 Montpellier,France

Early in the cell cycle chromosomes become “licensed” to replicate once and only once byformation of pre-replication complexes (pre-RCs) onto DNA replication origins. The last stepin this licensing reaction is the essential Cdt1 mediated loading of the MCM2-7 complexonto DNA, the helicase necessary to open up the double stranded DNA during replicationinitiation. Precise regulation of Cdt1 activity by the multifunctional protein geminin is crucialto prevent re-firing of origins and subsequent re-replication. Geminin was thought to act asa negative regulator of DNA replication by binding Cdt1 and thus blocking its ability to loadthe MCM2-7 complex onto DNA. Here, we show that a recombinant Cdt1-geminin complex is fully active in MCM2-7 loading

and initiation of DNA replication. Moreover, we show that only free Cdt1, but not thelicensing active Cdt1-geminin complex leads to re-replication and checkpoint activation ifadded in excess to Xenopus egg extract. We further demonstrate, that the Cdt1-geminincomplex exists in different stoichiometries, of which one is active in loading the MCM2-7complex and origin licensing, whereas only the other containing more geminin per moleculeof Cdt1 renders Cdt1 inactive. Our data give rise to a model in which a complex of Cdt1 andgeminin is the normal MCM2-7 loader, which - in contrast to free Cdt1 - can be efficientlyregulated. Besides its function in controlling Cdt1, geminin was also shown to possess neuralizingactivities and to control embryonic development and transcriptional programs by interactingwith several key transcription factors like Hox-and polycomb proteins as well as Brg1, amember of the SWI/SNF chromatin-remodeling complex.Interestingly, origin selection and –activation are in a poorly understood way linked todevelopmental- and transcriptional programs. Our findings, that geminin-complexed Cdt1activates replication origins makes it tempting to speculate, that the DNA-replicationindependent recruitment of geminin to chromatin might also take part in the selection oforigins to be activated. This could help to explain, how transcriptional programs andchromatin structure are linked to the spatial and temporal organization of S-phase.

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Dale Mackay Abstract 65Identification of an acetyltransferase of the DNA bindingproteinDale T. Mackay, Malcolm White and Garry Taylor

University of St Andrews, BMS Building, North Haugh, St. Andrews, Fife KY16 9ST, U.K.

Members of the crenarchaeal kingdom have a variety of small DNA binding proteins insteadof histones. Alba (Sso10b) is an architectural DNA binding protein from Sulfolobussolfataricus, which is controlled by acetylation and deacetylation. Acetylation of a singlelysine residue results in weaker binding to DNA, opening the way for transcription,replication and repair. Deacetylation by the enzyme Sir2 leads to transcriptional silencing ofchromatin in vitro. The search for a specific acetyltransferase of Alba commenced bysearching the Sulfolobus genome for genes encoding a putative acetyltransferase. Twogenes sso0082 and sso0209 were identified. From multiple sequence alignments, itappears that Sso0209 has eukaryotic N-acetyltransferase homologues as well as archaealN-acetyltransferase homologues. This is interesting because the degree of conservationand similarity in most of these cases is high (~70%), indicating that Sso0209 may be aconserved protein from archaea through to humans. Sso0082 shows sequence similarity toacetyltransferase enzymes belonging to other prokaryotes. Both Sso0082 and Sso0209share at least 25% identity with each other. This poster will describe the initial cloning of thetwo genes and the biochemical characterisation and crystallisation of Sso0209.

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Asoke Mal Abstract 66Linking HDAC1 and MyoD function in RhabdomyosarcomaAsoke K. Mal

Lerner Research Institute, Cleveland Clinic Foundation NE-20 Molecular Genetics, 9500Euclid Avenue, Cleveland, Ohio 44195, U.S.A.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and is asubstantial problem in pediatric oncology. RMS thought to arise from myogenic precursorcells and expresses myogenic transcriptional regulator MyoD. The expression of thisregulator is necessary for skeletal muscle cell development and differentiation duringdevelopment, postnatal growth and regeneration in injury and disease. Although MyoD isexpressed in RMS cells, it fails to activate differentiation-responsible gene expression inthese tumor cells. Our previous work demonstrated that HDAC1 association with MyoD isfunctionally linked to the inhibition of MyoD-mediated differentiation-responsible geneexpression and differentiation in muscle cells that undergo differentiation process (EMBO,2001; 20: 1739-1753). We investigated whether HDAC1 is responsible for MyoD failure toinduce differentiation program in RMS cells. We have observed that HDAC1 is over-expressed in these tumor cells and its expression level remains unchanged when thesecells are cultured in condition that is permissible for normal muscle cells to differentiate. Inaddition, HDAC1 is associated with MyoD in these tumor cells cultured in both growth anddifferentiation-permissible conditions. These findings are in opposite to the normal musclecells that undergo differentiation. ChIP and Re-ChIP experiments in RMS cells furthershows that MyoD-HDAC1 complex is occupied on the regulatory region of MyoD targetmuscle genes and histone H3 is in hypoacetylated state. However, RNAi-mediatedknockdown of HDAC1 in these tumor cells induces acetylated-H3 on the promoter ofmuscle genes and restores MyoD-mediated gene transcription. Together, these resultssuggest that over-expression of HDAC1 inhibits the ability of MyoD to induce differentiationprogram in RMS cells even under differentiation permissible environment.

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Roberto Mantovani Abstract 67Epigenetic changes to NF-Y controlled genes upon ERinduction.Giacomo Donati and Roberto Mantovani

Dipartimento di Scienze Biomolecolari U di Milano, DSBB, Via Celoria 26, Milano, 20133, Italy

The endoplasmic reticulum stress response leads to the induction of several genes. Most, ifnot all, require NF-Y, a conserved trimer that binds to CCAAT boxes. We evaluated thekinetic induction of several mRNAs, which can be divided in early and late response genes.By Chip experiments, we undertook a systematic analysis of in vivo binding of sequence-specific activators, cofactors and general transcription factors to a number of inducedpromoters. Furthermore, we evaluated the chromatin changes in terms of specific histonemodifications (acetylation and methylations). The picture emerging is one of remarkablecomplexity, with common themes, as well as peculiar promoters specific differences. Finally,we are evaluating the role of NF-Y in the ER-stress response, by using a dominant negativeNF-YA mutant. The results of our analysis will be presented.

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Jotin Marango Abstract 68The multiple myeloma SET domain (MMSET) protein is ahistone H3 and H4 methyltransferase with properties of atranscriptional co-repressorJotin Marango1, Manabu Shimoyama1, Boris A Leibovitch1, Ming Ming Zhou2,Yolanda Martinez1, Andres Sirulnik3, Marta Chesi4, P. Leif Bergsagel4 andJonathan D Licht1.1Division of Hematology/Oncology, Mount Sinai School of Medicine, New York, NY, U.S.A.,2Structural Biology Program, Mount Sinai School of Medicine, New York, NY, U.S.A., 3DanaFarber Cancer Institute, Boston, MA and 4Department of Hematology/Oncology, Mayo Clinic,Scottsdale, AZ, U.S.A.

Over 40% of cases of multiple myeloma (MM) are associated with translocations of theimmunoglobulin heavy (IgH) chain gene gene. The t(4;14) translocation, present in ca. 20% ofmyeloma cases, results in the overexpression of two potential oncogenes, MMSET and FGFR3, viajuxtaposition of their endogenous promoters to regulatory elements of the IgH locus. The presenceof t(4;14), and MMSET overexpression, is an adverse prognostic factor in MM irrespective ofFGFR3 expression. MMSET contains several conserved motifs found in proteins involved inchromatin function (PWWP, HMG, PHD domains) and in the epigenetic control of transcription (SETdomain). Accordingly, we found that the two main isoforms of the MMSET protein exhibit exclusivenuclear localization in both transfected fibroblasts and myeloma cells carrying t(4;14). Towards ourgoal of defining the ability of MMSET to affect gene regulation and contribute to the diseasepathogenesis, we found that the SET domain of MMSET possesses in vitro methyltransferaseactivity specific for core histones H3 and H4. Using a computational approach and theoreticalextrapolation from the solved NMR structure of vSET, we identified residues in the active site ofMMSET essential for catalysis, whose mutation drastically reduces enzymatic activity. Reporter assays using Gal4 fusion constructs showed that both the amino terminus of MMSET,containing the PWWP and HMG domains, as well as the SET-containing carboxy terminus act astranscriptional repressors. MMSET interacts physically and functionally with a number of known co-repressor molecules, such as HDAC1, HDAC2, Sin3a, and SIRT1, but not HDAC4 or HDAC6. Assuch, MMSET co-expression enhances HDAC1 and HDAC2-mediated repression in transcriptionalreporter assays, and MMSET repression is partially relieved by the addition of an HDAC inhibitor. Ayeast two hybrid screen identified a number of other functional partners of MMSET, includingZNF331/RITA (Rearranged in Thyroid Adenoma), a KRAB domain/zinc finger protein previouslyimplicated in malignancy. MMSET and ZNF331 co-localize in the nuclei of transfected fibroblasts, co-immunoprecipitate, and display cooperative repression in reporter assays. Collectively, these datasupport the idea that MMSET is a biologically active, bifunctional transcriptional mediator acting as aHMT enzyme in chromatin remodeling and as a complex adaptor in the recruitment of repressor species.Presently we are modeling the biological effects of MMSET through a conditional overexpressionsystem in a B cell line. While low levels of MMSET are ubiquitiously expressed, induction of highlevels of MMSET expression in the B cell line is associated with growth suppression and G1 arrest.While paradoxical for a presumed oncoprotein, such actions have been observed for other disease-associated proteins such as Runx1/MTG8. In contrast, a myeloma cell line harboring t(4;14)proliferates in the presence of high level MMSET expression. RNAi-mediated knockdown ofMMSET in these cells induces apoptotic cell death. This suggests that MMSET may be critical forgrowth and survival of myeloma cells. Profiling of gene expression changes in these systemsshould link the transcriptional and biological activities of MMSET.

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Philip Marsden Abstract 69The cell-specific and hypoxia-regulated expression ofendothelial nitric oxide synthase (eNOS) is controlled bychromatin-based mechanismsFish J.E., Matouk C.C., Yeboah E., Scott J.A. and Marsden P.A.

University of Toronto Room 7358, Medical Sciences Buillding, Room 7358, 1 King’s CollegeCircle, Toronto M5S 1A8, Canada

We have demonstrated that the eNOS gene is regulated in a cell-specific fashion byepigenetic mechanisms, namely proximal promoter DNA methylation (Chan et al., J. Biol.Chem., 2004). Here we present evidence that post-translational modifications of proximalpromoter histones also play an important role in the cell-specific and regulated expressionof eNOS in endothelial cells. We found that endothelial cells were highly enriched inacetylated histone H3 and H4 and lysine 4 di- and tri-methylation of histone H3 at the eNOSproximal promoter; marks of transcriptionally active chromatin. Endothelial cells werespecifically enriched in lysine 9 acetylation of histone H3 and lysine 12 acetylation ofhistone H4. Treatment of non-endothelial cells with trichostatin A (TSA), a histonedeacetylase (HDAC) inhibitor, increased steady-state levels of eNOS mRNA and wasaccompanied by increased histone H3 and H4 acetylation and lysine 4 methylation ofhistone H3, suggesting that HDACs repress eNOS transcription in non-endothelial cells.Lysine 4 methylation of histone H3 was found to be necessary for eNOS expression astreatment with methylthioadenosine, a lysine 4 methylation inhibitor, decreased expressionof eNOS in endothelial cells and also prevented the upregulation of eNOS in non-endothelial cells treated with TSA. To determine whether chromatin-based mechanismswere also important in the regulated expression of eNOS we assessed chromatinmodifications in endothelial cells grown in hypoxic conditions. Hypoxia is known todecrease eNOS mRNA, in part by transcriptional mechanisms. Hypoxia dramaticallydecreased the amount of RNA Polymerase II present at the eNOS proximal promoter andresulted in decreased histone acetylation by more than 50%, especially on H4. We positthat chromatin-based mechanisms play an essential role in the cell-specific expression ofthe eNOS gene and may be important in disease processes characterized by a decrease ineNOS expression in endothelial cells, including hypoxia.

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Darin McDonald Abstract 70Polymeric nuclear actin in chromatin modification anddouble-strand break repairDarin McDonald, Christi Andrin and Michael J Hendzel

Cross Cancer Institute Room 3341, Cross Cancer Institute, 11560 University Avenue,Edmonton, Alberta T6G 1Z2, Canada

Although it is now well established that beta-actin is an important functional component intranscription and chromatin remodeling, it is largely assumed that this function is mediatedby monomeric actin. Using fluorescence recovery after photobleaching (FRAP), wedemonstrate that the nuclear actin pool, like the cytoplasmic actin pool, is a dynamicequilibrium between polymeric and monomeric species. We have subsequently identifiedhistone deacetylases and components of the DNA double-strand break repair machinery asnuclear proteins and protein complexes that directly associate with F-actin in vitro. This isconsistent with our previously reported experiments where we demonstrated thatdepolymerizing actin enabled the solubilization of complexes containing histonedeacetylases 1, 2, and 3 that were otherwise insoluble in 0.42 M salt solutions. We have also performed experiments to examine the requirement for polymeric actin inDNA double-strand break repair. We find that a brief and reversible treatment of cells withthe inhibitor of actin polymerization, latrunculin B, significantly inhibits the phosphorylation ofhistone H2A.X, a hallmark of sites of DSB repair. We subsequently examined the ability ofcells to ligate double-strand breaks following irradiation. By examining the repair of double-strand breaks using the comet assay, which resolves broken DNA from intact DNA byelectrophoresis, is completely inhibited when actin is depolymerized by a brief treatmentwith latrunculin B. In summary, our results provide evidence that actin has importantnuclear functions that require polymerization rather than the mere presence of nuclear actinmonomers.

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Dominik Mojzita Abstract 71Pdc2, the yeast homologue of CENP-B, is a part of thecentromere-kinetochore complexDominik Mojzita and Stefan Hohmann

Department of Cell and Molecular Biology/Microbiology, Medicinaregatan 9C, S-405 30Göteborg, Sweden

Pdc2, initially identified in a screen for mutations affecting pyruvate decarboxylase activity inyeast, was characterized as regulatory gene. Pdc2 is necessary for expression of pyruvatedecarboxylase structural genes PDC1 and PDC5 and the set of THI genes involved inthiamine synthesis in yeast. Apparently, the production of thiamine and the enzymes using itas a cofactor is coordinated by Pdc2. In a homology search we have found considerablesequence similarity of Pdc2 with centromere binding proteins related to the human CENP-B.In S. pombe there are three homologues, Abp1, Cbh1 and Cbh2. All of these proteins arealso involved in formation of centromere. Two-hybrid screen revealed Pdc2 interacting withproteins Mif2 and Spt4. Mif2 is an analogue of human CENP-C and Spt4 is one of the unitsof functional kinetochore. Interactions found by 2-H screen were confirmed by in vitro assay(co-immunoprecipitation). It was shown that the human homologues CENP-B and CENP-Cinteract during assembly of centromeres, which makes our observations more relevant.These findings are interesting evidence of Pdc2 involvement in chromosome segregation asa structural protein. In addition to already known facts about Pdc2 function, our results mayserve as a good example of a protein acting as transcriptional factor and structural proteinat the same time. Pdc2 function is indeed remarkable from an evolutionary point of view.Moreover, some observations of CENP-B localization in the euchromatine, which areunclear, could be possibly clarified by the Pdc2 model. Our results could be an indication foran additional function also for this well studied mammalian protein.

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David Mosser Abstract 72Chromatin remodeling across the proximal promoter regionof the interleukin-10 gene in macrophagesXia Zhang, Shanjin Cao, Justin Edwards and David M. Mosser

Department of Cell Biology and Molecular Genetics, University of Maryland at College Park,College Park, MD 20742, U.S.A.

We previously showed that activation of macrophages in the presence of immunecomplexes induces the production of high levels of IL-10, through an up-regulation oftranscription. We now show that chromatin modifications at the proximal IL-10 promoterregion occur following this activation. A rapid phosphorylation at Ser10 of histone 3 (H3)with a peak at 30 min was followed by a modest and slower accumulation of acetylatedhistones. These modifications were specific for the IL-10 promoter and were not observedas little as 1kB upstream of the transcription start site. The magnitude of thephosphorylation and acetylation of H3 at each of the 12 successive nucleosomes along theproximal promoter region of IL-10 gene was quantitated. Histone phosphorylation directlycorrelated with sensitivity of chromasomal DNA to micrococcal nuclease and DNase Idigestion. Importantly, the remodeling of chromatin permitted the recruitment of twotranscription factors, SP-1 and STAT3, to their corresponding DNA binding sites. Insummary, we show that the IL-10 gene locus undergoes specific chromatin remodeling in aspatiotemporal manner along the proximal promoter region, which allows the recruitment oftranscription factors, SP-1 and STAT3, to their respective binding sites. These events arecritical for swift initiation of IL-10 gene transcription and subsequent mRNA accumulation.

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Toshinori Nakayama Abstract 73Long range histone modification of the Type2 cytokine geneloci in developing Th2/Tc2 cellsToshinori Nakayama and Masakatsu Yamashita

Graduate School of Medicine, Chiba University, 4F, Igakubu honkan, 1-8-1 Inohana Chuo-ku, Chiba, 260-8670, Japan

Chromatin remodeling of type 2 cytokine gene loci occurs during differentiation of naiveCD4 and CD8 T cells into type 2 helper (Th2) and cytotoxic (Tc2) T cells. IL-4 productionand histone hyperacetylation in IL-4-associated nucleosomes in developing Tc2 cells weresignificantly lower than those of Th2 cells, however, cytokine production and histonehyperacetylation of IL-5 and IL-13 genes were equivalent. Developing Tc2 cells expressedlower GATA3 levels and dramatically increased levels of repressor of GATA (ROG). A ROGresponse element in the IL-13 gene exon 4 displayed Tc2-specific binding of ROG, HDAC1and HDAC2, and exhibited repression of IL-4 gene activation. Thus, ROG may confer CD8T cell-specific repression of histone hyperacetylation and activation of the IL-4 gene locus.

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James Nicholson Abstract 74Structural studies of the oxidised histone octamerChristopher M. Wood1, Sirirath Sodngam1, James M. Nicholson2, StanleyJ. Lambert1, Alan Brown1, Colin D. Reynolds1 and John P. Baldwin2.1School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street,Liverpool, L3 3AF, U.K., 2College of Biology and Medicine, CCLRC Daresbury Laboratory,Warrington, Cheshire, WA4 4AD, U.K.

Oxidizing free radical molecules and their related compounds are present in the humanbody naturally and may also be produced environmentally by, for example, pollutants andsmoking. Oxidized proteins have been implicated in a number of human conditionsincluding: Parkinson’s disease, Alzheimer’s disease and ageing processes, where theamount of oxidized protein increases exponentially with age. The body will seek to maintaina balance between oxidation and reduction by the use of redox reactions. In principle,oxidized proteins should be degraded by proteosomal and lysosomal pathways, but inreality some products either avoid or survive these routes and build up in the cell. To investigate any possible effects of oxidation on the histone proteins, intact octamerspurified from chick erythrocyte nuclei were crystallized in the presence of S-nitrosoglutathione (GSNO). GSNO was chosen as the oxidizing agent as it occurs naturallyin the human body, and thus any effects seen in the crystallised octamers at 2.1Å resolutioncan be considered to be a good indicator of that which may occur in vivo. Thecrystallographic results show that GSNO induces changes in the histone octamer (HO),namely an alteration of the secondary structure by the removal of hydrogen bonds from α-helices. This loss of hydrogen bonds results in a reduction in surface area when comparedto the native high-resolution octamer structure [1] and also alterations to the acid-baseprofile of the octamer’s molecular surface. The changes in the acid-base profile of theoxidized HO may be of fundamental importance to the higher-order chromatin structure, asthere are changes in the acid-base patches identified previously [2]. The crystal structure ofthe oxidised HO has been deposited at the Protein Data Bank (PDB) and has referencenumber 2ARO. To quantify the degree of secondary structure change and to correlate this with the degreeof concentration of oxidizing agent, experiments were carried out using SynchrotronRadiation Circular Dichroism (SRCD). These results show that there is a sudden andpronounced loss of secondary structure within a particular range of concentration ofoxidizing agent. Furthermore, the histone octamer secondary structure is restored afterdialysis against histone-octamer buffer without the oxidising agent.

[1] Wood, C. M., Nicholson, J. M., Lambert, S. J., Chantalat, L., Reynolds, C. D. and Baldwin, J. P.(2005) Acta Cryst. F61, 541-545.[2] Chantalat, L., Nicholson, J. M., Lambert, S. J., Reid, A. J., Donovan, M. J., Reynolds, C. D., Wood, C.M. and Baldwin, J. P. (2003). Acta Cryst. D59. 1395-1407.

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Barbara Nikolajczyk Abstract 75The IL-1 beta gene is transcribed from a poised promoterarchitecture in monocytesMichael D. Liang, Yue Zhang, Daniel McDevit, Sylvia Marecki andBarbara S. Nikolajczyk

Boston University School of Medicine, L-516 Department of Microbiology, 715 Albany Street,Boston MA 02118, U.S.A.

IL-1 beta is a key pro-inflammatory cytokine implicated in the generation or exacerbation ofmultiple diseases. In general, cytokines are transcriptionally regulated by chromatinremodeling occuring concomitantly with mRNA synthesis. However, new data show the IL-1beta promoter assembles into a poised chromatin structure only in monocytes, asevidenced by accessible chromatin and modification of histones packaging the locus.These parameters do not change upon transcriptional activation. Furthermore, enhancerchromatin structure does not change upon activation. Analysis of transcription factorassociation with either the promoter or the enhancer fails to uncover significant changes inbinding by two key transcriptional activators, PU.1 and C/EBP beta. Hence the IL-1 betapromoter is packaged into a transcriptionally silent poised promoter architecture. Monocytestimulation, known to activate the protein kinase CK2, results in recruitment of a third factor,IRF-4, to a composite PU.1/IRF site. This recruitment absolutely requires PU.1phosphorylation, strongly suggesting that IL-1 beta-associated PU.1 is post-translationallymodified by CK2 to achieve inducible gene transcription. Overall, the data show that aunique two-step mechanism activates IL-1 beta transcription in the context of cellularchromatin.

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Keisuke Nimura Abstract 76Preferential association of Dnmt3l with Dnmt3a2 onchromatin in ES cellsKeisuke Nimura, Yasufumi Kaneda and Kiyoe Ura

Division of Gene Therapy Science, Osaka University Medical School, 2-2 Yamadaoka Suita,Osaka, 565-0871 Japan

In mammals, DNA methylation is involved in fundamental processes such as the silencingof gene expression and transposable elements, X-inactivation, genomic imprinting andtumorigenesis. However, control mechanisms of DNA methylation are still largely unknown.Dnmt3l, a Dnmt3 family protein sharing homology with DNA methyltransferase 3a (Dnmt3a)and Dnmt3b but lacking enzymatic activity, has been shown to be required for correctestablishment of DNA methylation patterns in germ cells. In this study, we demonstrate aphysical link between Dnmt3l and one of Dnmt3 enzymes, Dnmt3a2. Whereas in wild-typeES cells endogenous Dnmt3l is concentrated at heterochromatic foci, it fails to localize tothese regions in the absence of only Dnmt3a. In Dnmt3a/Dnmt3b double deficient ES cells,Dnmt3l was defused over the nucleus and cytoplasm and expression of Dnmt3a2, but notDnmt3a1 or Dnmt3b, relocalized Dnmt3l at hetrochromatic foci. We also show biochemicalinteraction between Dnmt3l with Dnmt3a2 in ES cell nuclei. Our results demonstratepossible control mechanisms of DNA methylation in which Dnm3a2 recruits Dnmt3l ontospecific genomic regions, leading active DNA methylation.

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Esperanza Nunez Abstract 77Repetitive elements, transcriptional control and cell diversityEsperanza Nunez and Victoria Lunyak

University of California at San Diego, CMM-West, Room 345, 9500 Gilman Dr., La Jolla, CA92093-0648, U.S.A.

The published sequence of the human genome revealed that coding sequences compriseless than 5% of it, whereas repeating sequences account for at least 50% of the genome.Among different types of repeats dominate transposon-derived repeats (~45% of thegenome), and, in particular, retroelements including short interspersed nuclear elements,SINEs (~13%), long interspersed nuclear elements, LINEs (~20%), and LTR-containingretroelements (~8%). Provided that regulatory signals entering the nucleus encounterchromatin, not DNA, and the rate-limiting biochemical response that leads to activation ofgene expression in most cases involves alterations in chromatin structure; the key issuethen becomes that nucleosomes act as a barrier to recognizing binding sites on the DNA(for RNAP or for regulatory proteins). If nucleosomes are positioned so as to avoid thepromoter, then clearly nucleosome-mediated repression is avoided. This raises the questionof how nucleosomes are positioned, since at first view they would seem to be quite non-specific. Probably some regulatory proteins bind to the DNA before nucleosome assembly,thus phasing the nucleosomes and leaving key parts of the DNA exposed. This implies acompetition between regulatory proteins and nucleosomes during formation of newchromatin. The presence of SINEs, especially Alu sequences, in the protein coding regionshas been controversial as is their biological meaning and importance. We have preliminarydata suggesting the maintained expression of a SINE B2 repeat in the murine pituitarygland as early as E12.5 where it may play a role as a possible boundary determinant.Recent evidence suggests that a main effect of repetitive elements is their ability toinfluence transcription of neighboring genes. The SINE B2 contains a putative promoter thatcan initiate transcription of flanking genomic DNA, if not act as RNA regulator to represstranscription. Furthermore, its transcript may be susceptible to epigenetic silencing, which isoften stochastic and incomplete, resulting in complex patterns of transcription; which in thecase of the pituitary gland this may have acute effects in cell line fate and determination.

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Laura O’Neill Abstract 78Distinctive histone modifications mark specific chromosomesin both human and mouse modelsLaura P.O’Neill, Matthew ver Milyea, Milan Fernando and Bryan Turner

Birmingham University, IBR, Vincent Drive, Birmingham, West Midlands, B15 2TT, U.K.

The epigenetic code is becoming more complex, with diverse modifications to the N-terminal tails of the core histones correlating with specific aspects of gene regulation. Themodification itself, the histone involved and the amino acid residue are all importantdeterminants of these epigenetic marks. Using adult and embryonic stem cells (ES cells)from human and mouse model systems, we have continued to investigate the roles ofhistone acetylation and methylation in gene silencing and their distribution alongchromosome domains. Female ES cells inactivate genes on one of their two Xchromosomes as they differentiate in culture. Moreover it has been shown that the activeand inactive Xs are differentially methylated at H3 lysines 4, 9 and 27. Using antisera thatdistinguish lysines with different levels of methylation, we have now been able to show thatthe X chromosomes carry another mark, irrespective of activity, which distinguishes themfrom the other chromosomes. Moreover, other chromosomes also carry distinctivecombinations of histone marks. In particular chromosome 19, a chromosome rich in genes,shows a distinctive pattern of histone modifications, which are unique to this chromosome.Further development of the N-ChIP procedure has also allowed us to investigate whetherthe epigenetic marks that we see on specific genes within embryonic stem cells are thesame as those isolated from the inner cell mass of a blastocyst from which these cells wereoriginally derived.

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Hisanobu Oda Abstract 79Generation and characterization of Set9 and PR-Set7conditional gene knockout miceHisanobu Oda1, Michael M. Shen2 and Danny Reinberg1

1Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department ofBiochemistry, 2Center for Advanced Biotechnology and Medicine and Department ofPediatrics, UMDNJ/RWJMS, RWJMS Research Building, 683 Hoes Lane, Piscataway,Middlesex 08854, U.S.A.

The lysine methyltransferase, Set9, was isolated based on its ability to methylate histoneH3 lysine 4; yet Set9 cannot use nucleosomal substrates. We recently found that the tumorsuppressor protein p53 is a considerably better substrate for Set9 than is histone H3. Set9may therefore be involved in various biological processes other than chromatin modulationin vertebrates. Another methyltransferase, PR-Set7 mediates monomethylation of H4-K20.PR-Set7 mutants in Drosophila die at the larval-to-pupal transition and exhibited phenotypesconsistent with a failure to complete cell division in their imaginal discs. We have nowgenerated mice carrying a conditional deletion for the gene encoding Set9 or that for PR-Set7. Our goal is to define the function of each of these monomethyltransferases in vivo.

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Søren Ottosen Abstract 80Bromodomain protein 7 (Brd7) promotes nucleolar disruption in responseto DNA breaks

Søren Ottosen, Randy Luciano, Shahana Mahajan and Angus Wilson

Department of Microbiology, New York University School of Medicine, 550 First Avenue,New York, NY 10016, U.S.A.

HCF-1 is a chromatin-associated factor required for VP16 to activate herpes simplex virusimmediate-early promoters. HCF-1 is also involved in regulation of cellular gene expression,interacting with a number of DNA-binding proteins (LZIP, Sp1, GABP, Krox20), as well asthe mSin3/HDAC and Ash2/Set1/MLL methyltransferase complexes. Loss of HCF-1 leads toa G1/G0 cell cycle arrest and defects in chromatin compaction prior to mitosis.The HCF-1 protein is composed of two subunits (HCF-1N and HCF-1C) derived from asingle large precursor by internal proteolytic cleavage. We found that bromodomain-containing protein 7 (Brd7) interacts with the C-terminal fibronectin repeats of HCF-1C.Elevated expression of Brd7 leads to an accumulation of HCF-1 in the nucleoli, formingdistinctive subnucleolar granules. This coincides with visible changes in nucleolar structureand release of nucleolar proteins into the nucleoplasm. Similar changes occur in responseto DNA double-stranded breaks and can be blocked by depletion of either Brd7 or HCF-1.Brd7 expression also results in stabilization of p53 and enhanced activation of p53-responsive promoters. Deletion analysis identified separate domains within Brd7responsible for HCF-1 subnucleolar granule formation and nucleolar disruption.Brd7 contains a single acetyl-lysine binding bromodomain and treatment of cells withhistone deacetylase (HDAC) inhibitors prevents Brd7-mediated activation of p53-responsivegenes. This effect is alleviated by mutation of conserved residues within the acetyl-lysinebinding pocket. Increased levels of acetylation leads to accumulation of Brd7 in discretenucleoplasmic speckles and a marked decrease in the overall diffusion rate as measured byfluorescence recovery after photobleaching (FRAP). We propose that in undamaged cells,Brd7 is associated with acetylated chromatin and held in an inactive form. Recognition ofdouble-stranded DNA breaks leads to a change in the acetylation state of these tethers,releasing or relocalizing Brd7 so that it can initiate nucleolar disruption and p53 stabilization.

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Mahadeb Pal Abstract 81The role of TFIIB and the transcription bubble during theearliest stages of transcription by RNA polymerase IIMahadeb Pal and Donal Luse

LRI, Cleveland Clinic Foundation, NE/20, LRI, 9500 Euclid Avenue, Cleveland, Cuyahoga44195, U.S.A.

We have investigated the role of the transcription bubble in the earliest stages of RNAsynthesis using a series of transcription complexes stalled on TATA box promoters withvarying TATA to +1 spacing. We found that regardless of the spacing, the upstream edge ofthe bubble is set at ~20 bp downstream from the TATA box. This edge remains fixed whilethe bubble expands in the downstream direction, until the bubble grows to more than 17bases and the transcript length reaches at least 7 nt. At this point the upstream half of thebubble abruptly reanneals (bubble collapse). The bubble collapse transition restorestranscription complex stability, confers independence from the TFIIH helicase and greatlydiminishes an elongation block (in the region of +7 to +9 nt of RNA synthesis) apparentlyinduced by the B-finger domain of TFIIB. These studies revealed that (1) bubble collapse isa critical determinant of promoter clearance by RNA polymerase II and (2) TFIIB plays animportant part in promoter clearance (Pal et al., Mol. Cell 19, 101). Recent structuralstudies indicate that the B-finger of TFIIB initially occupies the RNA exit channel of pol IIand must be displaced by the growing transcript. Our data are consistent with release ofTFIIB following the bubble collapse transition. However, when we attempted to directlydetect TFIIB release using in vitro transcription with purified transcription factors and bead-bound templates, we found somewhat surprisingly that TFIIB is not released upon bubblecollapse or immediately downstream (i.e., +30). Thus far, when using purified componentswe have been able to demonstrate TFIIB release only when pol II has run off the template.We are currently exploring the possibility that supplementing our reactions with nuclearextract will drive TFIIB release in early elongation complexes.

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Maria V. Panchenko Abstract 82pVHL partner and transcriptional co-activator Jade-1 is anovel substrate for HAT TIP60.Maria V. Panchenko and Herbert T. Cohen.

Boston University School of Medicine, Evans Biomedical Research Center, 650 AlbanyStreet, Boston, MA 02118-2393, U.S.A.

Jade-1 was identified as a protein partner of the von Hippel-Lindau tumor suppressor pVHL.We recently demonstrated that PHD zinc finger protein Jade-1 is localized to the nucleusand possesses intrinsic transcriptional activity. Strikingly, over-expression of the full lengthJade-1 in epithelial cells resulted in a global and specific hyperacetylation of endogenoushistone H4. PHD zinc fingers appeared indispensable for both transcriptional and HAT-associated Jade-1 functions. Moreover, mutant Jade-1 possesses a dominant negativephenotype, suggesting that Jade-1 may be a part of a HAT complex. In support of thisnotion, we showed that TIP60 binds and further enhances the HAT-associated function ofwild type but not mutated Jade-1. Others have demonstrated that the PHD zinc finger ofCBP binds nucleosomal histones in vitro. We hypothesize that PHD fingers provide theassociation of the Jade-1-TIP60 complex with the nucleosome thereby promotingacetylation of nucleosomal histones by TIP60. We have further investigated outcomes ofJade-1 interactions with TIP60. The endogenous Jade-1 protein band visualized by westernblot has diffused appearance characteristic for a post-translationally modified protein. SinceJade-1 polypeptide has numerous lysine residues we investigated whether Jade-1 can beacetylated. Western blot analysis of total cell extracts revealed that the protein bandcorresponding to endogenous Jade-1 is readily recognized by the Acetyl-lysine antibody,suggesting that Jade-1 can be acetylated. The levels of endogenous Jade-1 acetylationwere increased after incubation of cells with Trichostatin A, an inhibitor of HDACs,suggesting that Jade-1 undergoes acetylation/deacetylation mediated by endogenousHATs/HDACs. Because Jade-1 interacts with TIP60, we examined whether Jade-1 can be asubstrate for TIP60. Ectopic expression of TIP60 resulted in enhanced levels of acetylatedendogenous as well as overexpressed recombinant Jade-1, suggesting that TIP60 canacetylate Jade-1. Surprisingly, Gal4-Jade-1-mediated transcriptional activation of a viralpromoter was completely abolished in the presence of wild type TIP60. The potentialrelationship between TIP60-mediated acetylation of Jade-1 and inhibition of Jade-1transcriptional function is currently being investigated and will be discussed.

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Tej Pandita Abstract 83Mammalian ortholog of Drosophila MOF is critical forembryogenesis and essential for DNA damage responsesA. Gupta1, T. Geraldine Guerin-Peyrou2, M. Agarwal1, G. G. Sharma1, R.K.Pandita1, T. Ludwig2 and T. K. Pandita1

1Department of Radiation Oncology, Washington University School of Medicine, Saint Louis,MO 63108, U.S.A., 2College of Physicians and Surgeons, Columbia University, New York,NY 10032, U.S.A.

Tumor cells and normal cells have significant differences in their chromatin structure.Understanding the role of chromatin modifying factors in cellular responses to DNA damageat the molecular level will provide invaluable insights into questions of both how cancersstart and how to cure cancers. Recently, we have identified a chromatin-modifying factorhMOF the human ortholog of Drosophila MOF gene which interacts with ATM. Cellularexposure to IR enhances hMOF-dependent acetylation of its target substrate, lysine 16(K16) of histone H4, independent of ATM function. Inactivation of hMOF results inabrogation of ATM function. Based on the fact that hMOF is involved in ATM function, andtumors show loss of histone H4K16 acetylation, we hypothesize that hMOF is involved inthe oncogenic transformation. Since MOF is involved in DNA damage response as well astranscription, its function is indispensable for development, because Mof-deficiency inmouse embryos result in early embryonic lethality. We will discuss the function of mouseMof (mMof), and the functional links among mMof, histone H4K16 acetylation andspontaneous as well as IR-induced tumor formation.

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Biranchi Patra Abstract 84Genomewide localization of histone modifications duringmeiosisBiranchi Patra and Animesh Ray

Keck Graduate Institute, 535 Keck, 535 Watson Dr., Claremont CA, 91711 India

Chromatin remodeling by the posttranslational modification of histones has emerged as amajor mechanism of eukaryotic gene expression. A diversity of histone modifications suchas acetylation, methylation, ubiquitylation, sumoylation are known to dynamically regulategene activity. Recent studies indicate that histone modification may be important forselection of double strand break sites during meiotic recombination. However, the exactrole of histone modification in selecting the DNA break sites is not fully understood.Although much progress has been made on characterization of histone modifyingcomplexes with relation to transcriptional activation and repression in the yeastSaccharomyces cerevisiae and other model systems, much less is known about thechromatin condensation states and transcriptional activity genomewide. In particular, theubiquitin and SUMO modification of histones are dynamic and reversible. SUMOmodification of histone H4 is generally suggested to repress gene expression and ubiquitinmodification of histone H2B has generally been associated with gene activation. We reporthere our progress in genome-wide mapping of sites of H2B ubiquitylation and H4sumoylation during sporulation of the budding yeast Saccharomyces cerevisiae.

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Rushad Pavri Abstract 85A fully reconstituted chromatin transcription system for thedetailed study of epigenetic mechanismsRushad Pavri, Bing Zhu and Danny Reinberg

University of Medicine and Dentistry of New Jersey , School of Public Health, 683 HoesLane, Piscataway, Middlesex 08854, U.S.A.

We have developed a fully reconstituted activator-dependent chromatin transcription systemusing the endogenous promoter of the RARbeta2 gene and the RSF chromatin assemblysystem. We previously showed that ligand-dependent transcription from this promoterrequires PARP-1 as a novel cofactor. These studies employed crude S190 Drosophilaextract to assemble chromatin, limiting our ability to fully characterize the system. We havenow fully reconstituted the chromatin assembly reaction using recombinant RSF. Incombination with our established reconstituted transcription system comprised of highlypurified general transcription factors, RNA polymerase II, Mediator and PARP-1, this fullyreconstituted system allows us, for the first time, to study the effects of covalent histonemodifications on gene expression in vitro both activation and repression - using RARbeta2as a paradigm. The high purity of this system enables us to determine the exact contributionof each modification and the minimal set of factors required for gene expression. We findthat ligand-dependent transcription is dependent upon histone acetylation and chromatinremodeling as well as upon the histone chaperone FACT. We have also investigated theinterplay between histone methylation and ubiquitination on ligand-dependent transcription.Using in vitro ChIP assays, we have begun to correlate RARbeta2 gene expression with thepresence or absence of specific histone modifications on the RARbeta2 promoter. Our datademonstrate a link between specific histone modifications and ligand-dependent geneexpression with novel mechanistic implications.

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Inka Pawlitzky Abstract 86A novel regulatory region 5’ of the mouse IgH locusInka Pawlitzky, Christina Angeles, Andrea Siegel, Michelle Stanton, RoyRiblet1 and Peter Brodeur2

Immunology Program, Sackler School of Graduate Biomedical Sciences, Tufts UniversitySchool of Medicine, Boston MA 02111, U.S.A., 1Torrey Pines Institute for Molecular Studies,San Diego, CA 92121, U.S.A., 2Department of Pathology, Tufts University School ofMedicine, Boston MA 02111, U.S.A.

The IgH locus is encoded as gene segments that are assembled during B cell developmentby a series of recombination events to form continuous heavy chain coding sequences.The targeting of gene segments within Igh is controlled at the level of accessibility to therecombinase machinery. Regulated changes in the chromatin structure that cause aparticular region or gene segment to become accessible for recombination ultimately controlwhich gene segment can be targeted for rearrangement. These processes involve cis-acting elements, most notably the E and the 3IgH regulatory region, located near the 3 endthe locus. The presence of additional control elements has been postulated to regulaterearrangements of the Vh gene array that extends to the 5 boundary of Igh. To search fornovel Igh elements, we physically mapped the most D-distal Vh segments and scanned the5 flanking region for DNase I hypersensitive sites. Our studies revealed a cluster ofhypersensitive sites approximately 30 kb upstream of Igh. Using cell lines and normal Bcell populations we show that the detection of one site, HS-1, is restricted to pro-B cells, thestage defined by actively rearranging Igh-V loci. Sequence motifs within HS-1 for PU.1 andPax5 specifically bind these factors in vitro and these proteins are recruited to HS-1sequence in a pro-B cell-specific manner. In addition, the Pax5 site is required forrepression of transcriptional activity observed in HS-1 containing reporter constructs. Takentogether, the position, pro-B cell specificity, and interaction with PU.1 and Pax5 stronglysuggest that the identified hypersensitive sites mark a novel 5 IgH regulatory regionpotentially involved in Vh gene rearrangements.

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Laura Perez-Burgos Abstract 87Domain organization at the chicken β-globin locusLaura Perez-Burgos1, Thomas Jenuwein1, Michael D. Litt2 and GaryFelsenfeld3

1Institute of Molecular Pathology (I.M.P.), Dr. Bohr Gasse, 7, 1030 Vienna, Austria, 2IndianaUniversity School of Medicine - Muncie at Ball State University, 221 N. Celia Ave., MT 201,Muncie, IN 47306, U.S.A., 3N.I.H., Building 5, Room 212, Bethesda, MD 20892-0540. U.S.A.

Histones are subject to a series of post-translational modifications on their N-termini,leading to different phyisological outcomes. For instance, dimethylated lysine 4 of histoneH3 (H3-K4) has been found at active loci, whereas trimethylated lysine 9 (H3-K9) is ahallmark of pericentric heterochromatin and trimethylated lysine 27 (H3-K27) is enriched atthe inactive X chromosome (Xi). The chicken β-globin locus is an ideal system to study thedynamics of histone modifications within different chromatin domains during development. Itencompasses a folate receptor gene, a condensed chromatin region, an insulator (HS4), aseries of enhancers (HS3-1) and the globin genes, which are subject to developmentalregulation during erythropoeisis. Initial ChIP analysis over this region correlated enhancedH3-K4 dimethylation and H3- and H4 acetylation with expression of the mature β-globingenes, while H3-K9 dimethylation barely changed during development (Litt et al., 2001). Wehave expanded this study by performing ChIP analysis with our antibodies that are highlyspecific for mono-, di- and trimethylation at either H3-K9 or H3-K27. In 6C2 cells (animmature progenitor cell line that does not express any globin genes), a coexistence ofrepressive marks (di- and tri-H3-K9 and di- and tri-H3-K27) was observed over thecondensed chromatin region and the silent β-globin genes, while the active folate receptorgene was devoid of these marks. Strinkingly, in red blood cells, which express the mature β-globin chains, H3-K27 di- and tri-methylation were absent from the actively transcribedgenes, while persisting over the condensed chromatin region (together with H3-K9dimethylation). These observations clearly define an anti-correlation between di- andtrimethylated H3-K27 and the levels of transcription at the chicken β-globin locus. Moreover,we have deconstructed the β-globin locus into two distinct domains. First, a region of‘constitutive’ heterochromatin that is enriched in repressive marks (dimethylated H3-K9 anddi- and trimethylated H3-K27) at all stages of development. Second, a region of ‘facultative’heterochromatin, where these repressive marks disappear to allow transcription at theappropriate time during development. Our observations were confirmed with anotherprogenitor cell line (HD24) and with 10-day chick embryo brain cells.

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Yuri Postnikov Abstract 88Modulation of histone modifications by chromatin-bindingarchitectural proteinsYuri Postnikov and Michael Bustin

National Institutes of Health, 37 Convent Dr., Bethesda, MD 20892, U.S.A.

The linker histone H1 and members of the high mobility group (HMG) proteins alter thestructure of chromatin and modulate the interaction of regulatory factors with theirnucleosomal targets. We find that histone H1 inhibits the ability of PCAF to acetylate H3K14in chromatin, but not in purified histones. HMGN1, but not HMGN2, inhibits thephosphorylation of H3S10 and enhances the acetylation of H3K14 both in vivo and in vitro.HMGN1- dependent alterations in posttranslational modifications can be detected bothgenome-wide, and at the single gene level. The results suggest that the binding of HMGN tonucleosomes induces steric changes that alter the ability of histone modifying complexes tomodify specific residues in histone tails. FRAP analysis of living cells indicates that H1 andHMG proteins function within a network of proteins that compete for nucleosome bindingsites. We suggest that the competitive interactions among members of this network affectthe ability of modifying enzymes to access and modify their nucleosomal targets.

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Brendan Price Abstract 89The Tip60 histone acetyltransferase is essential for theacetylation and activation of the ATM protein kinaseBrendan D. Price

Dana-Farber Cancer Institute JF222, 44 Binney St., Boston, MA 02115, U.S.A.

The ATM protein kinase is essential for the repair of DNA strand breaks. The repair of DNAstrand breaks involves the remodeling of the chromatin structure. Structural changes inchromatin during DNA repair have been linked to post-translational modification of histonesby phosphorylation, methylation or acetylation. These histone modifications can createbinding sites for DNA repair complexes, including 53BP1, NuA4 and INO80. In addition,activation of the ATM protein kinase can be brought about by changes in chromatinstructure. However, the signal transduction pathway linking DNA strand breaks to activationof ATMs kinase activity is not clearly defined.Here, we investigated the role of the Tip60 Histone Acetyltranferase (HAT), a component ofthe NuA4 complex, in the activation of the ATM protein kinase. ATM is rapidly acetylated ina Tip60-dependent manner following DNA damage. Suppression of Tip60 blocks theactivation of ATMs kinase activity and prevents the ATM-dependent phosphorylation of p53and chk2. Further, inactivation of Tip60 sensitizes cells to DNA damage. ATM and Tip60exist as a preformed complex in cells; however, interaction between ATM and Tip60 is notregulated in response to DNA damage. Instead, the HAT activity of the ATM-Tip60 complexis specifically activated by DNA damage. This activation of Tip60 requires thechromodomain of Tip60, a domain which can associate with methyl-lysine residues. Finally,the ATM-Tip60 complex is biochemically distinct from the NuA4-Tip60 complex, andfunctions upstream of NuA4.The results demonstrate that Tip60 is required for the acetylation and activation of the ATM-Tip60 complex following DNA damage. This activation requires the chromodomain of Tip60,suggesting that the ATM-Tip60 complex associates with exposed methyl-lysine residues onhistones at sites of DNA damage. These results describe a mechanism to link changes inchromatin structure to the activation of ATM-dependent DNA damage response pathways.

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Michael Rehli Abstract 90Genome-wide profiling of CpG-methylation by methyl-CpGiimmunoprecipitationClaudia Gebhard, Lucia Schwarzfischer, Thu-Hang Pham, ReinhardAndreesen and Michael Rehli

University Hospital, H1 Forschungsbau, F.-J.-Strauss Allee 11, Regensburg, Bayern 93042,Germany

Methylation of CpG islands is associated with transcriptional repression and, in cancer,leads to the abnormal silencing of tumor-suppressor genes. We developed a novel androbust technique that allows the detection of CpG-methylation in limited DNA samples,without applying methylation-sensitive restriction endonucleases or bisulfite-treatment. Theapproach is based on a recombinant, methyl-CpG binding, antibody-like protein thatefficiently binds native CpG-methylated DNA depending on its degree of CpG methylation.Its application in methyl-CpG immunoprecipitation (MCIp) facilitates the monitoring of CpG-island methylation either on single candidate gene level (in combination with real-time PCR)or on a genome wide level (in combination with CpG-island promoter microarrays). Genomewide methylation profiling of myeloid leukemia cell lines identified a large number of geneswith aberrantly methylated CpG islands. Strikingly, more than half of the identified genesshow extremely low or no expression in normal cells, suggesting that hypermethylation incancer may be largely independent of the transcriptional status of the affected gene.Several individually tested genes were also affected in primary blast cells from AMLpatients, suggesting that our approach can identify novel potential disease markers.

