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The Evolution of a Heterochromatic The Evolution of a Heterochromatic

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The Evolution of a Heterochromatic - PPT Presentation

Domain in Drosophila Investigating the Strange Dot Chromosome Sarah C R Elgin January 2012 A collaborative investigation involving former members of the Elgin Lab Lee Silver Carl Wu TC James Joel Eissenberg Lori Wallrath Fang Lin Sun Karmella Haynes ID: 811092

genes chromosome dot heterochromatin chromosome genes heterochromatin dot chromatin dna gene fourth heterochromatic hp1 melanogaster silencing hp1a white tss

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Slide1

The Evolution of a Heterochromatic

Domain in

Drosophila

:

Investigating the Strange

Dot Chromosome

Sarah C R Elgin

January 2012

Slide2

A collaborative investigation involving:

- former members of the Elgin Lab:

Lee Silver, Carl Wu, TC James, Joel Eissenberg, Lori Wallrath, Fang Lin Sun, Karmella Haynes

-

current members of the Elgin Lab

: Nicole Riddle,

Tingting Gu, Chris Shaffer, Wilson Leung

-

modENCODE

: Gary Karpen, Mitzi Kuroda, Vincenzo Pirrotta,

Peter Park, and their colleagues

-

Faculty and students of the Genomics Education Partnership

Goal: to understand the organization and functioning of the dot

chromosome in Drosophila, an unusual heterochromatic domain.

Funding: HHMI Professors Program

NIH General Medical Sciences, National Human Genome Research Institute

Slide3

3

Minimum Haploid DNA Content - the C Value Paradox

Britten and Davidson, 1969 Science 165:349

Slide4

4

Allis et al:

Epigenetics

2007

Larger genomes reflect high levels of repeats -

retroviral and DNA transposon remnants (TEs)

Slide5

5

Considerations for Genome Sequencing

Satellite DNA, a sequence of tandem repeats, is very difficult to sequence, as there are few markers to help order

subclones

; hence

centromeric

regions of the chromosomes are usually left

unsequenced

.

Other repetitious DNA, derived from transposable elements, also causes difficulties; because one finds nearly identical sequences located in different regions of the genome, mistakes can be made in assembling sequence data. High quality discrepancies can identify these.

Much of the

repetitous

DNA is packaged into heterochromatin, which maintains these regions in a compact and

transcriptionally

silent form.

However, in many higher organisms, protein-coding genes are found embedded in repetitious DNA. Check out your favorite human gene on the UCSC Browser by taking off

RepeatMasker!

SCR Elgin

Slide6

Coding exons

1.5%

Conserved noncoding - regulatory? 3.5%

Human Genome 3 Gb

~2 m/cell !

Key Questions

:

Is it junk or garbage?

How is DNA packaged into a nucleus?

How is silencing maintained – while

allowing appropriate transcription ?

Eukaryotic genomes are very large – and most of that DNA is non-coding!

TEs –

retroviruses,

DNA transposons

Slide7

What determines phenotypes?

It

s not just your DNA….

Phenotype

Development

Environment (diet)

(grey bars = folate)

Epigenetics

?

Genotype

(Waterland and Jirtle 2003)

Slide8

Chromatin structure = epigenetics !

What sets and maintains tissue-specific gene expression patterns? Differences are heritable through mitosis, but

independent of DNA sequence

. DNA modification (mC)Chromatin structureNuclear localization

It

s all about silencing!

How is chromatin assembled?

When, where and how does

gene silencing occur?

Incorrect silencing can lead

To genetic disability, as seenIn Fragile X syndrome

Zoghbi and Beaudet 2007

Fragile X Foundation

Slide9

Felsenfeld

et al. Nature 2003, 421: 448

Chromatin formation:

First step - packaging in a nucleosome array

Second - differential packaging into heterochromatin & euchromatin

DNA

Chromatin

Lodish

et.al

., Molecular Cell Biology, 4th Edition

Chromosome

(metaphase)

Histone

protein core

Slide10

10

Electron Micrograph of Chromatin Fibers

(rat thymus nucleus)

Olins

et. al., 1975 J. Cell

Biol

, 64:528

0.1

m

m

Slide11

11

A eukaryotic chromosome made out of self-assembling 70A units, which could perhaps be made to crystallize, would necessitate rewriting our basic textbooks on cytology and genetics! I have never read such a naïve paper purporting to be of such fundamental significance. Definitely it should not be published anywhere!

Anonymous review of paper submitted by C.F.L. Woodcock, 1973, showing EM pictures of nucleosome arrays.

