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Home , Heterochromatin, Sarah Elgin

The fourth : targeting formation in

Drosophila melanogaster

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

1 DNA packaging domains

HATs

• • Heterochromatin – Less condensed – Highly condensed – Chromosome arms – and – Unique sequences; – Repetitious sequences; gene rich gene poor – Replicated throughout S – Replicated in late S – Recombination during – No meiotic recombination

Transcriptional activators Heterochromatin Protein 1 complex

Hyper-acetylated tail Hypo-acetylated histone tail; methylated H3/K9

But- - the banded 1.2 Mb of the dot chromosome containes 82 genes- a normal gene density for the Drosophila chromosome arms

- and a 10-fold higher concentration of repetitious elements

- suggesting interspersed euchromatin and heterochromatin

2 HP1

HP1: a banded pattern on

HP2 Merge

HP1

3 HP1 distribution is coincident with H3-mK9 but opposed to H4-acK8

P element transposon: variegating P-element Construct

-1917 +490 T hsp26-plant hsp70-white

4 Chromosome four has interspersed heterochromatic and euchromatic domains

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

*Many variegating reporters lie within genes.

Chromosome four has interspersed heterochromatic and euchromatic domains

• This variegation is suppressed by loss of HP1.

5 Mobilization of the P element

Local transposition

Local deletion

Local duplication

Local Deletions Produce a Switch in Eye Phenotype

6 Local deletions induce a change in structure 6 2 8 5 2 2 8 4 2 3 2 1 X - - 3 3 3 3 1 C C M M M M M 9 9 - - - - - 3 3 4 4 4 4 4

Prt

D XbaI P XbaI

100% 100% 90% 81% 79% 80% 74% 67% 70% 63% 60% 50% 42% 40% 30% 20% 10% 0% 39C-X 39C-12 M382 M348 M325 M33 M1226

SalI D XbaI P XbaI SalI

probe

A shift in histone acetylation correlates with shift in eye phenotype 39C-12 5 kb

BEST Hcf CG2052

7 Heterochromatic vs euchromatic domains

Heterochromatin Euchromatin Barrier?

HATs

HP1 complex Transcriptional activators

Methylated tail Acetylated histone tail

Duplications also cause a switch in eye phenotype

8 Local duplications change the distance between the P element and 1360 remnants

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

60

50

40

30

20

10

0

Distance to Nearest 1360 Element (Kb) -10 3-M48 5-M93 5-M29 4-M33 4-M979 6-M365 6-M421 4-M637 2-M010 4-M344 6-M300 2-M802 4-M382 6-M193 5-M340 2-M626 6-M180 5-M345 5-M275 6-M397 39C-12 5-M244 5-M263 6-M528 4-M325 4-M993 6-M156 2-M530 6-M534 4-M348 4-M271 4-M529 4-M779 4-M703 6-M114 6-M350* 4-M1266 6-M350** 2-M59A.R

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

A Model for Targeting Heterochromatin Formation

10 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 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

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

Our research goal: To compare finished sequence from the dot chromosomes of D. melanogaster with D. virilis

12 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 the Baylor 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 situ hybridizations

• Ten fosmids confirmed by in situ hybridization to the polytene chromosomes of D. virilis • Done in the lab of Dr. Mary- Lou Pardue at MIT • Will not tell us what gene the fosmid contains, but will tell us where some of the DNA from the fosmid is localized

*example in situ, M-L Pardue

13 Comparison of repeat densities in D. virilis (Dv) and D. melanogaster (Dm) ) 25 Dm % ( Simple Repeats A N

D 20 DNA Transposons s u

o Retrotransposons i t i t

e 15 p Dv e R f o

10 e g a Dm t n Dv e c 5 r e P

0 Dot Chromosomes Long Arms

14 Project Participants

Students ‘04 Jim Bogenpohl, Seth Bloom, James Dee, Emiko Morimoto, Jenny Myoung, Andrew Nett, Fatih Ozsolak, Mandy Tittiger, Andrea Zeug

Faculty and Staff , Prof Biology Elaine Mardis, Co-Director, GSC & Asst Prof Chris Shaffer, Biology, Senior Teaching Fellow Staff of the Sequencing Center Jeremy Buhler, Asst Prof Computer Science Michael Brent, Asso Prof Computer Science 04 TA’s Libby Slawson and Colin Malone Thanks to Rachel Shevchek, Elmer Kellman, Carolyn Craig, Mary Lou 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 job in 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?

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