Fribourg 120224 -120316

Chromatin Structure and Function in

Transcription, Replication, Repair

1FT Fribourg12F

Chromatin Structure and Function

Fritz Thoma Institute of Molecular Health Science

(previous Institute of Cell Biology) ETH-Zürich

Hönggerberg HPM-E42 +41-44-6333323

thoma@cell.biol.ethz.ch http://www.cell.biol.ethz.ch/research/thoma/

120309 1615 -1900

FT Fribourg12F 2

(Ball 2003, Nature)

Packaging Genomes

Space Questions

Volume of the DNA?

Volume between chromatin ("interchromatin" space")?

Volume of the chromatin?

Thoma111018 3

Chromatin Dynamics

How and how tighty are histones bound to DNA ?

How and how tighty NHCPs bound to DNA ?

1 2 3 4 5 6 1

Thoma and Koller(1981) J Mol Biol 149, 709

Protein PAGE (coomassy blue)

Thoma111018 4

H1 is removed at about 0.5 M NaCl H2A, H2B, H3, H4 remain bound to DNA

H1

H3 H2B H2A

H4

(1) Soluble rat liver chromatin (Histones; Non-Histone Chromosomal Proteins, NHCP). Histones are the major protein components of chromatin.

Stoichiometry: about H1:H3:H2B:H2A:H4 = 1:2:2:2:2

(2) Supernatant after high speed centrifugation of chromatin in 300 mM NaCl (NHCP). NHCP are loosely bound and dissociate from chromatin < 300 mM NaCl

(3) Supernatant after high speed centrifugation of chromatin in 500 mM NaCl: H1 and NHCP are released.

(4) Chromatin fraction in a sucrose gradient in 500 mM NaCl: contains all core histones

Chromosomal Proteins

Chromatin of rat liver nuclei was fractionated on sucrose grdients containing different ionic strength

Thoma1110185

Van Holde, K. E. (1989) Chromatin, Springer Verlag, Berlin

Stepwise Histone Binding to DNA

DNA binding strength: H1 < H2A, H2B < H3, H4

Ionic Interactions

Dissociation

Histone Basic Amino Acids

Acidic Amino Acids

MW

Lys Arg

H1 29% 1% 5% 23000

H2A 11% 9% 15% 13960

H2B 16% 6% 13% 13774

H3 10% 13% 13% 15342

H4 11% 14% 10% 11282

Assembly

H2A H2B are loosely bound towards the ends H3H4 are firmly bound in the center

Thoma111018 6

How to investigate localization and histone-DNA binding in

living cells?

Chromosomal Proteins

Thoma111018 7

Dynamic binding of histone H1 to chromatin in living cells Misteli et al (2000) Nature, 408, 877.

FRAP: Fluorescence Recovery After Photobleaching Cell expressing GFP-tagged protein

Photobleaching (seconds)

Distroys fluorescence, but not the proteins

Recovery of fluorescence (time)

Redistribution of proteins

HMG14-GFP: High Mobility Group 1 („Transcription“ Factor), fast recovery

H1-GFP: recovery in minutes strong binding (long residence time)

GFP alone: fast recovery

Thoma111018 8

Method • Cell lines expressing histones and HMGs (a High Mobility Group Protein; NHCP) tagged with green fluorescent protein (GFP) • Photobleaching (FRAP)

Results

HMG14-GFP: exchange in seconds

H1c-GFP : exchange in minutes

H2B-GFP: 3% of H2B exchanged within minutes, 40% did so slowly (t1/2 approximately 130 min).

H3-GFP: exchange many hours H4-GFP: exchange many hours

Dynamic binding of HMGs and histones to chromatin in living cells Misteli et al (2000) Nature, Kimura and Cook (2001) J Cell Biol, Kimura(2005) DNA Rep.

Dynamic binding: HMGs < H1 < H2B < H3, H4

dito: nucleotide excision repair factors, transcription factors, glucocorticoid receptor

Thoma111018 9

General Properties of Histones

Histones are highly conserved in evolution

Histones are basic proteins and positively charged

Bind to negatively charged DNA by ionic interactions

DNA binding strength: H1 < H2A, H2B < H3, H4

Histones are abunant (DNA/histones = 1/1 (w/w))

Histone stoichiometry about H1:H2A:H2B:H3:H4 = 1:2:2:2:2

Histone form (H2A H2B) and (H3 H4) dimers, (H3 H4)2 tetramers, octamers...