Quoted in “Chromatin” by K.D. van

Holde

, 1989

Slide12

12

The Structure of the Nucleosome Core

Rhodes, 1997 Nature 389:231, after

Luger et. al., 1997 Nature 389:251

Resolution: 2.8 Å

Half of the nucleosome structure is shown

One turn of the DNA helix is visible (73 bp)

View is down the superhelix axis

Protein - DNA contact: white hooks

Slide13

DNA packaging domains

EuchromatinLess condensedChromosome armsUnique sequences;

gene rich

Replicated throughout SRecombination during meiosis

Heterochromatin

Highly condensed

Centromeres and telomeres

Repetitious sequences; gene poorReplicated in late S

No meiotic recombination

Transcriptional activators

Hyper-acetylated histone tail

Heterochromatin Protein 1 complex

Hypo-acetylated histone tail; methylated H3/K9

Slide14

Heterochromatin formation – silencing counts!

How is heterochromatin organized and packaged to promote silencing?

The fourth chromosome appears heterochromatic

but has ~80 genes:

- do these genes have unusual characteristics?

- how has the chromosome evolved?

-- how do these genes function?

1

2

Slide15

Short life cycle, easily maintained: good

genetic tools Polytene chromosomes: excellent cytologyBiochemical approaches

Simple genome, good

reference sequence PEV – reporter for gene silencing, heterochromatin formation Metazoan useful for behavioral, developmental and human disease research

expressed silenced

euchromatin heterochromatin

Fruit Flies!

Mary Lou

Pardue

, MIT

Slide16

16

Using a

white

transgene to sample chromatin environments

mobilize P element

by crossing to stock

with

transposase

inject

transposon

carrying

white

gene

P[

white

+

]

white

67c23

insertion into

euchromatin

(99%)

insertion into

heterochromatin

( 1%)

Elgin Lab

Slide17

Transposition of a P element reporter allows sampling

of euchromatic and heterochromatin domains

X

2L

3L

2R

3R

4

Silenced

1%

Active

99%

Wallrath and Elgin, 1995

And the Y chromosome

Slide18

Assessing chromatin structure-

same gene, different environmentsAnalysis based on nuclease digestion of chromatin

The euchromatic

hsp26

transgene:

- DH sites: accessibility at the TSS, upstream regulatory region

- irregular nucleosome array

The heterochromatic

hsp26

transgene:

- loss of DH sites

- regular nucleosome array

Slide19

Looking for heterochromatic proteins by immunofluorescent staining of the polytene chromosomes: discovery of HP1a

C

C

HP1

Phase

James & Elgin,1986; James et al 1989

Slide20

Heterochromatin-associated gene silencing is dependent on HP1

Eissenberg et al, 1990, PNAS 87: 9923

Mutations in

gene for HP1a

Mutations recovered by T Grigliatti as suppressors of PEV.

Dosage dependent response.

Slide21

HP1 interacts with both the modified histone H3K9me2/3

and the modifying enzyme

HP1:

Shadow

Chromo

SU(VAR)3-9

Histone 3

methyl-Lys9

H3 K9 methyl

transferase

[(SU(VAR)3-9 identified in screen by Reuter;

H3 interaction first shown from work in mammals – Jenuwein, Kouzarides;

demonstrated in flies by Imhof.]

Slide22

Model for spreading of heterochromatin

Slide23

Establishing silencing: a multi-step process

w

m4

reporter (screens by Reuter, Grigliatti, others)

Loss of euchromatin marks

Gain of heterochromatin marks

Slide24

Heterochromatin formation on the dot chromosome…

The fourth chromosome appears heterochromatic

but has ~80 genes:

- do these genes have unusual characteristics?

- how has the chromosome evolved?

- how do these genes function?

2

Slide25

The Drosophila melanogaster

fourth chromosome exhibits an amalgam of euchromatic and heterochromatic properties (HP1a association)

C

C

HP1

Phase

James & Elgin,1986; James et al 1989

Heterochromatic properties:

late replication, lack of recombination

high repeat density (30%)

antibody staining of HP1, H3K9me2/3

But…

the fourth has ~ 80 genes in distal 1.2 Mb

these genes are transcriptionally active!

Slide26

Most

hsp70-white

reporters exhibit variegation

on insertion into the fourth chromosome

2-M1021

39C-12

2-M390

39C-52

Sun et al 2004; Riddle et al 2007

Slide27

Use comparative genomics to learn more

about heterochromatic domains, analyzing the

dot chromosomes and a control euchromatic

region of Drosophila genomesOur GEP Research Goal:

FlyBase

:

http://flybase.org

Reference

Status

Completed

Annotation

Sequence Improvement

New Project

Slide28

Genomics Education Partnership (GEP)

Partners are generally PUI schools; faculty join by

attending one-week workshop at WU. Shared

work organized on GEP website.

