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RIP-seq for SCML2 and SCML2 mutants in 293T-REx and K562 cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE38274 | GEO | 2014/07/02

SECONDARY ACCESSION(S): PRJNA167642

REPOSITORIES: GEO

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ChIP-seq for SCML2 and SCML2 mutants in 293T-REx and K562 cells

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

2014-07-02 | E-GEOD-38273 | biostudies-arrayexpress

RIP-seq for SCML2 and SCML2 mutants in 293T-REx and K562 cells

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Scml2 establshes germline-sepcific epigenome

Project description:we identify Scml2, a subunit of a germ cell-specific polycomb repressive complex 1 (PRC1), as a critical epigenetic modifier that establishes the germline-specific epigenome through two distinct functions. One of these functions is in the stem cell phase of spermatogonia and the other is on meiotic sex chromosomes. During the stem cell phase of spermatogonia, Scml2 establishes Rnf2- dependent ubiquitination of H2A (Rnf2-ubH2A) as an epigenetic memory that subsequently ensures programmed repression of somatic genes during the late stages of spermatogenesis. Additionally, during meiosis, Scml2 interacts with M-NM-3H2AX and works downstream of the DNA damage response factor Mdc1 on the sex chromosomes and, contrary to autosomes, suppresses Rnf2-ubH2A for proper epigenetic programming of the sex chromosomes. Taken together, Scml2 positively regulates Rnf2-ubH2A on autosomes and negatively regulates Rnf2-ubH2A on the sex chromosomes to establish the germline-specific epigenome in spermatogenesis. Our study reveals a novel layer of epigenetic regulation in the male germline and adds further insight into the functionality of the polycomb proteins. RNA-seq and ChIP-seq analyses using wild-type and Scml2 KO spermatogenic cells

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SCML2 Establishes the Male Germline Epigenome

Project description:Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells.

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Polycomb protein SCML2 mediates paternal epigenetic inheritance through sperm chromatin

Project description:Sperm chromatin retains small amounts of histones, and the chromatin states of sperm mirror gene expression programs of the next generation. It remains largely unknown how paternal epigenetic information is transmitted through sperm chromatin. Here we developed a novel mouse model of paternal epigenetic inheritance in which deposition of Polycomb repressive complex 2 (PRC2) mediated-repressive H3K27me3 is attenuated in the paternal germline. By applying modified methods of assisted reproductive technology, we rescued infertility of mice absent of Polycomb protein SCML2, which is the regulator of germline gene expression through the establishment of H3K27me3 on bivalent promoters with other active marks H3K4me2/3. In F1 males of Scml2-knockout mice, which has wild-type genotype, gene expression is dysregulated in the male germline during spermiogenesis. These dysregulated genes are targets of SCML2-mediated H3K27me3 in mature sperm. Thus, SCML2 mediates intergenerational inheritance of paternal epigenetic information through the regulation of sperm chromatin.

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ChIP-seq and RIP-seq for SCML2 and SCML2 mutants in 293T-REx and K562 cells

Project description:ChIP-seq and RIP-seq for SCML2 and SCML2 mutants in 293T-REx and K562 cells

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Polycomb protein SCML2 mediates paternal epigenetic inheritance through sperm chromatin

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Project description:The chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on the prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to the recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. Genetic ablation of catalytic subunit of the PRC1 complex (RINGA/B) and ChIP-seq analysis of PRC1 and PRC2 components confirmed genome-wide decreases in PRC2 occupancy and H3K27me3 levels at PRC target sites. This activity is restricted to variant PRC1 complexes and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for polycomb domain formation and normal development. Together these observations provide a surprising new PRC1-dependent logic for PRC2 occupancy and polycomb domain formation. RING1A-/-;RING1Bfl/fl ES cells were treated with 800M-BM-5M tamoxifen for 48hours and compared to untreated control cells by ChIP-seq for RING1B, SUZ12, EZH2 and H3K27me3.

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