1

The fourth chromosome: targetingheterochromatin formation in Drosophila

Drosophila melanogaster chromosomes

modified from TS Painter, 1934, J. Hered 25: 465-476.

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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

But-- the banded 1.2 Mb of the dot

chromosome containes 82 genes- anormal gene density for theDrosophila chromosome arms

- and a 10-fold higher concentration ofrepetitious elements

- suggesting interspersed euchromatinand heterochromatin

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HP1

HP1: a banded pattern on chromosome 4

HP1

HP2 Merge

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HP1 distribution is coincident with H3-mK9but opposed to H4-acK8

P-element Construct

T-1917

hsp26-plant hsp70-white

+490

P element transposon: variegating phenotype

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Chromosome four has interspersed heterochromatic andeuchromatic domains

2-M1021 39C-12 2-M390 39C-42

*Many variegating reporters lie within genes.

Chromosome four has interspersed heterochromatic andeuchromatic domains

• This variegation is suppressed by loss of HP1.

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Mobilization of the P element

Local transposition

Local deletion

Local duplication

Local Deletions Produce a Switch in Eye Phenotype

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100%

42%

81%

63%

74%79%

67%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

39C-X 39C-12 M382 M348 M325 M33 M1226

Local deletions induce a change in chromatin structure

Prt

D XbaIP XbaI

SalI D XbaI P XbaI SalI

probe

39C-

X

39C-

12

4-M

382

4-M

348

4-M

325

4-M

33

4-M

1226

39C-12

BEST Hcf CG2052

5 kb

A shift in histone acetylation correlates with shift in eyephenotype

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Heterochromatin Euchromatin

HP1 complex

Methylated histone H3 tail Acetylated histone tail

Transcriptional activators

Barrier?

HATs

Heterochromatic vs euchromatic domains

Duplications also cause a switch in eye phenotype

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Local duplications change the distancebetween the P element and 1360 remnants

Variegating P inserts lie within 10 kb of a copy of element 1360

-10

0

10

20

30

40

50

60

6-M

19

3

5-M

34

0

2-M

62

66

-M1

80

5-M

34

5

5-M

27

56

-M3

97

6-M

35

0*

5-M

93

5-M

29

39

C-1

2

5-M

24

45

-M2

63

6-M

52

8

4-M

97

94

-M3

25

6-M

36

5

6-M

42

14

-M9

93

6-M

15

6

4-M

63

72

-M5

30

6-M

53

4

4-M

34

84

-M2

71

4-M

52

9

4-M

77

94

-M7

03

6-M

11

4

4-M

33

2-M

01

0

4-M

34

4

4-M

12

66

6-M

30

0

2-M

80

2

3-M

48

2-M

59A

.R

4-M

38

2

6-M

35

0**

Dis

tance to N

eare

st 1360 E

lem

ent (K

b)

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Model: element 1360 (hoppel) as an initiator of heterochromatin

A Model for TargetingHeterochromatin Formation

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siRNA generated from 1360

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

– But 82 genes in 1.2 Mb- normal gene density– Ten-fold higher levels of repetitious sequences

• Incomplete transposition of the P element on the fourth chromosome– Results in local deletions and duplications– Can cause a switch in phenotype– Argues against a fixed boundary– Supports an equilibrium model– Suggests competition between alternative packaging states , summed

by nucleosome modification– Proximity to a 1360 associated with heterochromatin formation

• A role for RNAi? - mutations in RNAi machinery impact silencing, levels of H3-mK9

- observe 22 bp dsRNA from 1360– suggests RNAi may target assembly of HP1-associated heterochromatin

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The dot chromosome of D. virilis is not associated with HP1

To compare finished sequencefrom the dot chromosomes ofD. melanogaster with D. virilis

Our research goal:

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Selection of target genes• Small (dot) chromosome of many Drosophila species is known to have

several of the same genes (Podemski, 2001*)• Sequencing has recently been completed for D. pseudoobscura, a species

25-30 my diverged from D. melanogaster• Spring 2003, Rachel Shevchek did a BLAST comparison using cDNA

sequences from all the genes from the dot chromosome of D.melanogaster to the genomic sequence of D. pseudoobscura from theBaylor website

• She looked for >200bp chunks of genes that were very highly conserved(>80%) and designed PCR primers using Primer3(http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi)

• Library screened summer 2004 by Elmer Kellman & Libby Slawson;identified fosmids used in Bio 4342 in spring 2004.

*Podemski, Ferrer, Locke (2001) “Whole arm inversions of chromosome 4 in Drosophila species,”Chromosoma 110, 305-312

In situhybridizations

• Ten fosmids confirmed by insitu hybridization to thepolytene chromosomes of D.virilis

• Done in the lab of Dr. Mary-Lou Pardue at MIT

• Will not tell us what gene thefosmid contains, but will tellus where some of the DNAfrom the fosmid is localized

*example in situ, M-L Pardue

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Comparison of repeat densitiesin D. virilis (Dv) and D. melanogaster (Dm)

0

5

10

15

20

25

Simple Repeats

DNA TransposonsRetrotransposons

Dot Chromosomes Long Arms

Dv

Dv

Dm

Dm

Perc

enta

ge o

f Rep

etiti

ous D

NA

(%)

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Project ParticipantsStudents ‘04

Jim Bogenpohl, Seth Bloom, James Dee, Emiko Morimoto, JennyMyoung, Andrew Nett, Fatih Ozsolak, Mandy Tittiger, Andrea Zeug

Faculty and StaffSarah Elgin, Prof Biology

Elaine Mardis, Co-Director, GSC & Asst Prof GeneticsChris Shaffer, Biology, Senior Teaching Fellow

Staff of the Genome Sequencing CenterJeremy Buhler, Asst Prof Computer ScienceMichael Brent, Asso Prof Computer Science04 TA’s Libby Slawson and Colin Malone

Thanks to Rachel Shevchek, Elmer Kellman, Carolyn Craig, MaryLou Pardue (MIT), David Lopatto (Grinnell)

and to many other WU faculty & staff for guest lectures.

,

Big Questions• As we complete more of the D. virilis dot chromosome, will the same

conclusions hold?– Gene identity, synteny, evidence of rearrangements?– Size of genes, gene density?– Levels and kinds of repetitious sequences?

• Given sequence data from other Drosophila species, can we do a better jobin defining genes? What about patterns of repetitious sequences?– Previous- primarily identified coding regions– Start sites for transcription? Regulatory motifs?– Will D. mohavaensis look more like D. virilis than D. melanogaster?

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

- How does our finished sequence compare to unfinished strain? - Other questions?