Slide29

The

D.melanogaster

&

D. virilis

dot chromosomes

are 25% - 30% repetitious DNA

(typical – but up to 80% in

D. ananassae)

D mel D vir D mel D vir D vir D mel D vir D vir D vir

Leung et al 2010

Slide30

Dot chromosome genes: introns are larger, exons show less codon bias

Heterochromatic

Dot

Euchromatic

D. melanogaster

Het.

D. melanogaster

Euch.

D. virilis

Euch.

D. melanogaster

Dot

D. virilis

Dot

Intron

Size

Codon

Bias

Leung

et al

.

2010 Genetics 185:1519-1534

Slide31

Almost all of the same genes are present (27/28), but rearrangements within the chromosome are common!

Initial analysis of Drosophila virilis

dot chromosome fosmids

Slawson

et. al., 2006 Genome Biology, 7(2):R15.

Slide32

Comparison of gene order and orientation

72 genes on both the

D.

virilis and D. melanogaster

dots.

A minimum of 33 inversions are needed to convert order and orientation!

D. virilis

D. melanogaster

Leung et al 2010

Slide33

dot:

D. virilis

dot:

D. melanogaster

CG9935

CG5367

rho-5

CG4038

CG11076

CG1732

CG5262

CG11077

dot

Wanderer

genes move between the dot chromosome and a

euchromatic site in the long arms; they adopt the

properties (gene size, codon bias) of their local environment

Leung et al 2010

Slide34

Is there a homologous gene in D. melanogaster

?Is it on the dot chromosome? Are all of the isoforms found in D melanogaster

present?

How many exons? Any unusual splice sites?What is the order and orientation of genes compared to D. melanogaster

?

Are there repetitious elements nearby?

Check out your gene on FlyBase – what is the pattern of expression in

D. melanogaster

? Has a function been described?

Many dot chromosome genes are expressed at a high level - how can

this occur in a heterochromatic domain?

Some things to look for while annotating dot chromosome genes….

Slide35

Ch

romatin

I

mmuno-

p

recipitation - ChIP

(cells or nuclei)

qPCR

ChIP-chip*

ChIP-seq

1. Crosslink proteins to DNA

2. Isolate chromatin and sonicate

3. Incubate with antibody

4. Isolate AB/chromatin complexes

5. Isolate DNA from complexes

Slide36

Mapping chromatin marks by ChIP-chip:

Chromosome arm 3L shows a distinct shift between heterochromatin and euchromatin

HP1a

Su(var)3-9

H3K9me2

H3K9me3

genes

genes

Euchromatin

Heterochromatin

Centromere

S2 cells

Euchromatin / heterochromatin transition point from Flybase

Pink boxes show significant enrichment (0.1% false discovery rate)

Enrichment (log intensity ratio values)

Slide37

Chromosome 4 is largely heterochromatic, but shows distinct

peaks of H3K4me2/3, indicating transcription start sites

HP1a

H3K9me2

H3K9me3

H3K4me2

genes

Centromere

Telomere

Enrichment (log intensity ratio values)

Slide38

A model of 9 chromatin states, based on clustering of histone modification marks, identifies large-scale genomic domains

Heterochromatin (dark blue; H3K9me3)

TSS (red; H3K4me3 rich)

 Polycomb (grey; H3K27me3 rich)

Slide39

10 Mb

chr3L

BG3 cells, chromatin states:

Pericentric heterochromatin

1

2

3

4

5

6

7

8

9

chr4

500 kb

chr4

Red

Variegating

An expanded view of the fourth chromosome reveals

TSS (state 1, red) and Pc (state 6, grey) domains

interspersed within heterochromatin (states 7 & 8, blue).

Slide40

Might fourth chromosome genes function early, and be silenced later? No, fourth chromosome genes show a variety of

expression patterns, including expression in the adult

S Celniker, modENCODE

Slide41

Most 4

th

chromosome genes lie in heterochromatic space (blue),

but active genes achieve state 1 (red) at the TSS

1360

Slide42

Active fourth chromosome genes show depletion of HP1a and

H3K9me3 at the TSS, but enrichment across the body of the gene

Transcription levels are similar to

euchromatic

genes!

Average enrichment

TSS-relative position

RNA pol II

H3K4me3

HP1

H3K9me2

H3K9me3

chromosome 4

Slide43

The fourth chromosome: a repeat rich domain with

“heterochromatic” genes

1360

Future: try to determine what feature drives 4

th

chromosome

gene expression that is absent from euchromatic genes (hsp70).

Slide44

Eight new genomes

Expanded dot

chromosomes?

44

Slide45

Heterochromatin formation on the dot chromosome…

Heterochromatin formation changes chromatin at

the nucleosome level, eliminating HS sites at the TSS of euchromatic genes; silencing is dependent on HP1a

Fourth chromosome genes are larger, have more introns, and less codon bias than euchromatic genes

Fourth chromosome genes show high levels of HP1a and H3K9 methylation over the body of the gene,

but maintain access at the TSS.

