Project description:We report the evolutionary behaviour of Polycomb group proteins, their recruitment factors and their underlying sequences by performing ChIP-seq analysis in 4-5 different Drosophila species. We demonstrate an extremely high conservation of Polycomb repressive domains across Drosophila species We validate few cases of PRE divergence that shows that cis-driven PRE evolution is a rare event. We further show that PHO recruitment to Polycomb domains is evolutionarily robust to motif changes and that PRC1 stabilizes binding of its key recruiter ChIP-seq analysis of histone marks and chromatin associated factors across 4-5 Drosophila species
Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome. ChIP-seq of AGO2 in two Drosophila cell types (S2 and S3)
Project description:We report the evolutionary behaviour of Polycomb group proteins, their recruitment factors and their underlying sequences by performing ChIP-seq analysis in 4-5 different Drosophila species. We demonstrate an extremely high conservation of Polycomb repressive domains across Drosophila species We validate few cases of PRE divergence that shows that cis-driven PRE evolution is a rare event. We further show that PHO recruitment to Polycomb domains is evolutionarily robust to motif changes and that PRC1 stabilizes binding of its key recruiter
Project description:Polycomb-mediated chromatin repression modulates gene expression during development in metazoans. Binding of multiple sequence-specific factors at discrete Polycomb Response Elements (PREs) is thought to recruit repressive complexes that spread across an extended chromatin domain. To dissect the structure of PREs, we applied high-resolution mapping of non-histone chromatin proteins in native chromatin of Drosophila cells. Analysis of occupied sites reveal cooperative interactions between transcription factors that stabilize Polycomb anchoring to DNA, and implicate the general transcription factor Adf1 as a novel PRE component. By comparing two Drosophila cell lines with differential chromatin states, we provide evidence that repression is accomplished at multiple steps in transcription, including inactivation of distant enhancers, enhanced Polycomb recruitment to PREs and target promoters, and elevated stalling of RNAPII in repressed genes. These results suggest that the stability of complexes bridging promoters, enhancers, and PREs is a crucial aspect of developmentally regulated gene expression. Native chromatin immunoprecipitation of histones, transcription factors and Polycomb protein in Drosophila cell lines.
Project description:The bithorax complex (BX-C) in the fruit fly, Drosophila melanogaster, is a cluster of homeotic genes that determines the identities the body segments. Expression of these genes is governed by cis-regulatory domains, one for each parasegment, which are arranged on the chromosome in the order of the parasegments they affect. Stable repression of these domains depends on the functions of the Polycomb Group, including its ability to methylate lysine 27 of histone H3. To learn whether PcG proteins generate parasegment-specific chromatin signatures, we have used transgenes to mark and isolate nuclei from single parasegments. These nuclei were profiled for histone modifications and chromatin proteins. The H3K27me3 profiles across the BX-C in successive parasegments show a striking “stairstep” pattern that reveals sharp boundaries of the BX-C regulatory domains. The borders of H3K27me3 modification domains are sharp, and align precisely with binding sites for the CCCTC-binding protein (CTCF). H3K27ac is broadly enriched across active domains, in a pattern complementary to K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely localized H3K27 modification as a central determinant of segment identity. Nuclei from single parasegments of the Drosophila embryo were isolated and ChIP-seq performed for H3K27me3, CTCF, H3K27ac, H3K4me3, Ph, Pol2, Pc, and Suz12. For each sample, two biological replicates were performed.
Project description:Polycomb repressive complex-1 (PRC1) is essential for the epigenetic regulation of gene expression. SCML2 is a mammalian homolog of Drosophila SCM, a Polycomb-group protein that associates with PRC1. Here, we show that SCML2A, an SCML2 isoform tightly associated to chromatin, contributes to PRC1 localization and also directly enforces repression of certain Polycomb target genes. SCML2A binds to PRC1 via its SPM domain and interacts with ncRNAs through a novel RNA-binding region (RBR). Targeting of SCML2A to chromatin involves the coordinated action of the MBT domains, RNA binding, and interaction with PRC1 through the SPM domain. Deletion of the RBR reduces the occupancy of SCML2A at target genes and overexpression of a mutant SCML2A lacking the RBR causes defects in PRC1 recruitment. These observations point to a role for ncRNAs in regulating SCML2 function and suggest that SCML2 participates in the epigenetic control of transcription directly and in cooperation with PRC1. This is the ChIP-seq part of the study
Project description:Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development.
Project description:We report the evolutionary behaviour of Polycomb group proteins, their recruitment factors and their underlying sequences by performing ChIP-seq analysis in 4-5 different Drosophila species (GSE60428) and HiC analysis in Drosophila melanogaster. We demonstrate an extremely high conservation of Polycomb repressive domains across Drosophila species We validate few cases of PRE divergence that shows that cis-driven PRE evolution is a rare event. We further show that PHO recruitment to Polycomb domains is evolutionarily robust to motif changes and that PRC1 stabilizes binding of its key recruiter HiC experiments in wild type drosophila embryos
Project description:Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. We show that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin, like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells silence genes in traditional Polycomb domains and in generally inactive chromatin like embryonic somatic cells. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly-induced Scm concentrates PRC2 activity on traditional Polycomb domains. Our results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin.