Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A key role for Mbd3 in 5-hydroxymethylcytosine-dependent gene regulation in embryonic stem cells

ABSTRACT: Numerous chromatin regulators are required for embryonic stem (ES) cell self-renewal and pluripotency, but few have been studied in detail. Here, we examine the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We find that Mbd3 and Brg1 antagonistically regulate a common set of genes by regulating promoter-proximal nucleosome occupancy and recruitment of RNA polymerase II. Furthermore, both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC): Mbd3 colocalizes with both Tet1 and 5hmC in vivo, its localization is Tet1-dependent, and binding of Mbd3/NURD to DNA in vitro is inhibited by methylcytosine, but not hydroxymethylcytosine. Finally, both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo. Together, our results identify an effector for 5hmC, and reveal that control of gene expression by antagonistic chromatin regulators is a surprisingly common regulatory strategy in ES cells. Genomic binding profiles of Mbd3 in normal ES cells, along with ES cells depleted of additional chromatin regulators, Brg1 and Tet1 were performed by ChIP-seq. In addition, we performed mapping of RNA Polymerase II in control (EGFP), Mbd3 and Brg1 knockdown ES cells by ChIP-seq.

ORGANISM(S): Mus musculus

SUBMITTER: Jui-Hung Hung

PROVIDER: E-GEOD-31690 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Mbd3/NURD complex regulates expression of 5-hydroxymethylcytosine marked genes in embryonic stem cells.

Yildirim Ozlem O Li Ruowang R Hung Jui-Hung JH Chen Poshen B PB Dong Xianjun X Ee Ly-Sha LS Weng Zhiping Z Rando Oliver J OJ Fazzio Thomas G TG

Cell 20111201 7

Numerous chromatin regulators are required for embryonic stem (ES) cell self-renewal and pluripotency, but few have been studied in detail. Here, we examine the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We find that Mbd3 and Brg1 antagonistically regulate a common set of genes by regulating promoter nucleosome occupancy. Furthermore, both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC): Mbd3 colocalizes with Tet1 and ...[more]

PMID: 22196727

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Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development. To determine the genome-wide distribution of Tet1 and 5hmC in mouse ES cells, as well as identify the gene transcription changes after Tet1 depletion. GSM706669-GSM706671: We used GST pull-down followed by deep sequencing to map the DNA bound by the Tet1 CXXC domain in vitro. We made two mutants that have a single point mutation (Cys574 to Ala or Cys586 to Ala) in the core CXXC domain to ascertain the essential role of the CXXC domain in DNA binding by comparing the sequencing profile of DNA bound by wild type CXXC with the profiles of the CXXC mutants. GSM706672-GSM706673: Tet1 ChIP-seq was performed to identify the genome-wide distribution of Tet1 in mouse ES cells. GSM706674-GSM706679: We performed hydroxymethylated DNA immunoprecipitation (hMeDIP)-seq combined with a shRNA-mediated gene depletion strategy. To identify the loci specific 5hmC regulation by Tet1, we compared the 5hmC genome-wide distributions in control (Luc shRNA) and Tet1-depleted (Tet1 shRNA2863) mouse ES cells. GSM706680-GSM706682: To identify the gene regulation network by Tet1, we compared the gene expression profiles of control (scramble shRNA) and Tet1-depleted (Tet1 shRNA 2863 and Tet1 shRNA 3387) mouse ES cells determined by mRNA-seq.

Project description:Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. Recent demonstration that members of the Ten-eleven translocation (Tet) family proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell self-renewal and maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), here we show that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite a general increase in levels of DNA methylation at Tet1 binding-sites, Tet1 depletion does not lead to down-regulation of all the Tet1 targets. Interestingly, while Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also required for repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators. Mouse ES cells infected with control knockdown (KD) or Tet1 KD lentiviruses were FACS-sorted for RNA extraction and hybridization on Affymetrix microarrays. We also investigated the effect of Nanog overexpression (OE) in Tet1 KD mouse ES cells on dys-regulated Tet1 targets. We have collected four biologically independent replicates for each treatment.

Project description:TET-family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem (ES) cells. ES cells depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1, and display hyperactive Nodal signalling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions that favor derivation of trophoblast stem (TS) cells, Tet1-depleted ES cells activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in mid-gestation embryo chimeras. Consistent with these findings, Tet1-depleted ES cells form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm and ectopic appearance of trophoblastic giant cells. Thus Tet1 functions to regulate the lineage differentiation potential of ES cells. Here, we performed whole-genome transcriptome profiling of ES cells stably depleted of Tet1 by shRNA knockdown (Tet1-kd) cultured in either standard ES cell or in TS cell culture conditions. Gene expression changes in Tet1-kd ES cells were fairly modest compared to control (GFP-kd) cells, although gene ontology (GO) analysis of differentially expressed genes yielded many terms related to embryonic development and cell cycle regulation. In TS cell culture conditions, a core set of genes defining trophectodermal cell differentiation, including Cdx2, Eomes and Tead4, was enriched in Tet1-kd compared to GFP-kd cells. A total of 27 samples were analysed with at least 3 replicates in each group. Control/reference samples comprise ES cells cultured in standard ES cell media (true ES cells) and TS cells cultured in Fgf4/heparin-supplemented (TS) media (true TS cells). We compared Tet1-kd ES cells with GFP-kd ES cells cultured in either ES or TS cell media. In addition, we included a set of Tet1-kd subclones (Tet1-kd-sc) cultured in TS cell condition and a set of untransfected ES cells (parental control) cultured in TS cell condition.

Dataset Information

A key role for Mbd3 in 5-hydroxymethylcytosine-dependent gene regulation in embryonic stem cells

Publications

Mbd3/NURD complex regulates expression of 5-hydroxymethylcytosine marked genes in embryonic stem cells.

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