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: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 RIP-seq part of the study
Project description:SFMBT1 is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1M-bM-^@M-^SLSD1M-bM-^@M-^SCoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells and their chromatin binding activity is regulated during spermatogenesis. RNA-seq in HeLaS3 cells ctrl compared to triple knockdown for SFMBT1, CoREST, and LSD1
Project description:SIRT3 is a member of the Sir2 family of NAD+-dependent protein deacetylases that promotes longevity in many organisms. The processed, short form of SIRT3 is a well-established mitochondrial protein whose deacetylase activity regulates various metabolic processes. However, the presence of full-length (FL) SIRT3 in the nucleus and its functional importance remains controversial. Our previous studies demonstrated that nuclear FL-SIRT3 functions as a histone deacetylase and is transcriptionally repressive when artificially recruited to a reporter gene. Here, we report that nuclear FL-SIRT3 is subjected to rapid degradation upon cellular stress, including oxidative stress and UV-irradiation, whereas the mitochondrial, processed form is unaffected. FL-SIRT3 degradation is mediated by the ubiquitin-proteasome pathway, at least partially through the E3 activity of SKP2. Finally, we show by chromatin immunoprecipitation that some target genes of nuclear SIRT3 are derepressed upon the degradation of SIRT3 caused by stress stimuli. Thus, SIRT3 exhibits a previously unappreciated role in the nucleus modulating the expression of some stress-related and nuclear-encoded mitochondrial genes. ChIP-seq with a SIRT3 antibody in untreated, UV-irradiated, and stable SIRT3 knockdown (KD) U2OS cells
Project description:The heterogeneous nature of mammalian PRC1 complexes has hindered our understanding of their biological functions. Here, we present a comprehensive proteomic and genomic analysis that uncovered six major groups of PRC1 complexes each containing a distinct PCGF subunit, a RING1A/B ubiquitin ligase, and a unique set of associated polypeptides. These PRC1 complexes differ in their genomic localization and only a small subset co-localize with H3K27me3. Further biochemical dissection revealed that the six PCGFM-bM-^@M-^SRING1A/B combinations form multiple complexes through association with RYBP or its homolog YAF2, which prevents the incorporation of other canonical PRC1 subunits such as CBX, PHC and SCM. Although both RYBP/YAF2- and CBX/PHC/SCM-containing complexes compact chromatin, only RYBP stimulates the activity of RING1B toward H2AK119ub1, suggesting a central role in PRC1 function. Knockdown of RYBP in ES cells compromised their ability to form embryoid bodies, likely because of defects in cell proliferation and maintenance of H2AK119ub1 level. ChIP-seq experiments of different PRC1 components were performed either on HA-tagged transgenic stable 293T-REx lines or on endogenous subunits using specific antibodies.
Project description:Naturally occurring variations of Polycomb Repressive Complex 1 (PRC1) comprise a core assembly of Polycomb group proteins and additional factors that include, surprisingly, Autism Susceptibility Candidate 2 (AUTS2). While AUTS2 is often disrupted in patients with neuronal disorders, the underlying mechanism is unclear. We investigated the role of AUTS2 as part of a previously identified PRC1 complex (PRC1-AUTS2), and in the context of neurodevelopment. In contrast to the canonical role of PRC1 in gene repression, PRC1-AUTS2 activates transcription. Biochemical studies demonstrate that the CK2 component of PRC1-AUTS2 thwarts PRC1 repressive activity and AUTS2-mediated recruitment of P300 leads to gene activation. ChIP-seq of AUTS2 shows that it regulates neuronal gene expression through promoter association. Conditional CNS targeting of Auts2 in a mouse model leads to various developmental defects. These findings reveal a natural means of subverting PRC1 activity, linking key epigenetic modulators with neuronal functions and diseases. mRNA profiles of P1 brain from wild type mice were generated by deep sequencing
Project description:This experiment sought to determine the chromatin structure and PRC2 occupancy at the promoters of all genes in mouse ESCs ChIP-seq for EZH2, SUZ12, and H3K27me3 were performed on WT E14 cells in 2 biological replicates each. In addition H3K27me3 ChIP-seq was also performed on a clone of E14 cells expressing a tagged version of EZH2, which for the purpose of this study was used as replicate #3.
Project description:SFMBT1 is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1M-bM-^@M-^SLSD1M-bM-^@M-^SCoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells and their chromatin binding activity is regulated during spermatogenesis. ChIP-seq in HeLaS3 at different phases of the cell cycle and primary germ cells at different stages of spermatogenesis using antibodies against endogenous proteins
Project description:In this experiment we sought to identify region with differential PRC2, JARID2, and H3K27me3 occupancy in human induced pluripotent stem cell lines with silenced or expressed MEG3 locus to indirectly determine the effect of this ncRNA on PRC2 function. 8 hIPSC lines, 5 MEG3+ and 3 MEG3-; ChIP-seq for EZH2, JARID2, and H3K27me3. Some done in replicates.