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Kristen Riley Abstract 91Involvement of the TAC1 complex in transcription elongationKristen M. Riley1, Sheryl T. Smith2, Svetlana Petruk1, Sususma Hirose3,Hugh W. Brock4, Alexander Mazo1

1Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA, 2WistarInstitute, Philadelphia, PA 19107, U.S.A., 3Department of Developmental Genetics, NationalInstitute of Genetics and Department of Developmental Genetics, Graduate University forAdvanced Studies, Mishima, Shizuoka-ken 411-8540, Japan, 4Department of Zoology,University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada, V6T1Z4

The Drosophila heat shock genes are rapidly induced upon heat shock, a result of aspecial, nucleosome-free promoter structure. For elongation to proceed, transcriptionthrough nucleosomes downstream of the start site requires the action of a number ofelongation factors. A possible role for the TAC1 chromatin-modifying complex in theelongation process has recently been described (Smith et al., 2004). TAC1 localizes to thecoding region of hsp70 following heat shock, where it methylates and acetylates histonesand actively promotes transcription. We have examined how TAC1 functions in the networkof elongation factors by determining how other factors affect its recruitment and activity. Wehave found that recruitment of TAC1 to hsp70 requires the elongation factor FACT, thekinase Cdk7, which phosphorylates RNA Polymerase II and is required during earlyelongation steps, and the epigenetic regulator Asx. Interestingly, Asx also requires TAC1 forits recruitment to hsp70, indicating that there is a cooperative mechanism of TAC1 and Asxrecruitment. The ability of TAC1 to perform its methylation activity requires the enzymePARP, which is thought to facilitate loosening of chromatin structure. In addition,examination of TAC1 recruitment in another transcriptional model, the homeotic gene Ubx,shows an almost complete convergence with the heat shock model. The Ubx model alsoshows that TAC1 is required for recruitment of Cdk7 and FACT, suggesting that there mayalso be a cooperative recruitment mechanism for these factors with TAC1. Furthermore,staining of polytene chromosomes from larvae lacking Trx shows that TAC1 is required on abroader scale for association of Cdk7 and FACT with their target genes. Our data togethersuggests that TAC1 is part of the network of interacting factors involved with promotingelongation.

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Flavio Rizzolio Abstract 92Chromatin alteration on POF associated X/A balancedtranslocation.F. Rizzolio1, S. Alboresi1, S. Gilli1, C. Sala1, S. Bione1,2, T. Pramparo3,M.Goegan1, O .Zuffardi3 and D.Toniolo1. 1DIBIT Ospedale San Raffaele, Milano, 2Istituto di Genetica Molecolare-CNR, Pavia,3Dipartimento di Biologia Umana ed Ereditaria, sezione di Biologia Generale e GeneticaMedica, Universit di Pavia, Pavia, Milan, Lombardia 20132, Italy

Premature ovarian failure (POF) is a heterogeneous disorder characterized by menopausebefore the age of 40 years. Different chromosome anomalies associated with POF involvethe X chromosome. In our laboratory we have mapped 25 X/A balanced translocationsinterrupting the X chromosome, in the Xq21 critical region. Since only three genes werefound interrupted, a position effect on flanking genes was considered. In this work we haveanalyzed a region of 5 Mb in Xq21 were 13 breakpoints were mapped. RNA in situhybridization showed that none of the X linked genes is highly or specifically expressed inovarian follicles, suggesting that the ovary expressed genes responsible for the phenotypemay be located on autosomes.We mapped by FISH the autosomal breakpoints of 4patients, on chromosome 1p35.3, 2q14.2, 3p21 and 5q35. Expression analysis by RNA insitu hybridization demonstrated that many of the genes flanking the autosomal breakpointswere expressed in the ovary and indicated that the phenotype could be due to a positioneffect of the X chromosome on autosomal genes. To confirm the possible position effect ofthese breakpoints, we analyzed H3 lysine 9/14 acetylation, H4 lysine 5/8/12/16 acetylationand H3 Lysine 4 methylation on the promoter regions of flanking genes, in lymphoblastoidcells of patients and controls. The preliminary results of this analysis showed that thechromatin modifications were altered in all patients cells compared to controls andtentatively confirmed that the X chromosome critical region may have a chromatinorganization or may contain regulatory elements that could alter the expression ofautosomal genes involved in the breakpoints.

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Orit Rozenblatt-Rosen Abstract 93Paf1 complex is associated with mRNA processingmachinery.Orit Rozenblatt-Rosen1, Suraj J Nannepaga1, Matthew Hoffman2, KristineNordick2, Syuzo Kaneko3, Takashi Sugimoto3, Christina M Hughes1,James H. Resau4, Jim Manley3, Judith Jaehning2 and Matthew Meyerson1.1Department of Medical Oncology, Dana-Farber Cancer Institute, and Department ofPathology, Harvard Medical School, Boston, Massachusetts, U.S.A., 2Department ofBiochemistry and Molecular Genetics, Molecular Biology Program, University of ColoradoHealth Science Center, B121, 4200 East Ninth Avenue, Denver, CO 80262, U.S.A.,3Department of Biological Sciences, Columbia University, New York, NY 10027, U.S.A.,4Analytical, Cellular and Molecular Microscopy Laboratory, Van Andel Institute, GrandRapids, Michigan, U.S.A.

Human Cdc73 (hCdc73), the product of the HRPT2 tumor suppressor gene is part of thehuman Paf1 complex. Paf1 complex is known to be important or histone modifications andother co-transcriptional events. Yeast and human Paf1 complexes both contain Cdc73, Ctr9,Paf1 and Leo1protein subunits. The yeast complex contains an additional subunit Rtf1 andthe human complex was recently reported to include the Ski8 protein.We now show that hCdc73 is co-purified with mRNA processing factors. Immunoprecipitationand immunodepletion experiments as well as glycerol gradient fractionation further confirmthat these complexes interact. Taken together these results suggest that the Paf1 complexserves as a platform between RNA polII and factors modulating mRNA processing.

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Ingemar Rundquist Abstract 94Analyses of linker histone - chromatin interactions in situ indifferent cell systemsIngemar Rundquist1 and Herbert Lindner2

1Division of Cell Biology, Linkpings universitet, Sweden, 2Division of Clinical Biochemistry,Biocenter, Innsbruck Medical University, Austria

The linker histones, commonly referred to as H1, are involved in the formation andmaintenance of the higher order structure of the chromatin fiber and most likely also inepigenetic modulation of gene expression. In mammals, this family consists of eightsubtypes, H1.1-H1.5, H1t, H1 and H1oo. The highly specialized isoforms H5 (in avianspecies) and H1 accumulate in some terminally differentiated cells. In avian and amphibianerythrocytes, expressing H5 and H1 respectively, these proteins have been linked tocessation of cell proliferation and condensation of chromatin. We have studied theinteraction between linker histones and chromatin in a number of cell types, using 46-diamidino-2-phenylindole (DAPI) as an indirect cytochemical probe for linker histone affinityin situ, in combination with high performance capillary electrophoresis and reverse-phasehigh performance liquid chromatography. Significant differences were detected betweensome cell types. The results show that linker histones have a substantially higher affinity forchromatin in mature chicken erythrocytes than in frog erythrocytes. This difference maypossibly be explained by the high content of arginine-rich H5 in chicken erythrocytes. Ourresults also indicate that the affinity decreased in differentiating frog erythrocytes, showingthe lowest affinity in terminally differentiated cells with highly condensed chromatin.Furthermore, in cultured human fibroblasts the linker histones showed a relatively highaffinity for chromatin. However, in highly condensed metaphase chromosomes, the affinitywas significantly lower compared to interphase cells. We have also analyzed linker histoneaffinity for chromatin in H1-depleted fibroblasts reconstituted with purified linker histones.The results show that the exogenous linker histones were bound with somewhat loweraffinity than the native ones and that in vitro phosphorylated linker histones were bound withsubstantially reduced affinity. Our results also indicate a lack of correlation between linkerhistone affinity and chromatin condensation.

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Michael Scher Abstract 95SirT3 is A nuclear histone deacetylase that translocates tomitochondria upon stressMichael Scher1,3, Alejandro Vaquero1, Dong-Hoon Lee1,3, Yan Li1,3, HediyeErdjument-Bromage2, Paul Tempst2 and Danny Reinberg1.1HHMI-Robert Wood Johnson Medical School. University of Medicine and Dentistry of NewJersey, Piscataway, NJ, 08854, U.S.A.. 2Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021. 3UMDNJ-Graduate School of BiomedicalScience Piscataway, NJ, 08854, U.S.A..

Sir proteins are essential in the silencing and aging processes in yeast. Sir proteins werediscovered in budding yeast as factors required for the establishment of epigeneticrepression through specialized heterochromatin formation within portions of the genome,such as telomeric regions, mating type loci, and nucleolar rDNA. Sir2 is involved in thesilencing of all three of these regions dependent upon its enzymatic NAD+-dependentdeacetylase activity defined by a domain of approximately 250 residues. In humans, there are at least 7 Sir2-like proteins (SirT1-7). The human Sir2 related proteinsappeared to target mainly non-histone substrates: SirT1 deacetylates transcription factors,SirT2 localizes to the cytoplasm and deacetylates -tubulin, and SirT3 localizes to themitochondria. However, we have shown that SirT1 also functions in the formation offacultative heterochromatin through its deacetylation of H3-K9 and H4-K16 and interactionwith and deacetylation of histone H1.Contrary to reports that SirT3 localizes to the mitochondria, we have found that SirT3primarily localizes to the nucleus using highly specific antibodies against the endogenousprotein. In addition, certain stimuli trigger SirT3 translocation to the mitochondria. Usingmicroarray analyses together with chromatin immunoprecipitation assays, we also show thatSirT3 regulates a specific set of genes in the nucleus. Both the full-length and truncatedforms of SirT3 can target H4-K16 for deacetylation in vitro. Overexpression of SirT3 wasshown previously to increase the expression of genes involved in mitochondrial function,and promote aging. Our work expands on this and demonstrates a connection betweenSirT3 and chromatin.

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Gunnar Schotta Abstract 96H4-K20 methylation: a mark important for mousedevelopment?Gunnar Schotta, Stefan Kubicek, Roopsha Sengupta, Michaela Paganiand Thomas Jenuwein

Institute of Molecular Pathology, 7 IMP, Dr. Bohrgasse, Vienna, 1030 Austria

Histone lysine methylation is a central modification to mark functionally distinct chromatinregions. In particular, the combination of H3-K9 trimethylation and H4-K20 trimethylationhas emerged as a hallmark of pericentric heterochromatin in mammals.In previous studieswe identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases, which localizeto pericentric heterochromatin in a Suv39h-dependent manner. They act as nucleosomespecific H4-K20 trimethylating enzymes suggesting a sequential mechanism to establishH3-K9 and H4-K20 trimethylation at pericentric heterochromatin. In addition to generepression, H4-K20 trimethylation has recently been implicated in other biological processessuch as DNA damage repair and cancer progression. In order to address these potentialfunctions for mouse development we generated knock-out mice for both Suv4-20h1 andSuv4-20h2. Mice deficient for Suv4-20h1 die perinatally however; Suv4-20h2 null mice donot show obvious defects. The phenotype of Suv4-20h1 is more severe than that of Suv39hdouble null mice, which may suggest additional functions for this enzyme, independent ofpericentric heterochromatin organization.Immunofluorescence and mass-spec analyses ofwt vs. Suv4-20h1 and Suv4-20h2 null MEFs revealed that the enzymes could have differentin vivo activities. Suv4-20h2 appears to be the major enzyme responsible for pericentric H4-K20 trimethylation, while cells deficient for Suv4-20h1 show a reduction in H4-K20dimethylation. The different specificities of the enzymes could in part explain the phenotypesobserved. However, the identification of H4-K20 di- and trimethylation targets may providegreater insight into the function of these enzymes.

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Bernd Schuttengruber Abstract 97Effect of long-range chromosomal interactions mediated bythe Fab-7 element on its target chromatinBernd Schuttengruber, Fredric Bantignies and Giacomo Cavalli

Institut de Genetique Humeine, 141, rue de la Cardonille, Montpellier 34396, France

Proteins of the Polycomb group (PcG) are key players for the maintenance of cell identitythroughout development. In Drosophila, these proteins exert their function through bindingto specific DNA elements, known as PcG response elements (PREs). Fab-7 is a welldefined chromosomal element that contains a PRE and is involved in the regulation of thehomeotic gene Abdominal-B (Abd-B). In addition to an action in cis, homologous PREs caninteract over long distances, even if located on different chromosomes. In particular, weshow by two-colour DNA FISH that a transgene containing the Fab-7 element located onchromosome 2, can pair with the endogenous element at the Bithorax complex (BX-C)located on chromosome 3, leading to strong and stable repression of a mini-white markergene which is heritable through mitotic and meiotic cell division. The transgene can beswitched to a derepressed state by deletion of the endogenous Fab-7 element that leads tounpairing with the transgene. Here, we investigate what chromatin marks are associated tosilencing induced by long-range interactions and how these marks behave during changesin the nuclear localization of the transgene upon unpairing. We could not observe significantdifferences in the recruitment of PcG proteins to the transgene in the presence or absenceof Fab-7 pairing interactions. Similarly, methylation of histone H3 on lysine 27, a hallmark ofPcG-mediated silencing, and acetylation levels of core histones are not changed as afunction of long-distance interactions. We are currently investigating further chromatin marksas possible candidates to be involved in long-distance chromosomal interactions. Further,by combining DNA FISH with immunostaining of PcG proteins, we test whether the silencedFab-7 transgene co-localize with PcG foci, and how nuclear compartmentalization to thesedomains of polycomb action changes upon activation of the transgene.

