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Genomes, Heterochromatin,and the Dot Chromosome:
Comparative Genomics in DrosophilaSarah CR Elgin Lab
Genomics Education Partnership TA WorkshopWashington University in St. Louis
August, 2006
Minimum Haploid DNA Content - the C Value Paradox
from Saenger, after Britten and Davidson
Lack of Relationship Between Amount ofDNA and Organism Complexity
Alberts et. al., #3
In eukaryotes, genes are often much largerthan the coding region
Alberts et. al., #3
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Composition of the Mouse Genome:Frequency Distribution
after Britten and Kohne, Science 161, 529 (1969)
C0t curves of different DNA samples.Reassociation kinetics tells us how many different sequences are present.
Britten and Davidson, Carnegie Report
Experimental C0t CurveSome DNA sequences are repeated in the genome.
Davidson abd Hough, PNAS 63, 342. (1969)
Considerations for Genome Sequencing1. Satellite DNA is very difficult to sequence, as there are few
markers to help order subclones; hence centromeric regionsof the chromosomes are usually left unsequenced.
2. Middle repetitious DNA also causes difficulties; because onefinds nearly identical sequences located in different regionsof the genome, mistakes can be made in assemblingsequence data. High quality discrepencies can identify these.
3. Much of the repetitous DNA is packaged in heterochromatin,which maintains these regions in a compact andtranscriptionally silent form.
4. However, in many higher organisms, the protein-codinggenes are found embedded in repetitious DNA. Check outyour favorite human gene on the UCSC Browser by taking offRepeatMasker!
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Eukaryotic cells-coping with large genomes
Assembly into heterochromatin leads to- effective packaging- gene silencing
Lodish et.al., Molecular Cell Biology, 4th Edition
Mammals3,000,000,000 bp
2 meters of DNA /cell
Only 2% codes for proteins!
Much of the DNA isrepetitious
Felsenfeld et al. Nature 2003, 421: 448
CHROMATINDNA and its associated proteins.
The nucleosome is the basicsubunit of chromatin.• DNA: 167 bp + linker• Histone core: 1 H3/H4 tetramer + 2 H2A/H2B dimers• Linker histone H1 or H5
DNA
Chromatin
Lodish et.al., Molecular Cell Biology, 4th Edition
Chromosome(metaphase)
Histoneprotein core
Electron Micrograph of Chromatin Fibers (rat thymus nucleus)
Olins et. al., J. Cell Biol, (1975) 64, 528-537
0.1 µm
- Anonymous review of paper submitted by C.F.L.Woodcock, 1973, showing EM pictures ofnucleosome arrays
“A eukaryotic chromosome made out of self-assembling 70A units, which could perhaps bemade to crystallize, would necessitate rewritingour basic textbooks on cytology and genetics! Ihave never read such a naïve paper purporting tobe of such fundamental significance. Definitely itshould not be published anywhere!”
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The Structure of the Nucleosome Core
Rhodes, Nature (1997) 389, 231-233, after Luger et. al., Nature (1997) 389, 251-260
Resolution: 2.8 Å
Half of the nucleosome structure isshown
One turn of the DNA helix is visible(73 bp)
View is down the superhelix axis
Protein - DNA contact: white hooks
The Histone Octamer
Rhodes, Nature (1997) 389, 231-233
The complete histone octamer inthe absence of DNA.
The unstructured N-terminaltails are extensively modified.
Color code:
H2A
H2B
H3
H4
DNA packaging domains
• Euchromatin– Less condensed– Chromosome arms– Unique sequences;
gene rich– Replicated throughout S– Recombination during meiosis
• Heterochromatin– Highly condensed– Centromeres and telomeres– Repetitious sequences;
gene poor– Replicated in late S– No meiotic recombination
HATs
Transcriptional activators
Hyper-acetylated histone tail
Heterochromatin Protein 1 complex
Hypo-acetylated histone tail; methylated H3/K9
10 DAYS
Fruit flies are inexpensiveand easy to culture
•Short life cycle
•Easily visible phenotypes
•Polytene chromosomes
•Simple genome
•Fully sequenced genome
•Metazoan useful forbehavioral, developmental
and human diseaseresearchexpressed silenced
wt gene mutant
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Drosophila melanogaster chromosomes
modified from TS Painter, 1934, J. Hered 25: 465-476.
Distribution of Heterochromatin Protein 1 (HP1)
C C
HP1Phase
Comparisons of deduced amino acid sequences fromHP1 genes of D. virilis, D. melanogaster, mouse,
human and mealy bug
Position Effect Variegation in Drosophila
whiteWild Type
Inversion
Trans-acting modifiers of PEV:
E(var):Su(var):
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Mutations in the gene for HP1suppress PEV
HP1 distribution is coincident with H3-mK9but opposed to H4-acK8
Maintenance of heterochromatin
HP1: ShadowChromo
SU(VAR)3-9Histone 3methyl-Lys9
H3 methyltransferase
Model for spreading of heterochromatin
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PEV transition: loss of euchromatin marks
-and probably others!
Euchromatin Heterochromatin
PEV transition: gain of heterochromatin marks
- and probably others!
