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 HP1 Phase James et al, Mol Cell Bio 1986 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; could the genes lie in HP1-depleted domains? HP1 HP2 Merge Shaffer et al 2002 PNAS 99:  4 th chr Merge

Insertion sites resulting in a variegating phenotype identify heterochromatin domains X 2L 3L 2R 3R 4 Silenced 1% Active 99% 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-M102139C-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-M102139C-12 2-M390 39C 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 1360 OTHER 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 1360 OTHER 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 to nearest 1360 Reporter Riddle et al, Genetics, 2008 & Sun, et al., MCB, 2004

1360 is NOT the only target for heterochromatin formation on the fourth chromosome CG2316 CG2165 CG31992 bt CG32021 CaMKII sv CG32017 Caps ATPsyn-  hsp70-white5’P5’P 3’P3’Phsp26-pt 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 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: 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 Median 9bp sequence melting temperature 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 NcDistribution of CAI NcCAI 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 Muller F elementsMuller D elements D. melanogaster D. erecta D. melanogaster (base) D. erecta (base) D. erecta (extended) D. erecta (telomere) Nc Nc D. mojavensis D. grimshawi D. mojavensis (base) D. grimshawi (base) (GLEANR) 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. vir3273 D. mel / D. moj3172 D. vir / D. moj779 Relative gene order and orientation D. mel D. vir D. mel D. moj D. vir D. moj Inversion step Do clusters of genes in the same chromatin state stay together? Check with H3K9me2 H3K27me3, H3K4me2 for D. mojavensis

CG9935 CG5367 rho- 5 CG4038 CG11076 CG1732 CG5262 CG11077 dot PRY Y chromosome CG9935 y ellow-h Or13a CG11076 Or13 a Drosophila subgenusD. melanogasterD. mojavensisD. grimshawi Sopho.Dros. Sopho.replet a Sopho.Hawai. Dros. Movement: W Leung, WU, 2012 Wanderer genes have moved to/from the dot: Het Euchr

Drosophila subgenus GeneD. melD. mojD. virD. gri CG11076FAAE CG11077FAAA CG1732FDDD CG9935FEBB yellow-hFAFF CG4038CFFF CG5262DFFF CG5367BFFF rho-5BFFF Or13aAFscaffold E PRYchrYF 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 DY 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. Estelle Huang 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 PLoS Genet. 8:e

ChIP mapping shows that the fourth chromosome is largely heterochromatic, but has islands of H3K4me2 HP1a H3K9me2 H3K9me3 H3K4me2 genes CentromereTelomere Riddle et al 2010, Genome Res

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 Riddle et al Genome Res 21:147-63

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). W Leung & SCR Elgin

10 Mb chr3L BG3 cells, chromatin states : Pericentric heterochromatin chr4 500 kb chr4 Red Variegating 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 Slide from W Leung

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 Riddle et al PLoS Genet 8:e

A subset of 4 th 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, TSS Active genes, body Silent genes PC 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 = HP1a Red = POF Riddle et al PLoS Genet 8:e

Chr. 3L Chr. 4 pof D119 wt pof D119 wt Loss of POF (null)  Loss of HP1a from chr 4 gene bodies Maintain HP1a in pericentric heterochromatin, repeat clusters HP1a ChIP Riddle et al PLoS Genet 8:e

A speculative model… two methods of assembly with HP1a on the fourth: other partners? genes repeats no pausing silencing transcript elongation Note: EGG & SU(VAR)3-9 are both HMTs Both assemblies may be required to control TE remnants Riddle et al PLoS Genet 8:e

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?