Specialized properties and functions of histones, variants, modifications in chromosome structures, differentiation, development, transcription,

replication, repair, recombination

Histones

Chromatin Structures

Packaging Genomes

10Thoma111018

Nucleosomes

Higher Order Structures

Thoma MGE05-L04 11

Discovery and Characterization of Nucleosomes by Electron Microscopy

Miller Spreading, Negative Stain, Olins (1974), Science

(McKnight and Miller)

Miller Spreading, Negative Stain Miller Spreading, Metal Shadowing

(Hamkalo and Rattner)

„30 nm Fiber“, 150 m0 NaCl „beads - on - string“, low ionic strength

Le5 10-19

Unfolding of chromatin in „low salt conditions“ and high pH

"Beads-on-string", repetitive units, diameter: about 10 nm

Thoma111018 12

The first biochemical indication of a subunit structure

Biochemical Discovery and Characterization of Nucleosomes

1. Rat liver nuclei

2. Incubation with 1 mM Ca2+, 10 mM Mg2+

3. DNA purification and gel electrophoresis

Hewish and Burgoyne (1973) BBRC52,504

Thoma111018 13

Digestion of nuclear chromatin with micrococcal nuclease (MNase), a Ca2+ dependent endonuclease, cuts ss DNA, ds DNA, RNA, some preference for AT verus GC rich DNA

Method: Finch, Noll and Kornberg (1975)PNAS 1. Nuclei, add 1mM Ca2+ 2. Add MNase, digest for a limited time, stop reaction by removal of Ca2+ 3. Purify DNA 4. Agarose gel, stained with Ethidium Bromide

200 bp

400 bp

600 bp

800 bp

3 Nucleosomes

4 Nucleosomes

2 Nucleosomes

1 Nucleosome

MNase cuts DNA between nucleosomes ("linker DNA")

Nucleosomes: a repetitive subunit of chromatin

„Nucleosome repeat length“ of a genome

Nucleosomal DNA resists digestion by MNase

Nucleosomes – "repressive" structures

MNase

Packaging Genomes – Nucleosomes

Thoma111018 14

DNA-analysis in agarose gels

Analysis by EM

Soluble rat liver chromatin was generated by MNase digestion and fractionated on a sucrose gradient 1 2 3 4

Bottom Top A260

Finch, Noll and Kornberg (1975)Proc Natl Acad Sci U S A, 72, 3320-3322.

Nucleosomes - A Repetitive Subunit of Chromatin

Thoma111018 15

DNA-Agarose Gel, Färbung Ethidium Bromid

1. Rat liver nuclei 2. MNase digestion 3. DNA Purification 4. Agarose Gel

Noll and Kornberg (1977). J Mol Biol

Nucleosome Particles - Subunits of Chromatin

DNA

Core Histones

Linker Histone

168 bp: Chromatosome

Chromatosome

168 bp 2 turns

Octamer

H1

147 bp: Nucleosome Core

Nucleosome Core Particle conserved from yeast to man

147 bp 1.7 turns

Octamer 2x(H2A, H2B, H3, H4)

-

200 bp: Nucleosome

Nucleosome

> 168 bp 2 turns + extensions

Octamer

H1

Thoma111018 16

Noll (1974) NAR

1. Rat liver nuclei 2. DNaseI digestion 3. DNA purification 4. Polyacrylamide gel

Only the outside of DNA is accessible to proteins

Nucleosome structure regulates accessibility of

DNA for proteins

in transcription replication

repair

10 bp DNA repeat DNA is on the outside of

nucleosomes

Testing DNA-accessibility by Nucleases

DNaseI: endonuclease, binds DNA in the minor groove, generates ss cuts in ds DNA

EG12 Thoma 17

Nucleosome cores can be reconstituted

Agarose-Gel, „Band Shift“

DNA

Nucleosome

Histone Octamers Labelled DNA Histone Octamer (e.g. recombinant histones)

Dissociation Assembly by Stepwise dilution to 0.1M NaCl

(> 1M NaCl)

Thoma111018 18

11 nm 6 nm

H3 H4 H2A H2B

DNA 147 bp DNA (palindromic alpha-satellite DNA) 1.7 left handed supercoils Outside, bent, distorted

Histones: Octamer (recombinant Xenopus histones) (H3.H4.H4.H3) Tetramer 2 (H2A.H2B)-Dimers Histones, folded, inside,N-terminal tails protrude outside

Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal Structure of a Reconstituted Nucleosome Core

6 nm

DNA-Distortion in Nucleosome Cores

Richmond and Davey (2003) Nature 423, 145

Thoma111018 19

Structural and Functional Subunit of Chromatin Dynamic Particle

Controls DNA packaging, accessibility, protein

Some Properties of Nucleosome Cores Small disc shaped particles of 10 nm diameter, 5.7 nm high, pseudosymmetric

Abundant (about 107 nucleosomes / haploid human genome) Conserved structure from yeast to man

Histones inside – N-terminal tails protrude outside

Binding to DNA: 14 contact sites, binding strength: (H2A.H2B) < (H3.H4)

DNA 146 - 147 bp, 1.7 left-handed turns, one negative supercoil / nucleosomes, 80 bp/turn on

the outside, brings distant sites together inner surface is protected, outer surface is accessible, DNA is distorted - affects DNA recognition by proteins and damage formation

Nucleosome Heterogeneity Sequence

Histone Variants Histone Modifications

Nucleosome positioning & stability regulate DNA sequence accessibility

Nucleosome Cores

Chromatin Structures

Packaging Genomes

20Thoma111018

Nucleosomes

Higher Order Structures

Thoma111018 21

Method 1.  Nuclei 2.  MNase digestion 3.  DNA Purification 4.  Agarose Gel and

Ethidium Bromide Staining

200

400

600

800

1000

1200

1400

1 2 3 4 5 6 7

200

400

600

800

1000

1200

Nucleosomes

bp

200 bp / nucleosome

Average Nucleosomal Repeat / Genome

ca. 300 bp

ca. 400 bp Local variability of linker DNA

Nucleosome Repeat Length and Linker DNA

Thoma111018 22

Repeat bp

Seeigel Spermien ca 240 Seeigel Embryonen 222 Ratten Leber 196 Ratten Niere 196 Huhn Oviduct 196 Hühner Erythrocyten 207 Bäckerhefe S. cerev. 160

Linker bp 95 77 51 51 51 62

15

Linker DNA Commonly used: Linker DNA = DNA connecting nucleosome – cores Nucleosome-Core DNA is constant (147bp), but the linker DNA varies in different organisms, tissues, and between nucleosome cores.

Nucleosome Repeat Length and Linker DNA

What determines the linker length?

Not known. Possible contributions by histone H1

Thoma111018 23

30 nm Chromatin

Fibers

Thoma111018 24

chromatin with H1

Salt dependent condensation of chromatin and H1-depleted chromatin Roles of histone H1 in chromatin structure:

binds to the entry and exit site of linker DNA in nucleosomes and stabilizes nucleosomes stabilizes and organizes chromatin fibers

Structural Transitions In Soluble Rat Liver Chromatin Thoma, Koller, Klug (1979) J. Cell Biol. 83, 403

0 mM

open zig-zag nucleosome-filament

> 60 mM NaCl

"30 nm" compact fiber

unfolded nucleosomes

"beads-on-a-string" partally unfolded nucleosomes

compact clumps

chromatin without H1

removal of H1 on

sucrose gradients containing 0.5M NaCl

Dialysis against buffers containing different ionic strength, fixation by 0.1% glutaraldehyde, adsorbed on carbon coated grid and stained by rotary shadowing with Pt for EM

~ 5 mM

closed zig-zag nucleosome fiber

100 nm

Thoma111018 25

6-8 nucleosmes / turn (11 nm)

Histone H1 contacts stabilize the fiber

Path of the linker DNA is unknown

Salt-dependent folding of chromatin

"0" "~ 5"

"~ 5 " "~ 40 "

"~ 40 " "~ 100 " "~ 100 "

"~ 100 " "~ 100 " "~ 100 "

Structural Transitions In Soluble Rat Liver Chromatin Thoma, Koller, Klug (1979) J. Cell Biol. 83, 403

Irregular, heterogenous structures (due to different linker length, composition etc?)

Thoma111018 26

Reconstituted Tetranucleosome

DNA: 4 copies of a 147 bp long DNA sequence ("601") with strong nucleosome positioning properties, separated by 20 bp linker DNA

Histones: Recombinant core histones of Xenopus laevis

X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Schalch, T., Duda, S., Sargent, D.F. and Richmond, T.J. (2005) Nature, 436, 138-141.

Thoma111018 27

30 nm Fiber Models

Excluded by mass/unit length

and electric dichroism (McGhee et al.

(1980). Orientation of the nucleosome within

the higher order structure of chromatin.

Cell 22, 87-96.)

Thoma, Koller, Klug (1979) J. Cell Biol. 83, 403

"Solenoid" Finch and Klug (1976)

PNAS 73, 1897

"Helical-Ribbon" Woodcock et al.(1984).

J Cell Biol 99, 42-52.

a) depends on linker length

a)

"Crossed-Linker" Williams et al. (1986). Biophys J

49, 233

diameter depends on linker length

Thoma111018 28

In metaphase chromosomes & intephase

Structure(s) unknown

Diameter about 30-50 nm, irregular, dependent on linker length (?)

Nucleosomes arranged radially

Linker DNA path unknown

Packaging (mass / length) about 6-8 nucleosomes / 10 nm

H1 probably inside

Pronounced Heterogeneity structural, functional, compositin histone variants, histone modifications, NHCPs

Some properties of 30 nm fibers

Thoma111018 29

Loops

Chromosome Scaffold

Nuclear Matrix

Thoma101019 30Alberts8-16

Lampbrush chromosomes in amphibian oocytes

Interphase chromosomes in amphibian oocytes Paired homologous chromosomes, after replication, prior to meiotic divisions (4n)

Very active in transcription loops

open chromatin, transcribed condensed chromatin

(chromomeres)

Spreading on water

Thoma101019 31

Chromatin Loops - Chromosome Scaffolds – Nuclear Matrix

(Paulson & Laemmli, 1977)

Metaphase Chromosome

Removal of histones with heparin

Adsorption on EM grids Contrast by metal shadowing

Are chromatin loops attached to a nuclear matrix ? No components idenified so far MARs = Matrix Associated Regions in DNA

Are chromatin loops attached to a scaffold in metaphase chromosomes? Scaffoldprotein: Topoisomerase II & others ? SAR = Scaffold Associated Region (DNA)

Thoma MGE05-L06 32Holm et al (1985) Cell 41 553

DNA topoisomerase II is required at the time of mitosis in yeast

top2ts

26o

top2ts

35o

Yeast (top2ts), temperature sensitive mutation in the gene coding for topoisomerase II • Cells were blocked in G1 by alpha-factor • Released in fresh medium at 26° or 35° • Stained with DAPI

Role in segregation von ds-DNA after replication

Thoma111018 33

Metaphase

Chromosomes

Thoma101019 34

Indian Muntjac Cells Blocked in Metaphase with Colcemid

Chromosome 2

Saitho and Laemmli (1994)Cell 76, 609

Structure of Metaphase Chromosomes

Thoma101019 35

Metaphase Chromosome Structure(Saitho and Laemmli (1994)Cell 76, 609)

Bands arise from a differential folding path of the highly AT-rich scaffold. Evidence by differential staining approaches

Thoma111018 36

Packaging

P (smallest human chromosome) = 14 mm DNA / 2 µm = ca. 7000

Packaging Ratio (P) = Length of DNA (nm) / Length of chromatin

P (nucleosome) = (200bp.0.34nm/bp) / 10nm = ca. 6

P (30 nm fiber) = (6 to 8 nucleosomes) / 10nm = ca. 36 to 48

P (DNA) = 1

Thoma11101837

Roles of Packaging

(In-) accessibility of DNA/proteins

DNA

Nucleosomes

Chromatin Fibers

Loops / Domains

MetaphaseChromosome

Euchromatin

Hetero-chromatin

Few bp

one supercoil80 bp

6-8 nucleosomesabout 1 to 2 kb

one loop5 - 50 kb (?)

>>> Mb (?)