Euchromatin Heterochromatin
Using a white transgene to samplechromatin environments
injecttransposon
mobilize P elementby crossing to stockwith transposase
white67c23
P[white+]
insertion intoeuchromatin
(99%)
insertion intoheterochromatin
( 1%)
P-element Construct
T-1917
hsp26-plant hsp70-white
+490
P element transposon: variegating phenotype
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The fourth chromosome has interspersedheterochromatic and euchromatic domains
2-M1021 39C-12 2-M390 39C-52
200 kb
Local genomic deletions and duplications producea switch in eye phenotype
Silencing occurs within ~10 kb of 1360 fragments
Model: repetitious element 1360 as an initiator of heterochromatin
RNAi - dsRNA triggers gene silencing
Dicer
RISC
NucleosomeMethyl groupmRNA degradation
PTGSChromatin modification
or DNA methylation (TGS)
dsRNA
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Can we find 22 nt siRNA’s for 1360?
Size separate RNA in agel by electrophoresis,transfer the RNA to anylon membrane
Generate plus andminus strand probes for1360;
siRNA’s from bothstrands show up,suggesting they camefrom dsRNA
+ RNA
- RNA hsp70-white 5’P3’P FRT FRT1360
P[1360, hsp70-w] PEV depends on reporter’s position in the genome
FLP-mediated removal of 1360 suppresses PEV
+ FLP recombinase
hsp70-white 5’P3’P FRT FRT1360 hsp70-white 5’P3’P FRT
1360 excised
FLP14 FLP16 FLP4 FLP5
RNAi component mutations act as Su(var)’s
Heterochromatincomponents
RNAi components
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An RNAi model for targeting heterochromatin
HP1
PIWI1360
Distribution of Heterochromatin Protein 1in D. melanogaster
C C
HP1Phase
No association with HP1 indicates that thedot chromosome in D. virilis is euchromatic • GOAL: Students work as a research team through a large-scale
sequencing project to finish and annotate dot chromosome
• PROCESS: Data generation, finishing and quality control incollaboration with the WU Genome Sequencing Center; completeannotation and analysis collaborating with WU Computer Sciencefaculty.
Bio 4342: Research Explorations in Genomics
SpringSemester,2004
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Progress, 2004 - 2006
• Fosmids (~40 kb each) are selected from agenomic library
• Each student sequences and annotates one fosmid• Remaining gaps sequenced using a PCR-based
method, whole assembled (summer work)
Finished sequence, 2004, 2005
D. virilis dot chromosome, reference strain
Remaining gaps
(PCR, summer 06)
~12kb 15kb 8kb 9kb12kb 13kb 3kb
Finished fosmids 2006
Bio 4342, Spring 2005
Bio 4342, Spring 2005
Initial analysis of D. virilisdot chromosome fosmids
Almost all of the same genes are present (27/28), butrearrangements within the chromosome are common!
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Dot chromosomes genes have larger introns due to repetitious DNA
Legend: Perc. D. virilis DotPerc. D. virilis Other
Perc. D. melanogaster DotPerc. D. melanogaster Other
Intron Size Distribution: Dot Chromosome versus Other Chromosomes
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 200 400 600 800 1000 1200 1400
Intron Size
% I
ntro
ns T
his
Siz
e o
r S
maller
Other Chromosomes
Dot Chromosomes
Repeat Density Comparison of D. melanogaster and D. virilis using RepeatMasker/Superlibrary with Classification
0
5
10
15
20
25
30
DM: Dot DV: Dot DM: Arms DV: Arms
Species: Chromosome
Rep
eat
Den
sit
y (
%)
Dot chromosomes of both species have a high density of repetitious elements; types of elements differ
1360 ElementsDINESOther DNA Transposons
UnknownSimple RepeatsRetroelements
To compare finished sequence from the dotchromosomes of different Drosophilaspecies: next targets, D. erecta andD. mojavensis
Our research goal: Summing up…..Eukaryotic genomes are
- unexpectedly large, complex,- contain a high percentage of repetitious sequences.
Much of the genome is packaged in heterochromatin:- formation may be targeted by repetitious sequences
triggering an RNAi response;- leads to alternative chromatin packaging;- results in gene silencing;- can impact nearby genes.
Epigenetic inheritance and spreading are achieved by- a protein (e.g., HP1) or protein complex that interactswith a modified histone and with the modifying enzyme.
Organization of the genome, patterns of repetitious DNA arecritical for genome function.
- can be studied through analysis of the dot chromosome.
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AcknowledgementsWashington University
Elgin LaboratoryPast: T.C. James, Joel Eissenberg, Lori Wallrath,
Fang-Lin Sun, Boris Leibovitch, Cory Simpson, Amy Caudy, Libby Slawson, Michelle Itano
Present: Karmella Haynes, Jo Wuller, Brent Brower-Toland, Kate Huisinga, Chris Shaffer, Wilson Leung,
Taylor Cordonnier, Students of Bio 4342,Ale Figueroa-Clarevega, Stephen McDaniel, Shachar Shimonovich, Amy Wu
Elena Gracheva, Nicole Riddle, Gena Bloth,
CollaboratorsJeremy Buhler, WU Computer Science
Elaine Mardis, members of the WU Genome Sequencing CenterDavid Lopatto, Grinnell College
Financial support: NIGMS, NIH; HHMI