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The Evolution of a Heterochromatic 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 - 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 Minimum Haploid DNA Content - the C Value Paradox 3 Britten and Davidson, 1969 Science 165:349 Larger genomes reflect high levels of repeats retroviral and DNA transposon remnants (TEs) 4 Allis et al: Epigenetics 2007 Considerations for Genome Sequencing 1. 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. 2. 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. 3. Much of the repetitous DNA is packaged into heterochromatin, which maintains these regions in a compact and transcriptionally silent form. 4. 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! 5 SCR Elgin Eukaryotic genomes are very large – and most of that DNA is non-coding! Human Genome 3 Gb ~2 m/cell ! Coding exons 1.5% Conserved noncoding regulatory? 3.5% Key Questions: Is it junk or garbage? How is DNA packaged into a nucleus? How is silencing maintained – while allowing appropriate transcription ? TEs – retroviruses, DNA transposons What determines phenotypes? It’s not just your DNA…. Environment (diet) (grey bars = folate) Phenotype Genotype Epigenetics ? Development (Waterland and Jirtle 2003) 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 structure • Nuclear 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 seen In Fragile X syndrome Zoghbi and Beaudet 2007 Fragile X Foundation Chromatin formation: DNA First step - packaging in a nucleosome array Chromatin Second - differential packaging into heterochromatin & euchromatin Histone protein core Chromosome (metaphase) Lodish et.al., Molecular Cell Biology, 4th Edition Felsenfeld et al. Nature 2003, 421: 448 Electron Micrograph of Chromatin Fibers (rat thymus nucleus) 0.1 mm Olins et. al., 1975 J. Cell Biol, 64:528 10 “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 11 The Structure of the Nucleosome Core 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 Rhodes, 1997 Nature 389:231, after 12 Luger et. al., 1997 Nature 389:251 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 Transcriptional activators Heterochromatin Protein 1 complex Hyper-acetylated histone tail Hypo-acetylated histone tail; methylated H3/K9 Heterochromatin formation – silencing counts! 1 2 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? Fruit Flies! Short life cycle, easily maintained: good genetic tools Polytene chromosomes: excellent cytology Biochemical approaches Simple genome, good reference sequence Mary Lou Pardue, MIT euchromatin expressed heterochromatin silenced PEV – reporter for gene silencing, heterochromatin formation Metazoan useful for behavioral, developmental and human disease research Using a white transgene to sample chromatin environments inject P[white+] transposon carrying white gene white67c23 mobilize P element by crossing to stock with transposase insertion into euchromatin (99%) insertion into heterochromatin ( 1%) 16 Elgin Lab Transposition of a P element reporter allows sampling of euchromatic and heterochromatin domains X Silenced 1% 2L 2R 3L 3R 4 And the Y chromosome Active 99% Wallrath and Elgin, 1995 Assessing chromatin structuresame gene, different environments Analysis 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 C C Phase HP1 James & Elgin,1986; James et al 1989 Looking for heterochromatic proteins by immunofluorescent staining of the polytene chromosomes: discovery of HP1a Heterochromatin-associated gene silencing is dependent on HP1 Mutations in gene for HP1a Mutations recovered by T Grigliatti as suppressors of PEV. Dosage dependent response. Eissenberg et al, 1990, PNAS 87: 9923 HP1 interacts with both the modified histone H3K9me2/3 and the modifying enzyme HP1: Chromo Histone 3 methyl-Lys9 Shadow H3 K9 methyl transferase SU(VAR)3-9 [(SU(VAR)3-9 identified in screen by Reuter; H3 interaction first shown from work in mammals – Jenuwein, Kouzarides; demonstrated in flies by Imhof.] Model for spreading of heterochromatin Establishing silencing: a multi-step process Loss of euchromatin marks Gain of heterochromatin marks wm4 reporter (screens by Reuter, Grigliatti, others) Heterochromatin formation on the dot chromosome… 2 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? The Drosophila melanogaster fourth chromosome exhibits an amalgam of euchromatic and heterochromatic properties (HP1a association) But… - the fourth has ~ 80 genes in distal 1.2 Mb - these genes are transcriptionally active! C C Phase HP1 James & Elgin,1986; James et al 1989 Heterochromatic properties: - late replication, lack of recombination - high repeat density (30%) - antibody staining of HP1, H3K9me2/3 Most hsp70-white reporters exhibit variegation on insertion into the fourth chromosome 2-M1021 39C-12 2-M390 Sun et al 2004; Riddle et al 2007 39C-52 Our GEP Research Goal: Use comparative genomics to learn more about heterochromatic domains, analyzing the dot chromosomes and a control euchromatic region of Drosophila genomes Status Reference Completed Annotation Sequence Improvement New Project FlyBase: http://flybase.org Genomics Education Partnership (GEP) Partners are generally PUI schools; faculty join by attending one-week workshop at WU. Shared work organized on GEP website. 