Condensation of DNA

Distance between twobinding sites

(Dis-) favours (long-) distant interactions between proteins and/or DNA elements

Chromatin Structure and Function

Genes & Transcription TBP

TFIIB

RNA Polymerase II

38Thoma111018

Control - Regulation activation – inactivation - silencing

Transcription Cycle initiation – elongation - termination

Thoma111018 39

rRNA

mRNA Protein

5s-rRNA, t-RNAs, snR6, 7SLRNA ...

Nucleolus

Nucleus

Nucleus

50-70%

20-40%

Ca.10%

RNA-Polymerase I

„Pol-I-Genes“

RNA-Polymerase II „Pol-II-Genes“

RNA-Polymerase III

„Pol-III-Genes“

Product Location Activity Class

Eukaryotic Gene Classes (see textbook)

Thoma111018 40

5‘ Enhancers

Proximal and distal regulation sites (Elements, Enhancers)

Internal control regions

RNA-Polymerase I

„Pol-I-Gene“

RNA-Polymerase II „Pol-II-Gene“

RNA-Polymerase III

„Pol-III-Gene“

TFI A,B,C, UBF

TFII A,B,D (TBP), E,F,H

TFIII A,B,C

TFIIH, TBP

TBP

• RNA-Polymerases: Enzymes for DNA-dependent RNA-synthesis

• General Transcription Factors (GTF): Formation of the initiation complex with RNA-polymerases. TBP, TFIIH are GTFs involved in different classes RNA-Polymerases and GTFs are required for correct initiation and elongation

• A combination of specific transcription factors interact with specific DNA elements and regulate transcription by interactions with GTFs and RNAP (Combinatorial control)

Eukaryotic Gene Classes (see textbook)

Thoma111018 41

Genes Transcribed by RNA Polymerase II (see textbooks)

Yeast+1 -90 Coding Region

TATA INR Box Cap-Site Promoter

Proximal UAS, URS Upstream Activating /Repressing Sequence

500 - 100

CpGIslands

Coding Region

house keeping genes

regulatedgenes

Higher Eukaryotes exon intron premRNA

+1 (TSS, Transcription Start Site)30TATA INR Box Cap-Site Promoter

Core Promoter: Coordinate assembly of General Transcription Factors (GRFIIs) and RNAPII to initiate transcription. Only about 40% of genes have a TATA box.

downstreamupstream

Control Regions: Enhancers, UAS (Upstream Activating Regions), Silencers,..... • DNA-Element: binding site on DNA• Recruit sequence specific transcription factors for activation or repression

1 - 50kbDistant Control Region

+10 bis +50kbDownstream Control Region

Internal Control Region

200 - 400bpProximal Control Region

Thoma111018 42

Locus Control Regions (LCR)

Locus Control Regions •  a distant control region •  regulates expression of globin genes

Human β-Globin Gene Cluster

Alberts et al.

Thoma111018 43

Insulators and Boundaries

Bell(2001)Science291,447

HS4, 3ʻ Hs: Insulators, enhancer blocking elements

Insulators (HS4, 3'HS) mark the boundaries of chromatin domains by limiting the range of action of

enhancers and silencers

LCR, Locus Control Region, regulates

transcription of globin gene

Enhancer regulates transcription of

folate receptor gene

Thoma11101844

Long distance control - Genes transcribed by RNAPII

Transcription is controlled by various distant and proximal control elements ("enhancers") that recruit factors for activation/repression

DNA

Nucleosomes

Chromatin Fibers

Loops / Domains

MetaphaseChromosome

Euchromatin

Hetero-chromatin

Few bp

one supercoil80 bp

6-8 nucleosomesabout 1 to 2 kb

one loop5 - 50 kb (?)

>>> Mb (?)

Distance between twobinding sites

Promoter(DNA-)Elements

Regulatory(DNA-)Elements

Lodish et al. 5th edThoma111018 45

General Concepts: Genes transcribed by RNAPII

Chromatin-Remodelling

Thoma111018 46

Chromatin States of Genes

regulatory proteins regulatory elements RNAPolymerase Nucleosomes

Inactive in Euchromatin

Nucleosomal regions have reduced accessibility to nucleases (MNase, DNaseI)

MNaseDNaseI

Active in Euchromatin

MNaseDNaseI

Enhanced nuclease sensitivity in and around transcribed regionHypersensitive sites (HHS) in control regions: proteins disrupt nucleosomes