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Alexandra Schulmeister Abstract 98Histone H3.3 phosphorylation during mitosis and meiosis inthe urochordate Oikopleura dioicaAlexandra Schulmeister, Philippe J. Ganot and Eric M. Thompson

Sars International Centre, 55 High Technology Centre, Thormohlensgt. 55, Bergen,Hordaland N-5008, Norway

Histone H3.3 phosphorylation during mitosis and meiosis in the urochordate OikopleuradioicaSars International centre, University of Bergen, Thormhlensgt. 55, N-5008 Bergen,NorwayThe histone variant H3.3 differs from the major histone H3 (H3.1) in five aminoacids, where alanine at position 31 is replaced by serine. In contrast to H3.1, the variantH3.3 is expressed throughout the cell cycle and is deposited at transcriptionally active lociindependent of S-phase (1, 2). Recent data from mammalian cells suggest that, whenphosporylated at Ser-31, H3.3 also plays a role in mitosis (3). Our previous findings, thatH3.3 becomes a major H3 variant in the prominent endocycles of the planktonicurochordate Oikopleura dioica, have led us to investigate its state of phosphorylation atdifferent developmental stages. We find that phosphorylation of H3.3 at Ser-31 also occursin the miotic cells of the planktonic urochordate Oikopleura dioica, suggesting that thishistone modification and its function in mitosis is already present at the invertebrate-vertebrate transition. The spatial pattern differs from that of H3.1 phosphorylation at Ser-28.Whereas Ser-28 phosphorylation is enriched in the centromeric region, H3.3 Ser-31phosphorylation spreads throughout the condensed chromosomes. We further investigatedthe phosphorylation of H3.3 in meiosis during gametogenesis in Oikopleura dioica. H3.3phosphorylation occurs after Ser-10 phosphorylation, together with the phosphorylation ofSer-28 in diplotene and persists until oocytes are arrested in metaphase I. We are currentlyinvestigating whether the variant H3.3 is phosphorylated via the mitogen-activated proteinkinase (MAPK) pathway during meiosis.

1) Schwartz, B. E. and K. Ahmad (2005). Genes Dev 19(7): 804-14.2) Wirbelauer, C., et al. (2005). Genes Dev 19(15): 1761-6. 3) Hake, S. B., B. A. Garcia, et al. (2005). Proc Natl Acad Sci U S A 102(18): 6344-9.

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Jill Schumacher Abstract 99The Tousled kinase functions in mitosis as a substrate andactivator of the Aurora B kinaseZhenbo Han1, Gary M. Riefler1,2, Jennifer R. Saam3, Susan E. Mango3,and Jill M. Schumacher1,2

1Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center,Houston, Texas, 77030, U.S.A., 2Genes and Development Program, Graduate School ofBiomedical Sciences, The University of Texas-Houston, Houston, Texas, 77030, U.S.A.3Huntsman Cancer Institute, University of Utah, Salt Lake City 84112, U.S.A.

The Tousled kinases comprise an evolutionarily conserved family of proteins that have beenpreviously implicated in chromatin remodeling, DNA replication, and DNA repair. Tousledsubstrates include the Asf1 chromatin assembly factor and histone H3. We have previouslyreported that the C. elegans Tousled-like kinase TLK-1 is required for embryonictranscription and viability. Recently, we have found that TLK-1 also has an essential role inmitotic chromosome segregation. Embryos depleted of TLK-1 display abnormalities inmetaphase chromosome alignment, anaphase chromosome bridges, and polyploid nuclei.TLK-1 is specifically phosphorylated by the AIR-2 Aurora B kinase duringprophase/prometaphase, and this phosphorylation increases TLK-1 kinase activity in vitro.In addition, TLK-1 phosphorylation initiates a feedback loop that increases AIR-2 kinaseactivity. As predicted for an AIR-2 activator, depletion of TLK-1 enhances the mitotic defectsof a hypomorphic AIR-2 allele, revealing that AIR-2 and TLK-1 cooperate to ensure propermitotic chromosome segregation. These results demonstrate that Tousled kinases have apreviously unrecognized role in mitosis and represent a new class of Aurora B activatingproteins.

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David Schrump Abstract 100Gene expression profiling of primary lung cancers exposedto 5 aza 2’ deoxycytidine (DAC), depsipeptide FK228 (DP),or sequential DAC/DPZhao M.1, Li X.2, Hong J.A.1, Chen G.A.1, Kunst T.F.1, Hancox A.1, NguyenD.M.1 and Schrump D.S.1

1Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National CancerInstitute, Bethesda, MD, U.S.A., 2University of Chicago, Chicago, IL, U.S.A.

The emerging relationships between epigenetics and malignant transformation provide impetus forthe use of DNA demethylating agents and histone deacetylase (HDAC) inhibitors for cancertherapy. Presently, limited information is available concerning gene expression in primary cancersexposed to these chromatin remodeling agents. During the past several years, more than 70patients have participated in clinical trials in the Surgery Branch, NCI, examiningpharmacokinetics, toxicities, and potential efficacy of DAC, DP, and sequential DAC/DP infusionsin thoracic oncology patients. Recently, long-oligo array techniques were used to examine geneexpression profiles in RNA amplified from laser-captured tumor cells obtained from fine needleaspirates (FNAs) of 20 individuals receiving DAC, DP, or sequential DAC/DP treatment. Results ofpre- and post-treatment arrays from these patients were compared to data derived from analysisof laser-captured tumor cells and normal bronchial epithelia from 15 patients undergoing lungcancer resections at the NCI. A standardized pool of normal lung RNA was used as a referencefor all arrays, enabling comprehensive analysis of data within and across treatment groups.Pharmacokinetic analysis confirmed that plasma DAC and DP concentrations in these patientsapproximated threshold levels for modulation of gene expression in cultured lung cancer cells.Micro-array analysis demonstrated complex, heterogeneous responses to DAC, DP, andsequential DAC/DP in lung cancer cells. Approximately 735 genes were induced or repressed byDAC treatment in a statistically significant manner; 230 genes exhibited significantly alteredexpression following DP exposure. Interestingly, despite the apparent synergy of DAC and DP incultured lung cancer cells, only 54 genes were modulated significantly in primary lung cancersfollowing sequential DAC/DP therapy. Sixteen genes were modulated by DAC as well as DP. Nogenes were modulated by all three treatment regimens; only one gene was altered by DAC andsequential DAC/DP treatment, and no genes were modulated by DP as well as DAC/DP. Thirty-four genes, which exhibited significantly different expression levels in primary lung cancer cellsrelative to adjacent normal bronchial epithelia were modulated by DAC treatment, whereas 5genes were modulated by DP. Three genes, which were aberrantly expressed in untreated lungcancer cells, were modulated by DAC as well as DP. Overall, DAC and DP treatment tended toinhibit or induce expression of genes aberrantly over-expressed or silenced, respectively, inprimary lung carcinomas. The response to sequential DAC/DP appeared to be less evident,possibly due to pharmacodynamics of DAC and DP in tumor tissues, or effects of stromalelements on gene expression in lung cancer cells. Collectively, these data confirm that DNAdemethylating agents and HDAC inhibitors can modulate gene expression in primary lungcarcinomas. Despite the complexity of the data, comprehensive analysis of gene expressionprofiles in laser- captured tumor cells may facilitate rational development of chromatin remodelingagents for lung cancer therapy.

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Brian E. Schwartz Abstract 101Nucleosome assembly pathways discriminate between sitesin the H3 and H3.3 histone tails.Brian E. Schwartz and Kami Ahmad.

Harvard Medical School, 204 C1, 240 Longwood Ave., Boston MA 02115, U.S.A.

Nucleosome assembly can occur through at least two known pathways. The bulk of DNAis packaged during S phase via the replication-coupled (RC) pathway, while transcriptionallyactive chromatin can be rebuilt outside of S phase through the transcription-coupled (TC)pathway. The RC pathway can use both H3 and the conserved variant H3.3 for assembly,while the TC pathway exclusively uses H3.3. H3 differs from H3.3 by only four aminoacids, and three of these residues are responsible for targeting the histone to theappropriate assembly pathway. Using Drosophila cultured cells, we show here that lysinesin the N terminal tails of H3 also have distinct roles in assembly. In particular, a K4Rmutation disables RC assembly but not TC assembly. No other single lysine to argininemutation affects either pathway. These mutations show that assembly pathways use the N-terminal tail of the H3 subtype histones differently. Additionally, only the RC pathway isdisrupted if histone tails are maintained in a constitutively acetylated (neutralized) state withthe histone deacetylase inhibitor sodium butyrate. Taken together, our data suggests thatthe RC deposition machinery binds H3-lysine 4 and requires that N-terminal lysines aretransiently de-acetylated during nucleosome assembly. The TC pathway does not requirelysine 4, or even the entire N-terminus, as a tail truncation mutant is efficiently incorporated.However, the presence of four point mutations (H3.3-K4,9,14,18R) abrogates TC deposition.We argue that reversible lysine acetylation serves as a mechanism to shuffle histonesbetween chaperones and DNA by temporary disruption of charge interactions.

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Kristin Scott Abstract 102A heterochromatin barrier partitions the fission yeastcentromere into discrete chromatin domainsKristin C. Scott, Stephanie L. Merrett and Huntington F. Willard

Duke University Institute for Genome Sciences and 2383 CIEMAS, 101 Science Drive,Durham, 27708, U.S.A.

Centromeres are cis-acting chromosomal domains that direct kinetochore formation,enabling faithful chromosome segregation. Centromeric regions of higher eukaryotes arestructurally complex, consisting of various epigenetically modified chromatin types includingspecialized chromatin at the kinetochore itself, pericentromeric heterochromatin, andflanking euchromatin. Although the features necessary for the establishment andmaintenance of discrete chromatin domains remain poorly understood, two models havebeen proposed based either on the passive convergence of competing activities involved inindividual domain formation or, alternatively, on the action of specific genomic sequencesand associated proteins to actively block the propagation of one chromatin type intoanother. Here, functional analysis of centromeric sequences located at the intersection ofSchizosaccharomyces pombe central core chromatin and outer repeat heterochromatinidentified a chromatin barrier that contains a tRNA alanine gene. Deletion or modification ofthe barrier sequences result in the propagation of pericentromeric heterochromatin beyondits normal boundary. The tRNA alanine gene is transcriptionally active, and barrier activityrequires sequences necessary for RNA polymerase III transcription. Moreover, absence ofthe barrier results in defective meiotic chromosome segregation. The identification of DNAsequences with chromatin barrier activity at the fission yeast centromere provides a modelfor establishment of centromeric chromatin domains in higher eukaryotes.

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Judith Sharp Abstract 103XIST functions independently of BRCA1 in X chromosomeinactivationJudith Sharp and Barbara Panning

UCSF, Genentech Hall, 600 16th Street, Rm. S374, San Francisco, CA 94158, U.S.A.

In mammals, dosage compensation of the X chromosome is equalized between the sexesby the transcriptional inactivation of one X chromosome in female cells. Precise control ofX chromosome dosage is critical for the proper functioning of the embryo: mutant miceunable to enact X chromosome dosage compensation exhibit female specific earlyembryonic lethality. X inactivation during early female development is mediated by the XISTnoncoding RNA. XIST initiates silencing in cis from a region termed the X inactivationcenter. XIST RNA eventually coats the entire chromosome, resulting in silencing of themajority of genes on the Xi. In terminally differentiated cells, XIST coats the inactive Xchromosome but plays a less stringent role in the silencing mechanism. Humans withgermline mutations in the BRCA1 gene are predisposed to develop early-onset breast andovarian cancer. A recent report indicated that BRCA1 was required to maintain XISTlocalization on the inactive X chromosome in somatic cells and tumor cells, suggesting acorrelation between hereditary breast cancer and defects in Xi chromosome structure.Instead, our findings indicate that X chromosome dosage compensation is oftenmisregulated in female cancer cells independently of BRCA1 genotypic status. Further, wefound that BRCA1 is dispensable for XIST chromosome-coating activity in somatic cells,suggesting that XIST functions independently of BRCA1 during the maintenance phase of Xinactivation.

