The Strange Case of the Dot Chromosome of Drosophila

Sarah C R Elgin Bio 4342

Copyright 2014, Washington University

The Drosophila melanogaster fourth chromosome exhibits an amalgam of heterochromatic and euchromatic properties

C C

HP1Phase

Jam

es e

t al,

Mol

Cel

l Bio

198

6

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

HP1 has a banded pattern on chromosome 4; couldthe genes lie in HP1-depleted domains?

HP1

HP2 Merge

Shaffer et al 2002 PNAS 99: 14332

4th chr

Merge

Insertion sites resulting in a variegating phenotype identify heterochromatin domains

X

2L

3L

2R

3R

4

Silenced1%

Active99%

Wallrath and Elgin, 1995

Can we map heterochromatic domains on the fourth? Are there permissive domains on the fourth that allow full expression? Screen for lines giving variegating or red-eye phenotype with insertion sites on the fourth.

The fourth chromosome has interspersed silencing and permissive domains

2-M1021 39C-12 2-M390 39C-52

Each triangle indicates a line carrying a single P element reporter inserted at that site, with the eye phenotype shown, red or variegating, indicating a permissive (euchromatic?) or silencing (heterochromatic) environment.

C T

Sun et al 2004 Mol Cell Bio 24: 8210

But most fourth chromosome genes lie in heterochromatic domains

2-M1021 39C-12 2-M390 39C-52

200 kb

What are the sequence characteristics of the contrasting domains?Sun et al 2004 Mol Cell Bio 24: 8210

Local genome deletions and duplications produce a switch in eye phenotype

Element 1360OTHER TE’S

MAP (200 kb)

DELETIONS

DUPLICATIONS

Sun et al 2004 Mol Cell Bio 24: 8210

Mobilization of the P element

Local transposition

Local deletion

Local duplication

Illustration by Cory Simpson

Local deletions and duplications can change the distance between the P element and cis-acting

determinants of heterochromatin formation

Illustration by Cory Simpson

Cis-acting determinant

Element 1360OTHER TE’S

MAP (200 kb)

DELETIONS

DUPLICATIONS

Proximity to the 1360 element appears to be critical

Sun et al 2004 Mol Cell Bio 24: 8210

Most variegating reporters in the Hcf region are close to a 1360 element, a remnant of a DNA transposon

kb t

o n

ear

est

136

0

Reporter

Riddle et al, Genetics, 2008 & Sun, et al., MCB, 2004

1360 is NOT the only target for heterochromatin formation on the fourth chromosome

CG

2316

CG

2165

CG

3199

2

bt

CG

3202

1

CaM

KII

sv

CG

3201

7

Cap

s

AT

Psy

n-

hsp70-white 5’P3’P hsp26-pt

8-M2944-M1030

8-M1

1360s

Other TEs

Inserts

500kb 1Mb

101F 102A 102B 102C 102D 102E 102F

20kb

Genes

Syt

7

bip2

zfh2

39C-12

8-M22

7-M201

7-M1015/7-M547

7-M484/7-M1365

7-M1061

Riddle et al 2007, Genetics 178: 1177

Repetitious elements are recognized as such and silenced, an epigenetic change

• Petunias

• Extra copy of pigment gene leads to silencing by heterochromatin formation

• Important for genome stability/ transposon control

(Napoli et al. 1990)

Parent line

+transgene

+transgene

from N. Riddle

Use comparative genomics to learn more about heterochromatic domains, analyzing the dot chromosomes and a control euchromatic region of Drosophila genomes

Our GEP research goal:

FlyBase: http://flybase.org

Reference

Status

Completed

Annotation

Sequence Improvement

New Project

Muller F Elements

Muller D Elements

F elements have high levels of transposable elements (TEs)

Total CDS length

Size (bp)

D. melanogaster Muller F

D. erecta Muller F

D. mojavensis Muller F

D. grimshawi Muller F

D. melanogaster Muller D (base)

Number of CDS

D. erecta Muller D (base)

D. erecta Muller D (extended)

D. erecta Muller D (telomere)

D. mojavensis Muller D (base)

D. grimshawi Muller D (base)(GLEAN-R)

Count

F elements have larger genes with more exons

F element genes have a lower melting temperature

Translation start relative position

Me

dia

n 9

bp

se

que

nce

me

lting

tem

pera

ture

D. melanogaster: Muller F

D. erecta: Muller F

D. mojavensis: Muller F

D. grimshawi: Muller F

D. melanogaster: Muller D (base)

D. erecta: Muller D (base)

D. mojavensis: Muller D (base)

D. grimshawi: Muller D (base) (GLEANR)

D. erecta: Muller D (extended)

D. erecta: Muller D (telomere)

Distribution of Nc Distribution of CAI

Nc CAI

D. melanogaster Muller F

D. erecta Muller F

D. mojavensis Muller F

D. grimshawi Muller F

D. melanogaster Muller D (base)

D. erecta Muller D (base)

D. erecta Muller D (extended)

D. erecta Muller D (telomere)

D. mojavensis Muller D (base)

D. grimshawi Muller D (base) (GLEANR)

F element genes show low codon bias

Nc = deviation from uniform codon usage; CAI = deviation from codon usage observed in species.