Transcription and histone modifications ‚open ʻ chromatin

Nucleosomal regions have slightly more reduced accessibility to nucleases (MNase, DNaseI)

MNaseDNaseI

Silenced in Heterochromatin

Thoma111018 47

Hypersensitive Sites

indicate interactions

of regulatory proteins and remodelling factors with DNA

Gene

Thoma111018 48

Transcription

+ induced

- repressed + constitutive + induced

- repressed

Firtel et al. In Architecture of Eukaryotic Genes, ed. G. Kahl (1988), VCH

DNaseI Hyper Sensitive Sites around the Chicken Lysozyme Gene

Hypersensitive sites are an indication for interactions of regulatory and remodelling factors with DNA.

1. Nuclei2. Digest with DNaseI (limited)3. Extract DNA4.Cut with restriction enzyme5. Gel and Southernblot6. Hybridize with probe close to one restriction site ("indirect endlabeling")

Thoma111018 49

Promoters

Regulation

Initiation of Transcription

Thoma111018 50

Stepwise assembly of the preinitiation complex and initiation of transcription by RNA-Polymerase II (in vitro and in vivo)

TFIID: TBP (TATA Binding Protein) binds TATA box TAFs (TBP associated proteins) interacts with Inr in TATA less promoters TFIIA: 3 subunits TFIIB: positions RNA-PolII TFIIF: 2 subunits

TFIIE: 2 subunits RNAP-II: 12 subunits

TFIIH: 5 subunits including 2 Helicases (opening of promoter) 1 Kinase (phosphorylation of RNAP-II-CTD)

TFIIA

Preinitiation Complex Initiation & Promoter Clearance (initiation to elongation transition) Phosphorylation of RNAP-II-CTD (Serine 5)

Mediator Elongation Recruitment of elongation factors, RNA-processing factors Phosphorylation of RNAP-II-CTD (Serine2)

Lodish 5th ed

Thoma111018 51

a, Open promoters ("preset promoters") have a depleted proximal nucleosome adjacent to the transcription start site (TSS, black arrow), a feature common at constitutive genes. (NDR Nucleosome depeted region or Nucleosome Free Region). ACT, transcription activator

b, Covered promoters (also remodelling promoters) have a nucleosome adjacent to the TSS in their repressed state, a feature common at highly regulated genes. The figure depicts features more common in each contrasting promoter type, but most yeast genes blend the features shown to provide appropriate regulation. Green nucleosomes contain canonical H2A, whereas brown nucleosomes bear H2A.Z. Binding sites (BS) for transcriptional activators (ACT) are shown. These are mainly exposed for open promoters and mainly occluded by nucleosomes (in the repressed state) at covered promoters. Covered promoters typically have nucleosome positioning sequence elements of varying strength and locations that help define nucleosome positions (faded green) and promoter architecture. NDR, nucleosome-depleted region.

Chromatin Concepts at PromotersCairns, B.R. (2009). Nature 461, 193; Wallrath & Elgin (1994)

Thoma111018 52

The ARS1 (yeast origin of replication): Nuclease sensitive, non-nucleosomal ‚open‘

The URA3 Gene: • 6 positioned nucleosomes in coding region, • non-nucleosomal ‚open‘ promoter (5') • non-nucleosomal ‚open‘ 3‘end

URA3 : an example of an open (non-nucleosomal) promoter

Nuclease (Hyper-) Sensitive Regions (no nucleosomes)

Positioned Nucleosomes: • protected regions of 145 - 200 bp = footprint of histone octamer • In all cells, the histone octamers sit on the same DNA sequence of the minichromosome

Chromatin Structure of the Yeast Minichomosome YRpTRURAP Method:

1. Yeast nuclei and DNA 2. Partial digest with MNase or DNase I 3. Mapping of cutting sites by indirect endlabelling (Purify DNA, cut with restriction enzyme e.g. EcoRI; Agarose gel, Southernblot; hybridize with short probe close to EcoRI site)

ARS1 3‘

URA3

TRP1

5‘

Thoma (1986) J. Mol.Biol.

Chr

om.

DN

A

Chr

om.

DN

A

Chr

om.

