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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