Next steps: what makes fourth chromosome

genes robust? Lets look for fourth chromosome motifs!

1

2

Slide46

Question Slides

Slide47

A role for POF? A different configuration for HP1a?

Are there chromosomal proteins unique to the fourth chromosome? Yes – POF (Painting of Fourth) is uniquely associated with active genes on the fourth chromosome

(

See J Larsson,

PLoS

Genet. 11:e209 for more on POF)

Green = HP1a Red = POF

Slide48

Drosophila melanogaster: 1/3 heterochromatin.

Pericentric heterochromatin is under-replicated in polytene chromosomes; the arms fuse in the chromocenter

Drawing of polytene chromosomes modified from TS Painter, 1934, J. Hered 25: 465-476.

Slide49

HP1 sequence from Drosophila, mouse, human and mealy bug identifies chromo domain & chromo shadow domain

Clark and Elgin, 1992 Nucleic Acids Res. 20:6067

HP1 from mammals can rescue mutations in flies and yeast!

Slide50

Position Effect Variegation in Drosophila:

an assay for heterochromatic packaging

white

Wild Type

Inversion

HP1 is a trans-acting modifier of PEV

E(var)

(3 copies HP1)

Su(var)

(1 copy HP1)

i

Eissenberg et al

Slide51

The heterochromatic hsp26

transgene: - loss of accessibility at the TSS when in heterochromatin

- reversed in an HP1 mutant background

Cryderman et al 1999 (Wallrath & Gilmour labs)

Slide52

Chromosome 4 short arm is a unique heterochromatic domain

Haynes et al. 2007

Slide53

Translocation away from the chromocenter results in loss of silencing – spatial organization plays a role

Slide54

Define chromatin states by K-means clustering

(using enrichment values for 1 kb chromatin fragments)

Select

k

means as starting points.

Assign each data point to closest mean

.

The

centroid

of each cluster is the new mean

.

Repeat the last two steps.

H3K9me3

H3K4me3

Slide55

Chromatin states are highly interspersed-a folded view of the chromosome (Bg3 cells)

Polycomb

Pericentric heterochro-matin

Slide56

Chromatin states reveal cell type specific patterns

(note facultative heterochromatin – light blue)

Chromosome 3L

BG3

S2

Slide57

A folded view of the chromosome reveals TSS and Pc states within chromosome 4

(Bg3 cells)

Pericentric heterochromatin

4th chromosome

Slide58

Chromosome 4 shows a distinct subset of Polycomb sites in a cell-type specific pattern

H3K9me3

(S2 cells)

H3K9me3

(Bg3 cells)

Polycomb

(S2 cells)

Polycomb

(Bg3 cells)

genes

genes

Slide59

A subset of 4

th

genes is associated with Polycomb;

these domains are permissive for reporter expression

(red eyed fly).

Slide60

Heterochromatin formation on the dot chromosome…

How are specific domains targeted for

heterochomatin formation?

Repetitious elements such as

1360

are targeted by a mechanism involving the RNAi system

3

Slide61

hsp70-white

5

P

3

P

FRT

FRT

1360

Analysis of chromosome 4 identified

1360

as a target.

P

[

1360, hsp70-w

] silencing depends on the

reporter

s position in the genome

Haynes et al 2006 Curr Biol 16: 2222

Slide62

FLP-mediated removal of

1360

results in loss of silencing

+ FLP recombinase

hsp70-white

5

P

3

P

FRT

FRT

1360

hsp70-white

5

P

3

P

FRT

1360

excised

FLP14

FLP16

FLP4

FLP5

Haynes et al 2006 Curr Biol 16: 2222

Slide63

Mutations in RNAi components impact PEV

spn-E (hls)

Dmp68/Lip

dFMR1

Results from Birchler, Elgin, Schedl, others;

note also esiRNA pathway, Siomi, Hannon & others

Slide64

y w

; 118E-10/+

y w

; 39C-12/+

piwi

aub

hls

Mutations in RNAi components

piwi, aubergine

and

homeless

suppress PEV

y w

; 118E-10/+

y w

; 39C-12/+

piwi

1

+

aub

QC42

+

hls

∆125

+

+

+

B Leibovitch in Pal-Bhadra et al, 2004

Slide65

A tentative model for heterochromatin targeting –

HP1a – PIWI interaction (piRNA)

PIWI

transposon

HP1

Aub

piRNA

Ago3

PIWI

Heterochromatin

PIWI (binds piRNA) is an argonaute family member

and nuclear protein which interacts with HP1a

Cytoplasm

Nucleus