MNase

2 5 6

5 1 2

7 6 8

1 0 2 4 1 2 8 0 1 5 3 6 1 7 9 2 2 0 4 8 2 3 0 4 2 5 6 0

DNA Size

Marker

Yeast

ARS1 3'

5'

TRP1

URA3

5'

3'

H

H

YRpTRURAP

RI

Thoma111018 53

PHO5: "Classic Example" of Chromatin Remodeling in a Promoter (I)

Nuclei isolated from cells that had been grown in YPDA (- Induction) or no phosphate medium (+ Induction) were digested with 3, 6, 10 and 3 U/ml micrococcal nuclease (MNase) (lanes 2-5), or with 1, 1, 2 and 4 U/mol DNase I for 20 min (lanes 7-10, respectively). DNA was isolated, cut with ApaI, separated in a 1.5% agarose gel, blotted and hybridized with probe D ("indirect endlabelling"). The ApaI site used is about 1300 bp upstream of the PHO5 gene. A control digest of free DNA with 0.05 U/ml micrococcal nuclease, subsequently cleaved with ApaI, is shown in lane 1. Lane 6 contains three double digests (BamHI, HaeII, ClaI) and lane 11 the pBR322 reference fragments. The arrow denotes a prominent fragment characteristic of the active state that is not observed with free DNA (compare lanes 1 and 2). HS, hyper sensitive site.

MNase DNaseI "Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements."Almer et al. (1986). EMBO J. 5: 2689.

TATA

high phosphate (inactive)

low phosphate (active)

HS2

UASp1 and UASp2: Upstream Activating Sequences, binding sites for Pho4 activator. UASp1 is in a hypersensitive site (HS2), not folded in nucleosomes and readily accessible for Pho4.

Thoma111018

low phosphate (active)

2. Pho4 recruits SAGA complex (containing Histone Acetyl Transferase Gcn5) 3. Gcn5p acetylates promoter region

TATA

SAGA

Gcn5

4. Gcn5p (bromodomain, binds acetylated histones) and recruits/stabilizes binding of SWI/SNF to newly hyperacetylated histones

TATA

SWI/SNFSAGA

Gcn5

5. SWI/SNF uses ATP hydrolysis to remodel promoter nucleosomes. 6. Histones are evicted (lost). 7. RNAPII and GTF bind promoter and initiate transcription TATA

Syntichaki et al. (2000) Nature, 404, 414) Barbaric (2001) Embo J, 20, 4944-4951.

1. Transcription activator Pho4 binds UASp1 in linker between nucleosomes

TATA

Pho4

high phosphate (inactve)

TATA

UASp1 UASp2

54

PHO5: "Classic Example" of Chromatin Remodeling in a Promoter (II)

Thoma111018 55

Remodeling of the PHO5 promoter occurs by histone dissociation and reassemby and not by nucleosome displacement in cis (mobility)

soluble histone pool

3. During phosphate starvation cells were shifted to galactose to express H3-FLAG and generate a soluble pool of H3-FLAG.

high phosphate (repression)

4. Phosphate was added to repress PHO5

5. Chromatin was analysed by ChIPs Promoter closure occurs very rapidly (minutes). The incorporated histones originate from the histone pool, H3-FLAG. TATA

TATA

TATA

TATA

low phosphate (actvation)

histone eviction? or nucleosome shift?

Schermer, U. J., Korber, P., and Horz, W. (2005). Mol Cell 19, 279 Strain with two differently tagged H3:, GAL-H3-FLAG expressed in glactose and H3-Myc expressed from the histone promoter in S-phase.

1.  Cells were grown in glucose and high phosphate expressing H3-Myc.

2.  Cells were starved for phosphate to induce PHO5. Starved cells are arrested in cell cycle and do not replicate. Chromatin contains only H3-Myc.

• Experiments with mutants showed that the histone chaperones Asf1 and Hir1 as well as the SWI/SNF nucleosome remodeling complex appear to be important for rapid reassembly of nucleosomes at the PHO5 promoter.

Asf1 Hir1 SWI/SNF

PHO5: "Classic Example" of Chromatin Remodeling in a Promoter (III)

Thoma111018 56

In one model (left), a combination of acetylation and chromatin remodeling directly results in the loss of Htz1-containing nucleosome, thereby exposing the entire core promoter to the GTFs and Pol II. SAGA and mediator then facilitate PIC formation through direct interactions. In the other model (right), which represents the remodeled state, partial PICs could be assembled at the core promoter without loss of Htz1. It is the binding of Pol II and TFIIH that leads to the displacement of Htz1-containing nucleosomes and the full assembly of PIC

Models of Chromatin Regulation during Transcription Initiation Li, B., Carey, M. and Workman, J.L. (2007) Cell, 128, 707-719.