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 Dot chromosome genes: introns are larger, exons show less codon bias % Introns This Size or Smaller 100% Euchromatic 90% 80% Leung et al. 2010 Genetics 185:1519-1534 70% Dot 60% Codon Bias 50% 40% 30% 20% Heterochromatic 10% 0% Intron Size D. melanogaster Dot D. virilis Dot Intron Size (bin) D. melanogaster Euch. D. virilis Euch. D. melanogaster Het. Initial analysis of Drosophila virilis dot chromosome fosmids Almost all of the same genes are present (27/28), but rearrangements within the chromosome are common! Slawson et. al., 2006 Genome Biology, 7(2):R15. Comparison of gene order and orientation D. melanogaster D. virilis 72 genes on both the D. virilis and D. melanogaster dots. A minimum of 33 inversions are needed to convert order and orientation! Leung et al 2010 “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 CG5262 CG9935 CG5367 rho-5 CG1732 dot CG4038 dot: D. virilis CG11077 CG11076 dot: D. melanogaster Leung et al 2010 Some things to look for while annotating dot chromosome genes…. • • • • • • • 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? 1. Crosslink proteins to DNA Chromatin Immunoprecipitation - ChIP (cells or nuclei) 2. Isolate chromatin and sonicate qPCR 3. Incubate with antibody ChIP-chip* 4. Isolate AB/chromatin complexes 5. Isolate DNA from complexes ChIP-seq Mapping chromatin marks by ChIP-chip: Chromosome arm 3L shows a distinct shift between heterochromatin and euchromatin Centromere Enrichment (log intensity ratio values) S2 cells HP1a Su(var)3-9 H3K9me2 H3K9me3 genes genes Euchromatin Heterochromatin Euchromatin / heterochromatin transition point from Flybase Pink boxes show significant enrichment (0.1% false discovery rate) Chromosome 4 is largely heterochromatic, but shows distinct peaks of H3K4me2/3, indicating transcription start sites HP1a H3K9me2 H3K9me3 H3K4me2 genes Telomere Enrichment (log intensity ratio values) Centromere A model of 9 chromatin states, based on clustering of histone modification marks, identifies large-scale genomic domains TSS (red; H3K4me3 rich) Polycomb (grey; H3K27me3 rich) Heterochromatin (dark blue; H3K9me3) 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). Pericentric heterochromatin 10 Mb chr3L chr4 Red Variegating chr4 500 kb BG3 cells, chromatin states: 1 2 3 4 5 6 7 8 9 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 Most 4th chromosome genes lie in heterochromatic space (blue), but active genes achieve state 1 (red) at the TSS 1360 Active fourth chromosome genes show depletion of HP1a and H3K9me3 at the TSS, but enrichment across the body of the gene Average enrichment chromosome 4 TSS-relative position RNA pol II H3K4me3 HP1 H3K9me2 H3K9me3 Transcription levels are similar to euchromatic genes! 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). Eight new genomes Expanded dot chromosomes? 44 Heterochromatin formation on the dot chromosome… 1 Heterochromatin formation changes chromatin at the nucleosome level, eliminating HS sites at the TSS of euchromatic genes; silencing is dependent on HP1a 2 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! Question Slides Are there chromosomal proteins unique to the fourth chromosome? Yes – POF (Painting of Fourth) is uniquely associated with active genes on the fourth chromosome Green = HP1a Red = POF A role for POF? A different configuration for HP1a? (See J Larsson, PLoS Genet. 11:e209 for more on POF) 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. Clark and Elgin, 1992 Nucleic Acids Res. 20:6067 HP1 sequence from Drosophila, mouse, human and mealy bug identifies chromo domain & chromo shadow domain HP1 from mammals can rescue mutations in flies and yeast! Position Effect Variegation in Drosophila: an assay for heterochromatic packaging white Wild Type Inversion i HP1 is a trans-acting modifier of PEV Su(var) (1 copy HP1) E(var) (3 copies HP1) Eissenberg et al The heterochromatic hsp26 transgene: Cryderman et al 1999 (Wallrath & Gilmour labs) - loss of accessibility at the TSS when in heterochromatin - reversed in an HP1 mutant background Chromosome 4 short arm is a unique heterochromatic domain Haynes et al. 2007 Translocation away from the chromocenter results in loss of silencing – spatial organization plays a role H3K4me3 Define chromatin states by K-means clustering (using enrichment values for 1 kb chromatin fragments) H3K9me3 Select k means as starting points. Assign each data point to The centroid of each cluster is closest mean. the new mean. Repeat the last two steps. Chromatin states are highly intersperseda folded view of the chromosome (Bg3 cells) Polycomb Pericentric heterochromatin Chromatin states reveal cell type specific patterns (note facultative heterochromatin – light blue) BG3 S2 Chromosome 3L A folded view of the chromosome reveals TSS and Pc states within chromosome 4 (Bg3 cells) 4th chromosome Pericentric heterochromatin 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 A subset of 4th genes is associated with Polycomb; these domains are permissive for reporter expression (red eyed fly). Heterochromatin formation on the dot chromosome… 3 How are specific domains targeted for heterochomatin formation? Repetitious elements such as 1360 are targeted by a mechanism involving the RNAi system Analysis of chromosome 4 identified 1360 as a target. P[1360, hsp70-w] silencing depends on the reporter’s position in the genome 3’ P FRT 1360 FRT hsp70-white 5’ P Haynes et al 2006 Curr Biol 16: 2222 FLP-mediated removal of 1360 results in loss of silencing 3’P FRT 1360 FRT hsp70-white 3’P 5’P + FLP recombinase FLP14 FLP16 FRT hsp70-white 5’P 1360 excised FLP4 FLP5 Haynes et al 2006 Curr Biol 16: 2222 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 Mutations in RNAi components piwi, aubergine and homeless suppress PEV y w; 118E-10/+ + + piwi1 + aubQC42 hls∆125 + + y w; 118E-10/+ y w; 39C-12/+ y w; 39C-12/+ B Leibovitch in Pal-Bhadra et al, 2004 piwi aub hls A tentative model for heterochromatin targeting – HP1a – PIWI interaction (piRNA) Cytoplasm Aub piRNA PIWI Ago3 PIWI HP1 Nucleus Heterochromatin PIWI (binds piRNA) is an argonaute family member and nuclear protein which interacts with HP1a transposon