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Shivendra D. Shukla Abstract 104Histone modifications by ethanol in liverShivendra D. Shukla, Pil-Hoon Park, Jee-soo Kim and Robert Lim

Department of Medical Pharmacology and Physiology, University of Missouri School ofMedicine, Columbia, Missouri 65212, U.S.A.

Ethanol exposure increases expressions of various genes. Histone acetylation has beenshown to induce transcriptional activation. We have therefore investigated the effects ofethanol on histone H3 acetylation in rat liver cells. Ethanol treatment significantly increasedH3 acetylation at Lys9 with negligible effect at Lys14, 18 and 23 in primary cultured rathepatocytes. Treatment of rat hepatic stellate cells with ethanol produced a similar patternexcept that acetylation at Lys23 was also increased at high concentration of ethanol. Acutein vivo administration of alcohol in rats for 12 hrs (analogous to binge drinking) increasedacetylation of H3-Lys9 in the liver without affecting Lys14, 18 and 23, showing the sameresults as in vitro with hepatocytes. The in vivo effects of acute ethanol administrationoccurs in a tissue-specific manner; H3-Lys9 acetylation is seen in lung and spleen, but notthe kidney, brain, heart, stomach, colorectum, pancreas and vessels. Inhibitors of ethanolmetabolizing enzymes, alcohol dehydrogenase (4-methyl pyrazole) and aldehydedehydrogenase (cyanamide), diminished ethanol-induced H3 acetylation at Lys9 inhepatocytes, suggesting a role for ethanol metabolism, especially acetate, in histoneacetylation. Ethanol exposure increased nuclear histone acetyltransferase (HAT) activity inhepatocytes as monitored by ELISA or immunoblot assays. Chromatin immunoprecipitation(CHIP) assay demonstrated that ethanol increased the association of class I alcoholdehydrogenase (ADH I) gene with acetylated H3-Lys9 domain in the chromatin. Takentogether, emerging evidence support the view that post-translational modifications inhistones may underlie the mechanism for ethanol induced gene alterations and mayconstitute an important event in alcoholic liver damage. (Supported by NIH grant #AA14852)

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Robert Sims Abstract 105Regulation of mRNA biogenesis by the coordinator complex,which specifically recognizes methyl H3K4Robert J. Sims III, Chi-Fu Chen, Scott Millhouse, Patrick Trojer, SubhraS. Mandal, Paul Tempst, Robert P. Perry, Brian A. Lewis, James L.Manley and Danny Reinberg

RWJ-UMDNJ 1, RSB, 683 Hoes Lane, Piscataway, Middlesex 08854, U.S.A.

A contemporary view of mRNA biogenesis depicts an integrated process that incorporatesmultiple events simultaneously, encompassing transcript elongation and RNA processing,including CAP addition, pre-mRNA splicing, polyadenylation, in addition to mRNAsurveillance and export. How chromatin dynamics participate in this integrated process ispoorly understood. Here we identify a large protein complex termed the Coordinator, whichcontains factors that facilitate post-initiation events. This complex was isolated by its abilityto recognize the amino terminal tail of histone H3 specifically tri-methylated on lysine 4(H3K4me3). H3K4 methylation is tightly associated with active loci and corresponds to the 5region of human genes. The Coordinator contains the chromatin remodelers CHD1 andSNF2h, components of the U2 snRNP, and PAF, a complex implicated in multiple stages ofmRNA maturation, as well as chromatin modulation. Human CHD1 directly and selectivelybinds methylated H3K4, which in contrast to yeast requires two intact chromodomains intandem. In addition, H3K4me3 recruits factors that assist transcription on chromatinizedtemplates in vitro and methylate H3K4, perhaps identifying a mechanism to propagate thismark. While distinct from the Coordinator, our results also suggest a probable mode ofFACT recruitment spatially within an active gene. We hypothesize that the Coordinatorintegrates multiple processes during mRNA biogenesis, instigated by its recruitment toactivated genes through the interaction of CHD1 with H3K4me3.

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Jeffrey Smith Abstract 106Genetic identification of chromatin-related regulators ofribosomal RNA synthesis in Saccharomyces cerevisiaeRobert D. Hontz and Jeffrey S. Smith

University of Virginia Health System , Jordan Hall, Box 800733, Charlottesville, VA 22911,U.S.A.

Eukaryotic ribosomal RNA (rRNA) is transcribed from repetitive rDNA genes using RNApolymerase I (Pol I). Despite being responsible for the majority of overall transcription inactively growing cells, the regulation of Pol I, especially at the chromatin level, is poorlyunderstood. To facilitate the identification of chromatin-related factors that regulate thisprocess, we have developed a genetic assay in Saccharomyces cerevisiae that efficientlydetects whether a specific gene mutation or growth condition alters the strength of Pol I-mediated rDNA transcription. The assay is based on a modified URA3 reporter gene(mURA3) positioned next to the centromere-proximal (left) end of the rDNA tandem array.The leftmost rDNA repeat has been modified such that Pol I transcription termination doesnot occur, causing interference with the adjacent mURA3 reporter. Mutations that reduce thelevel of Pol I transcription therefore relieve the interference with mURA3, resulting in a Ura+phenotype, whereas mutations that promote Pol I transcription cause more interference withmURA3, resulting in a strong FOA-resistant phenotype. A genetic screen based on thesephenotypes has identified approximately 100 different non-essential genes that appear tofunction in Pol I regulation, including genes encoding components of the RPD3 and HOS2histone deacetylase complexes, and the SAGA histone acetyltransferase complex. Otherchromatin-related factors identified include the SWR1 histone exchange complex, andsurprisingly, the Sir1/Sir4 silencing proteins. Development of the new genetic Pol Itranscription assay and characerization of the above screen candidates as Pol I regulatorswill be presented.

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Yee Sun Tan Abstract 107Skp1 regulates transcription via mono-ubiquitylationYee Sun Tan, Wee Leng Siew, Tze Chin Tan, Debra Morley and NorbertLehming

Department of Microbiology, National University of Singapore, Block MD4, 5 Science Drive2, Singapore 117597

The Mediator complex is involved in transcriptional regulation, transducing signals fromactivators and repressors to RNA polymerase II. The mammalian Mediator complex iscomprised of up to 30 subunits, and Med21, previously known as the Suppressor of RNApolymerase B 7 (Srb7), is one of the subunits found in the middle module of the Mediatorcomplex. The amino acids in the N-terminal region of Med21 are highly conserved frombudding yeast to humans, and this subunit plays a role both for the activation and therepression of transcription. We have found that Med21 interacts with S-phase kinaseassociated protein 1A (Skp1A) in Saccharomyces cerevisiae and in human cells as well asin vitro. Skp1 is part of the nuclear SCF complex (Skp1-Cullin-F-box protein) E3 ligaseinvolved in ubiquitylation of nuclear proteins, and it also interacts with proteins like Ctf13that do not contain F-box motifs. Our work shows that knock-down of Skp1 and pointmutations in Skp1 reduced transcriptional activation in human and yeast cells, respectively.Thus, our results indicate that Skp1 plays a role in transcription. In addition, there isincreasing evidence of mono-ubiquitylation to act as a regulatory signal in cellular processeslike transcription. For instance, mono-ubiquitylation of K123 of H2B leads to methylation ofK4 of H3 in S. cerevisiae. As Skp1 is part of the SCF complex, we are investigating theinteractions of the various subunits of the SCF complex with numerous transcription factors,as well as the core histones. We will discuss the possibility of mono-ubiquitylation of specifictranscription factors by the nuclear SCF complex.

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Hannah Tims Abstract 108Spontaneous and catalyzed nucleosome conformationalchangesJonathan Widom and Hannah Tims

Northwestern University, 551 Hinman Ave., Evanston, IL 60202, U.S.A.

DNA wrapped in the nucleosome is sterically occluded, yet must be accessed for vitalcellular processes, including transcription, replication, recombination, and DNA repair. Weare studying spontaneous and also ATP dependent processes that provide access to buriedstretches of nucleosomal DNA. Studies of spontaneous accessibility have focused oncharacterizing the nucleosome conformational changes that make buried sites accessible,and the frequencies of these conformational changes. We find that the mechanism ofspontaneous accessibility is via partial DNA unwrapping starting from one end of thenucleosome and proceeding inward. Spontaneous unwrapping occurs rapidly (>>1 sec-1)and with no loss or exchange of histones. Our studies of the ATP-dependent processes, conducted in collaboration with the Beckergroup (ABI, Munich), focus on the Drosophila nucleosome remodeling factor ISWI. Studiesof the enzymes ATPase activity reveal that at low (nM) ATP concentration, ISWI is self-inhibited, hydrolyzing ATP but failing to release product efficiently. High (mM) ATPconcentration greatly stimulates the activity. Together with fluorescent anisotropy studies ofISWI binding to DNA and nucleosomes, these findings suggest that ISWI may be active asa dimer.

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Patrick Trojer Abstract 109A functional interplay between MLL and LSD1 Patrick Trojer1, Rushad Pavri1, Hedjye Erdjument-Bromage2, PaulTempst2, Ramin Shiekhattar3 and Danny Reinberg1

1University of Medicine and Dentistry New Jersey (UMDNJ), Robert Wood Johnson MedicalSchool (RWJMS), Department of Biochemistry, 683 Hoes Lane West, Piscataway, NJ08854-0009, U.S.A., 2Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, NewYork, NY 10021, U.S.A., 3The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104,U.S.A.

Histone H3 lysine 4 tri-methylation (H3-K4m3) is linked with activation of transcription.However, in the mammalian system there is a high redundancy of histone lysinemethyltransferases (HKMTs) specific for H3-K4. Knockout models have shown that H3-K4specific HKMTs are not functionally redundant implying an additional layer of regulationregarding their recruitment and respective role(s). For instance, MLL, a Trithorax groupprotein with H3-K4-HKMT activity, was shown to be required for the proper regulation of thedevelopmentally important Hox genes. Recently, genome-wide ChIP analysis demonstratedthat MLL is present on most active genes suggesting it functions globally in transcriptionalactivation.Intriguingly, the rise in the H3-K4m3 level upon gene activation is followed by its subsequentdecrease after loss of the transcriptional stimuli. This used to be explained by histoneexchange. This hypothesis was challenged by the discovery of a histone lysine demethylase(HKDM), LSD1 (BHC110/KIAA0601/PAO), which specifically removes mono- and di-methylgroups from histone H3-K4. Since H3-K4 methylation is an activation mark and LSD1 wasfound as a component of repressive multiprotein complexes, HKDMs appeared tocounteract H3-K4 specific HKMTs analogous to HATs and HDACs. Our data provide evidence that MLL and LSD1 are present in a protein complex of 1.5-2.0MDa. Each enzyme is active in histone methylation or histone demethylation assays,respectively. However, the complex is only capable of di-methylation, not tri-methylation ofH3-K4. The complex acts as a transcriptional repressor in our highly purified reconstitutedtranscription system. Together with our ChIP data, this suggests that the MLL-LSD1complex is already present at silenced genes. Upon activation, the LSD-corepressorcomplex is lost while MLL occupancy is increased concomitant with tri-methylation of H3-K4.Furthermore, based upon the identities of other subunits of this complex it may also play arole in the coordination of transcriptional initiation as well as processes downstream oftranscription.The interaction of these two enzymes that modify H3-K4 with opposing catalytic propertiesrepresents another critical facet of transcriptional regulation.