Thoma111018 57

Transcribing through

chromatin

Thoma111018 58

Loss of nucleosomes during transcription of the yeast GAL10 gene Cavalli and Thoma (1993)EMBO 12,4603

Nucleosomes In genome 1. Nuclei 2. MNase digestion (limited) 3. DNA purification 3. Agarose gel: reveals nucleosomal repeat of the genome in glucose and galactose

Southern blot / Probe: GAL10 gene

glucose galactose GAL10 gene

nucleosomal repeat of genome

glucose galactose Yeast genome GAL10 gene:

Repressed in glucose Transcribed in galactose

Nucleosomes on GAL10? Southern blot, probe with defined sequence (GAL10):

Loss of a regular nucleosome repeat upon transcription; (partial) loss of nucleosomes due to transcription (?)

Thoma111018 59

Transkription Elongation - Supercoiling - Topoisomerases

RNAP DNA

Topoisomerase I migrates with RNAP II and relaxes supercoils

DNA RNAP

DNA and RNAP can't freely rotate: RNAP generates 1 positive and 1 negative supercoil /10bp

Alberts et al.

Nucleosomes contain one negative supercoil Positive supercoils might destabilize nucleosomes (?)

Negative supercoils might favour reassembly of nucleosomes (?)

Thoma111018 60

Elongating Pol II requires HATs to acetylate the nucleosome in front of elongation machinery.

The passage of Pol II causes histone displacement. Subsequently, these histones are redeposited onto the DNA behind Pol II via concerted actions of histone chaperones. Alternatively, the free forms of histones in the nucleus are also available for reassembly.

Newly deposited nucleosomes are immediately methylated by Set2.

Methylation of H3K36 is then recognized by Eaf3, which in turn recruits the Rpd3S deacetylase complex.

Rpd3S removes the acetyl marks and leaves the nucleosome in a stable state.

Methylation of H3K36 is eventually eliminated by a histone demethylase when the gene turns off.

Transient Disruption and Reassembly of Nucleosomes During Transcription Elongation Li, B., Carey, M. and Workman, J.L. (2007) Cell, 128

Histone chaperone Asf1 binds H3.H4 dimers

Thoma111018 61

The distribution of histones and their modifications are mapped on an arbitrary gene relative to its promoter (5' IGR; Inter Genic Region), ORF (Open Reading Frame), and 3' IGR.

The curves represent the patterns that are determined via genome-wide approaches (ChIPs and microarrays "ChIP on Chip").

The squares indicate that the data are based on only a few case studies. With the exception of the data on K9 and K27 methylation, most of the data are based on yeast genes.

Genome-Wide Distribution Pattern of Histone Modifications from a Transcription Perspective

Li, B., Carey, M. and Workman, J.L. (2007) Cell, 128

"General" for active genes • acetylation of H3 and H4

"General" for inactive (often "heterochromatin"): • H3 K9me and H3 K27me

Thoma111018 62

"open chromatin domains"

Thoma111018 63

Active genes are more sensitive to DNase I than inactive genes

Active genes are in an „open“ chromatin conformation

1. Nuclei2. Digest with DNaseI (limited)3. Extract DNA4. Cut with BamH1 restriction enzyme5. Gel and Southernblot6. Hybridize with probe for adult globin gene

active inactive

globin gene

control

"Open" chromatin structure of active genes

Structural transitions occur during activation and inactivation of genes.

Thoma111018 64

Chromatin structure around the Chicken Lysozyme Gene Locus

Firtel et al. In Architecture of Eukaryotic Genes, ed. G. Kahl (1988), VCH

Transcribed Region

Open Domain (DNase I sensitivity)

The domain of DNase I sensitivity and hyperacetylation is larger than the transcription unit.

Histone hyperacetylation might destabilize chromatin structures.

Acetylated Histones ChIPs (Chromatin Immuno Precipitation) with antibodies against acetylated histones (H3)

Thoma111018 65 FT Fribourg12F 66

End of lecture120309