CAI CAI CAI CAI CAI

Muller F elements Muller D elements

D. melanogaster D. erectaD. melanogaster

(base)D. erecta

(base)D. erecta

(extended)

D. erecta (telomere)

30

40

50

60

0.0 0.2 0.4 0.6 0.8

Nc

30

40

50

60

0.0 0.2 0.4 0.6 0.8

30

40

50

60

0.0 0.2 0.4 0.6 0.8

30

40

50

60

0.0 0.2 0.4 0.6 0.8

30

40

50

60

0.0 0.2 0.4 0.6 0.8

30

40

50

60

0.0 0.2 0.4 0.6 0.8

Nc

D. mojavensis D. grimshawi

30

40

50

60

0.0 0.2 0.4 0.6 0.8

D. mojavensis (base)

30

40

50

60

0.0 0.2 0.4 0.6 0.8

30

40

50

60

0.0 0.2 0.4 0.6 0.8

D. grimshawi (base) (GLEANR)

30

40

50

60

0.0 0.2 0.4 0.6 0.8

Codon bias measurements indicate that the D. grimshawi F is under stronger positive selection than other F elements

Almost all of the same genes are present, but rearrangements within the chromosome are common!

Initial analysis of Drosophila virilisdot chromosome fosmids

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

Synteny analysis of the D. melanogaster, D. virilis, and D. mojavensis F elements shows large numbers

of gene rearrangements

Species

(from / to)

Est. # Inversions

# Shared Genes

D. mel / D. vir 32 73

D. mel / D. moj 31 72

D. vir / D. moj 7 79

Relative gene order and orientationD. mel

D. vir

D. mel

D. moj

D. vir

D. moj

Inversion step

Do clusters of genes inthe same chromatinstate stay together?Check with H3K9me2H3K27me3, H3K4me2for D. mojavensis

CG9935

CG5367rho-

5

CG4038

CG11076

CG1732

CG5262

CG11077

dot

PRYY chromosome

CG9935

yellow-h

Or13a

CG11076

Or13a

Drosophila subgenusD. melanogaster D. mojavensis D. grimshawi

Sopho. Dros. Sopho. repleta

Sopho. Hawai. Dros.Movement:

W Leung, WU, 2012

Wanderer genes have moved to/from the dot: Het Euchr

  Drosophila subgenusGene D. mel D. moj D. vir D. gri

CG11076 F A A ECG11077 F A A ACG1732 F D D DCG9935 F E B Byellow-h F A F FCG4038 C F F FCG5262 D F F FCG5367 B F F Frho-5 B F F FOr13a A F scaffold

13050E

PRY chrY F chrY chrY

Wanderer genes, Muller element locations

*scaffold_13050 in D. virilis has not been assigned to a Muller element

Time line? Problem – low sample size

W Leung, WU 2012

F

F

F

F

The D. mojavensis F element has a hot spot for wanderer genes

F

F

F

F

F

F

F

F

F

F

C

C

C

F

F

D

D

D Y

Y

Y

Y

Y

B

B

B

A

A

A

A

scaffold_13050

New Drosophila genomes available from modENCODE: several species with expanded Muller F elements

D. ananasse – Sanger sequencing, but fosmid library no longer available;New species – Solexa and 454 sequencing, no libraries.

GenScan predictions proved to be 1 orthologue and 4 pseudogenes, all from chromosomes 2 & 3

Dot matrix view from BLASTx alignment of Virilizer protein (predicted feature 3) – note inverted repeats

Estelle Huang, WU, 2012

Wolbachia DNA from D. ananassae endosymbiant (top) accounts for many of the unknown repeats (bottom)

GEP plans: continue D. ananassae, do other species with expanded F after long-read sequencing becomes available.

Est

elle

Hua

ng

E Huang & W Leung, WU, 2012

The chromatin state map shows chromosome four to be heterochromatic

1Mb

Mapping the nine chromatin states onto the Drosophila genome (Bg3 cells).