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Patrick Trojer Abstract 110L3MBTL1 – the chromatin lock?Patrick Trojer1*, Robert Sims III1*, Guohong Li1*, Alejandro Vaquero1,Piernicola Boccuni2, Nagesh Kalakonda2, Regine Losson3, HedjyeErdjument-Bromage2, Pierre Chambon3, Stephen Nimer2, Paul Tempst2,Yuh-Hwa Wang1 and Danny Reinberg1

*contributed equally1University of Medicine and Dentistry New Jersey (UMDNJ), Robert Wood Johnson MedicalSchool (RWJMS), Department of Biochemistry, 683 Hoes Lane West, Piscataway, NJ 08854-0009, U.S.A., 2Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY10021, U.S.A., 3Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), Parcd´Innovation, 1, rue Laurent Fries, 67404 Illkirch Cedex, C.U. de Strasbourg, France

Dynamic regulation of chromatin structure is essential for cellular processes like replication,transcription, differentiation, cell-cycle progression and DNA-repair. Posttranslationalmodifications of histones turned out to be important regulators of these biologicalprocesses. However, the histone modifications are not sufficient to carry the biologicalmessage. Rather, the histone “marks” serve as recognition modules for proteins which areable to distinguish between different modified residues and can even distinguish betweendifferent degrees of modifications on the same residue in the case of histone lysinemethylation. The chromodomain of HP1 was shown to bind methylated lysine 9 on histoneH3 (H3-K9m) and the tandem-tudor domain of 53BP1 was recently shown to recognize H3-K79m2. It is of great interest to find novel chromatin binding factors to understand howchanges of the chromatin structure are regulated.The MBT (malignant brain tumor) domain shows structural similarity with the chromo-, tudorand PWWP-domains and therefore was predicted to bind modified histones. L3MBTL1, thehuman homolog of the Drosophila tumor suppressor protein D-l(3)mbt, was previouslyshown to be a repressor of transcription and its mode of action was independent of HDACs We have purified a protein complex using a cell line that stably expresses L3MBTL1. Mass-spectrometry analysis confirmed L3MBTL1-binding to core histones. In addition our dataprovide evidence that L3MBTL1 binds to the linker histone H1 as well as to HP1 gamma.We show that L3MBTL1 binds to mono- and di-methylated H1-K26 in vitro and in vivo.Using a chromatin compaction assay we demonstrate that recombinant L3MBTL1 is able tocompact chromatin in a histone modification specific manner. This, together with theinteraction with HP1 suggests that L3MBTL1 functions in the process of chromatincompaction by two distinct mechanisms: (1) Binding of L3MBTL1 to repressive histonemarks of different histone tails “locks” histone tails to prevent access of proteins involved intranscriptional activation, (2) Recruitment of factors like HP1 could promote the propagationof chromatin compaction. This work is supported by a fellowship of the Austrian Science Fund (FWF, J2354-B12) toP.T. and NIH(GM-71166) to R.S.

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María Isabel Tussié Luna Abstract 111Pro-proliferative function of the long isoform of PML-RARαinvolved in acute promyelocytic leukaemiaMaría Isabel Tussié Luna, Liliana Rozo and Ananda L. Roy.

Tufts University School of Medicine. Pathology Department. 150 Harrison Avenue.Boston, MA 02111, U.S.A.

The promyelocytic leukemia (PML) gene codes for a tumor suppressor protein that isassociated with distinct subnuclear macromolecular structures called the PML bodies. ThePML gene is frequently involved in the t(15;17) chromosomal translocation of acutepromyelocytic leukemia (APL). The translocation results in a fusion gene product, PMLRARα, in which the PML gene fuses to the retinoic acid receptor α (RARα) gene. PMLRARα has been shown to promote transcriptional repression of genes involved in myeloidterminal differentiation and to disrupt the architecture of PML bodies, a phenotype reversedby treatment with all trans retinoic acid (ATRA). However, there are several alternativelyspliced isoforms of PML RARα. Here we addressed the differences between the short andthe long isoforms of PML RARα (L and S) since both are associated with APL. Wedemonstrate that PML RARαL but not PML RARαS, can directly promote cell growth bytranscriptionally activating the pro proliferative gene, c fos, in response to mitogenicstimulation. The activity of the PML RARL is completely sensitive to ATRA. We furthershow that this activation is not via direct recruitment of the protein to the c fos promoter butindirectly by altering the chromosomal environment of the c fos gene thereby rendering itmore accessible to the signal induced transcriptional activators. Our results suggest that inaddition to antagonizing the PML tumor suppressor or the PML pro apoptotic activity, PMLRARα proteins can also directly promote cell growth by activating c fos.

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Wim Vanden Berghe Abstract 112Differential interleukin-6 gene expression dynamics in benignand metastatic breast cancer cells reflects distinct chromatinsignatures at the promoter regionWim Vanden Berghe1, Matladi N. Ndlovu1, Nathalie Dijsselbloem1, LindaVermeulen1, Carine Van Lint2 and Guy Haegeman1

1Laboratory for Eukaryotic Gene Expression and Signal Transduction (LEGEST),Department of Molecular Biology, Ghent University, KL Ledeganckstraat 35, B-9000, Gent,Belgium. 2Université Libre de Bruxelles (ULB), Institut de Biologie et de MédecineMoleculaires (IBMM), Service de Chimie Biologique, Laboratoire de Virologie Moleculaire,Rue des Profs Jeener et Brachet, 12, 6041 Gosselies, Belgium.

Previously, we have demonstrated involvement of the mitogen- and stress-activated proteinkinase-1 MSK1 as a crucial kinase for NF-kB p65 and histone H3 phosphorylation in IL6gene expression during inflammatory stress (Vermeulen et al. EMBO J. 2003). As IL6 isalso an autocrine growth factor in various cancers, which accelerates tumor progressionand promotes metastasis, we have compared NF-kB signaling and chromatin dynamics in abenign and a metastatic breast cancer model. We have therefore analysed the promoterregion of the IL6 gene in metastatic MDA-MB-231 cells, producing high levels of IL6, andbenign MCF-7 cells, producing background levels of IL6, for the presence of hypersensitivesites (HS) with DNase-I, and for nucleosome positioning with micrococcal nuclease (MNase)and restriction accessibility assays. We detected that elevated IL6 expression levelscorrelate with increased number of HSs, constitutive NF-kB/DNA binding and elevatedMSK1 activity. Of particular interest, soy isoflavones, which are claimed to reduce breastcancer risk, were found to selectively inhibit the MSK1 pathway towards NF-kB and H3chromatin modifications. These results suggest that MSK1 is a critical trigger forestablishing a transcription-competent enhanceosome and a permissive nucleosomalstructure of the IL6 promoter, thus restricting NF-kB-driven gene dose responses to adefined physiological frame. Unlimited MSK1 activity may predestine cells towardstumorigenesis or chronic inflammatory disorders at the NF-kB/chromatin interface.

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Adam Wood Abstract 113The Bur1/Bur2 complex is required for histone H2Bmonoubiquitination by Rad6/Bre1Adam Wood, Jessica Schneider, Jim Dover, Mark Johnston and AliShilatifard

Saint Louis University. Doisy, 1402 South Grand Ave., Saint Louis 63104, U.S.A.

Transcriptional regulation is highly coupled to chromatin structure. Severalclasses of enzymes have been shown to affect transcription by catalyzing modification ofnucleosomes including the methyltransferases COMPASS and Dot1p. By way of our globalproteomic screen GPS, we have determined that deletion of BUR2, the gene encoding thecyclin component of the Bur1/Bur2 cyclin-dependent protein kinase, is required for histoneH2B monoubiquitination by the Rad6/Bre1 complex. We also demonstrate that the deletionof BUR2 results in a significant decrease in histone H3K4 methylation catalyzed byCOMPASS and H3K79 methylation by Dot1. The effect on histone monoubiquitination andmethylation are the result of defective Bur1/Bur2-mediated phosphorylation of Rad6 on itsserine residue 120 and the recruitment of the Paf1 complex to chromatin. We havedemonstrated that serine 120 of Rad6 is required for proper histone H2B monoubiquitinationand the regulation of gene expression in vivo. Our results identify in vivo substrates for theBur1/Bur2 kinase that links its role to transcriptional elongation and demonstrates apotential activation mechanism for histone H2B monoubiquitination by the Rad6/Bre1complex.

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Dag H. Yasui Abstract 114MeCP2 recruits SNF2h to the 15q11-13 imprinting controlregionDag H. Yasui and Janine M. LaSalle

Medical Microbiology and Immunology and Rowe Program in Human Genetics, UC DavisSchool of Medicine, Medical Microbiology and Immunology, Davis CA 95616, U.S.A.

Rett syndrome is an X-linked neurodevelopmental disorder cause by mutations in MECP2.MECP2 encodes methyl CpG binding protein 2 (MeCP2) that acts as a transcriptionalrepressor for methylated genes by recruiting factors such as HDAC1, Sin3a, and DNMT1.Recent evidence supports a role for MeCP2 in long-range mechanisms that control geneexpression, including chromatin looping and remodeling. We have previously demonstratedbinding of MeCP2 to the methylated promoter of SNRPN/Snrpn that acts as an imprintingcontrol region (ICR) for multiple genes on human 15q11-13 and syntenic mouse 7qB5. Toidentify additional factors specifically binding to the methylated 15q11-13 ICR, DNA affinitychromatography using a methylated or unmethylated SNRPN promoter sequence wasperformed on nuclear extracts from SH-SY5Y neuroblastoma cells. Proteins isolated fromcolumn fractions were identified by mass spectroscopy and confirmed by immunoblotanalysis. MeCP2 was isolated specifically from the methylated but not the unmethylatedaffinity column and co-fractionated with SNF2h, but not Sin3a. SNF2h is a chromatinremodeling ATPase of the SWI/SNF family that requires recruitment by DNA bindingproteins for long-range regulation of gene expression. Reciprocal co-immunoprecipitationusing anti-MeCP2 and anti-SNF2h confirmed the association in vivo. Immunofluorescenceof mouse cortex sections also demonstrated a partial colocalization of MeCP2 and SNF2hin a subset of developing neurons. To determine if MeCP2 binding is required for therecruitment of SNF2h to the methylated ICR, chromatin immunoprecipitation (ChIP) followedby quantitative PCR was performed in SH-SY5Y cells transfected with a methylatedoligonucleotide decoy versus a mutant unmethylated control. A decrease in SNRPN signalwithin both MeCP2 and SNF2h ChIP DNA was observed in SH-SY5Y cells treated with themethylated but not the unmethylated MeCP2-specific decoy, suggesting that MeCP2 bindingto methylated CpG sites is required for SNF2h binding to the SNRPN ICR. Experiments toconfirm this result in the Mecp2-null mouse model are ongoing. These results are the firstdemonstration of a chromatin remodeling ATPase associating with an ICR and have directrelevance to understanding the pathogenesis of Rett, Prader-Willi, and Angelmansyndromes.

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Notes

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Notes

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Radisson Hotel telephone numbers Hotel: 242-327- 6000

Hotel General Emergency: 33 (from inside hotel)

Concierge: 20 (from inside hotel)

Local Services: 0 (from inside hotel)

Police: 0 (from inside hotel)

Hotel Doctor Service: 242-327-7711(ask for Medi Center (9-5) not free service.)

Hotel Doctor Service (after 5pm):call operator above and a doctor can be arranged at a fee

Hotel Security: 242-327-6000 ext.6474

Local telephone numbers Police & Fire Dept: 242-327- 8800

Hospital: 242 322-2861

Walk in clinics: The Hotel recommendsthe 'Walkin Medical Clinic' (242-328-4014)

24hr pharmacy: 242-326-4629 (regular hours)

Taxi: 242- 327-2042

Airport: 242 -377-1759

OrganisersConference organisers: AbcamAt the resort from 13th November, +44(0)7968 024628

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Resort Information

Resort Information

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Radisson Hotel Map

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Sponsor of 2005 Chromatin Structure & FunctionKeynote Presentation

Chroma Therapeutics (Oxford, UK) was founded in 2001 and is focusedon the exploitation of chromatin biology to discover and develop a newgeneration of targeted treatments for cancer and inflammatory disorders.Chroma's founding scientists include Tony Kouzarides (University ofCambridge) and David Allis (Rockefeller Institute), leaders in the field ofchromatin regulation. In oncology, inhibition of chromatin modifyingenzymes (CMEs) such as HDACs, aurora kinases and histonemethyltransferases, impacts multiple aspects of tumour progressionincluding proliferation, angiogenesis and invasion while in models ofchronic inflammation, CME inhibitors show potent anti-cytokine activity,highlighting their potential as a new class of oral therapies.

Chroma's highly experienced management team aims to build a dominantposition in CME inhibitors as treatments for both oncology andinflammatory disorders. The company's resources are focused on thediscovery and early stage development of CME inhibitors which, whencombined with its access to leading academic institutions working in theCME area, aim to deliver a strong and renewable pipeline of advancedtherapeutics. The company is also developing a non-invasive biomarkertechnology based on the analysis of histone modifications on the tumour-derived chromatin which circulates in the blood of cancer patients.

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