Transcript PcG - IGH
The chromatin Yin / Yang Heterochromatin - Heavily condensed - Gene poor - Silent genes - Late replicating - DNA hypermethylated - Rich in histone H1 - Histones have repressive post translational marks Euchrochromatin - Less condensed - Gene rich - Active genes - Early replicating - DNA hypomethylated - Poor in histone H1 - Histones have specific, activating post translational marks Where on the chromosomes are condensed and open chromatin located? Condensed chromatin Constitutive Heterochromatin Telomeres Centromeres Open chromatin Typical of the active gene loci along the chromosomal arms Condensed chromatin is also found at several silent genes located in the chromosomal arms Polycomb group and trithorax group genes are important regulators of chromatin along the chromosomal arms Polycomb (PcG) and trithorax (trxG) group proteins: epigenetic regulators of genome function • Originally discovered in Drosophila as regulators of Homeotic genes, responsible for specification of the body plan, they also regulate many other targets involved in cell differentiation and proliferation • PcG proteins silence genes, trxG proteins activate them • Conserved throughout evolution • In human, they maintain the fates of differentiated cells, but they are also required for cell proliferation and the maintenance of several types of stem cells including ES cells. Finally, they regulate X-inactivation in females as well as genomic imprinting • Mutations in PcG and trxG genes induce many different cancers PcG, trxG, and maintenance of gene expression Early development Establishment of patterns OFF Ubx ON Maternal, Gap, Pair-rule, Segment polarity OFF Polycomb-Group OFF ON Maintenance phase Transmission of pattern after disappearance of early factors trithorax-Group ON Members of the PcG and of the trxG PcG Gene protein motifs PhoRC Complex Pleiohomeotic (pho) Zinc-finger (DNA binding) Enhancer of zeste (E(z)) SET (H3K27MTase) extra sex combs (esc) WD repeat Polycomb (Pc) chromo domain (Binding to H3 methyl K9 or K27) polyhomeotic (ph) Zinc finger, SAM domain Posterior sex combs (Psc) RING finger trxG Gene protein motifs FACT complex Trithorax-like (Trl) BTB/POZ (dimerization) Zinc finger (DNA binding) TAC1 complex trithorax (trx) SET (H3K4HMTase)/ PHD-finger Ash-1 SET (H3/H4HMTase)/ PHD-finger brahma (brm) bromo domain Esc/E(z) Complex PRC1 complex Brm complex (DNA dependent ATPase/helicase) PcG proteins bind to specific DNA elements, named PREs Bound by PcG proteins in vivo (in polytene chromosomes and by cross-linking experiments) Binding leads to maintenance of PcG-dependent repression of reporter genes Repression is enhanced by the presence of multiple PRE copies Action of PcG and trxG complexes on chromatin Ac Histone acetylation and methylation (TAC1 and ASH1 complexes) Nucleosome remodeling (BRM complex) trxG ON Maintenance of active states (open chromatin) Me K4 H3 Target gene PRE Deacetylation and methylation (ESC-E(Z) complex) Me K27 H3 - Chromatin compaction - H2A Ubiquitination (PRC1 complex) PcG OFF Maintenance of repressed states (compact chromatin) Ub H2A What does Polycomb do to chromatin ? Chromatin Condensation Recombinant PC-containing complexes can condense an array of 12 nucleosomes in vitro Condensation requires PSC (not PH) protein, and involves histones but does not necessitate histone tails Data from: Francis et al. (2004), Science 306, 1574 2. Silencing of a transgenic reporter gene depends on PcG and trxG proteins Fab-7 Fab-7 Pc -/+ Fab-7 Pc +/+ UAS-lacZ white Fab-7 trx -/+ Fab-7 trx +/+ PcG and trxG proteins associate to multiple genomic loci DAPI Merge PH Polytene chromosome staining shows around 100 bands for each PcG protein Recruitment of PcG proteins at PREs: chromatin analysis by chromatin immunoprecipitation (ChIP) and DNA microarrays (chips) Cross-link cells or embryos with formaldehyde to induce protein-DNA crosslinks Sonicate and purify chromatin (average size = 1 kb) Add antibody and purify antibody-chromatin complexes on Protein A Sepharose, purify DNA and amplify by Linker-mediated PCR Use amplified DNA as probe to hybridize DNA chips containing the genome. Extract the distribution profile of the proteins of interest ChIP on chip Example of the chromosomal distribution of PcG proteins at BX-C locus BX-C bx bxd iab2 * * * iab4 mcp * * Fab7 Fab8 * * PH PC H3K27me3 E(z) Suz12 Pcl Ubx abdA AbdB PcG proteins form nuclear compartments called PcG bodies Z axis maximum intensity projection PC / Heterochromatin We count about 30-50 PcG bodies in diploid nuclei, while ChIP on chip finds over 200 Polycomb domains We postulate that multiple Polycomb domains are clustered in the nucleus !! PcG proteins are required for the maintenance of ES cell fate, as well as for the fate of hematopoietic and neuronal stem cells and of differentiated cell types Genome-wide Chip on chip in ES cells Transcription factor family members occupied by the PRC2 protein SUZ12 in human ES cells Overlap between PRC1 and PRC2 targets in mouse ES cells Eed (831) PRC2 66 IRX BHLHB CDX PRC1 365 94 Suz12 (1271) HOX Rnf2 (1219) MEIS/EVX TBX 55 300 DLX 164 Phc (922) HES FOX ATOH 512 98 6 121 MYO 31 NEUROD EBF GATA POU RUNX 23 PAX SOX NKX Boyer et al. Nature. 2006 May 18;441(7091):349-53. Epub 2006 Apr 19 SIX LHX Lee et al. Cell. 2006 Apr 21;125(2):301-13 Regulation of transcriptional pathways is a major « raison d’etre » of PcG proteins Human PcG targets 240 18 Total No. of fly target genes: 260 98 target genes in fly, that have clear human and mouse homologs 26 6 Fly PcG targets Potassium channel activity (2.8%) Receptor activity (2.8%) Morphogen (2.8%) 29 48 Unknown (2.8%) Electron transporter activity (1.4%) 18 Mouse PcG targets Alkaline phosphatase activity (3.85%) Kinase activity (3.85%) coA ligase activity Receptor activity (3.85%) (3.85%) Dehydrogenase activity (3.85%) Oxidoreductase activity (3.85%) Transcription factor activity (38.5%) Nucleic acid binding (3.85%) N=72 Transcription factor activity (87.4%) common targets to fly and vertebrates Actin binding, cytoskeleton (15.4%) mRNA binding (11.45%) N=26 Unknown (7.7%) unique targets for fly PcG and trxG proteins regulate cell memory and dynamic patterns of gene expression stem cell maintenance and plasticity proliferation cell fate determination differentiation PcG and trxG proteins X-chromosome inactivation Vertebrates Fly spermatogenesis Genomic imprinting Cancer The ph0 clones show ectopic autonomous mitosis in eye discs Anti PHOSPHO-H3 wt ph0 clones Massive overgrowth of adult ph0 eyes Control Mutant escapers 90% lethal 10% of escapers Polyhomeotic is a tumor suppressor gene ! Allez, on a presque fini… une dernière question: alors, elle a quelle forme cette chromatine en fait ? Chromatine active Chromatine réprimée For more info… http://www.igh.cnrs.fr/equip/cavalli http://www.epigenome-noe.net Inserm Actualités N. 201, Octobre 2006 (http://194.254.71.5/basisdiaggen/IA/201/Mag201-dossier.pdf) Polycomb and cancer: http://www2.cnrs.fr/presse/communique/1672.htm Chromatin movie: http://www.dailymotion.pl/tag/nisic Website to download this course: http://www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.html [email protected]