Karchenko et al 2011 Nature 471: 480

Expression levels of genes in the different genomic domains are similar

Riddle et al. 2012 PLoS Genet. 8:e1002954

ChIP mapping shows that the fourth chromosome is largely heterochromatic, but has islands of H3K4me2

HP1a

H3K9me2

H3K9me3

H3K4me2

genes

Centromere Telomere

Rid

dle

et a

l 201

0, G

enom

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es

Active fourth chromosome genes show depletion of HP1a andH3K9me3 at the TSS, but enrichment across the body of the gene

Transcription levels are similar to euchromatic genes!

Ave

rage

enr

ichm

ent

TSS-relative position

RNA pol II H3K4me3 HP1 H3K9me2 H3K9me3

chromosome 4

Rid

dle

et a

l. 20

11 G

enom

e R

es 2

1:14

7-63

The fourth chromosome: a repeat rich domain with “heterochromatic” genes

1360

Future: try to determine what feature drives 4th chromosome gene expression that is absent from euchromatic genes (hsp70).

W L

eung

& S

CR

Elg

in

10 Mb

chr3L

BG3 cells, chromatin states:

Pericentric heterochromatin

1 2 3 4 5 6 7 8 9

chr4

500 kb

chr4

RedVariegating

An expanded view of the fourth chromosome: variegating reporters lie in domains that are red (#1, TSS) or blue (#7/8, heterochromatin);

red-eye reporters lie in grey domains (#6, Polycomb) in at least one cell type

Slid

e fr

om W

Leu

ng

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

Riddle et al. 2012 PLoS Genet 8:e1002954

A subset of 4th genes is associated with Polycomb; these domains are permissive for reporter expression

(red eyed fly).

W Leung & SCR Elgin

* * ** * *

POF Painting of Fourth

Active genes, TSSActive genes, bodySilent genesPC domains

Are there unique features of fourth chromosome genes? POF marks the 5’ end and gene bodies

(See J Larsson, PLoS Genet. 11:e209 for more on POF)

Green = HP1aRed = POF

Riddle et al. 2012 PLoS Genet 8:e1002954

Chr. 3L

Chr. 4

pofD119

wt

pofD119

wt

Loss of POF (null)

• Loss of HP1a from chr 4 gene bodies

• Maintain HP1a in pericentric heterochromatin, repeat clusters

HP1a ChIP

Rid

dle

et a

l. 20

12 P

LoS

Gen

et 8

:e10

0295

4

A speculative model…two methods of assembly with HP1a on the fourth:

other partners?

genes repeats no pausing silencingtranscript elongation

Note: EGG &SU(VAR)3-9are both HMTs

Both assemblies may be required to control TE remnants

Rid

dle

et a

l. 20

12 P

LoS

Gen

et 8

:e10

0295

4

Conclusions• The fourth chromosome of D. melanogaster is largely heterochromatic

-- ~30% repetitious DNA; higher in D. ananassae;– But ca. 80 genes in 1.2 Mb, a normal gene density (most species);– Ten-fold higher levels of repetitious sequences around genes

• Incomplete transposition of the P element on the fourth chromosome– Results in local deletions and duplications that can cause a switch

in phenotype, indicating a switch in chromatin packaging;– Argues against a fixed boundary, supports an equilibrium model;-- Proximity to a 1360 associated with heterochromatin formation;

requires high repeat density and/or proximity to chromocenter.

• Chromatin structure of fourth chromosome genes- Most are “heterochromatic genes,” dependent on HP1a for optimal

expression; active marks at TSS, but revert to het marks over the body of the gene.

- Permissive domains are those associated with Polycomb in at least one cell type; lack H3K9me3 under those conditions, can form DH site.

Challenges, Questions• Can we get a convincing assembly for the D. ananasae dot chromosome? -

Can we identify invading transposable elements, other repeats?- How do the gene structures compare?

• As we complete analysis of the D. grimshawi and D. mojavensis dot chromosomes, see consistent patterns for F element: – Gene identity (~90%), synteny evidence of rearrangements– Genes larger, larger number of exons -- Variation in levels /kinds of repeats; DINEs may be critical– Interspersion of genes and repetitious sequences? -- Comparison between D. ananassae and others

• Given sequence data from multiple Drosophila species, can we do a better job of defining the genes? (D. biarmipes work)– Previous- primarily identified coding regions; UTR’s?– Start sites for transcription? Regulatory motifs?

• Other features? - Can we look for conserved non-coding regions? Hairpins?

- How does our finished sequence compare to unfinished sequence?