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

ABSTRACT: To identify functional interactions among six chromatin regulators with key roles in embryonic stem cells, we examined gene expression changes in mouse ES cells with single and pair-wise combinations of knockdowns for those factors, along with control (EGFP) KDs.

ORGANISM(S): Mus musculus

PROVIDER: GSE31008 | GEO | 2011/12/01

SECONDARY ACCESSION(S): PRJNA146333

REPOSITORIES: GEO

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

Project description:To identify functional interactions among six chromatin regulators with key roles in embryonic stem cells, we examined gene expression changes in mouse ES cells with single and pair-wise combinations of knockdowns for those factors, along with control (EGFP) KDs. Effects on gene expression of RNAi-mediated knockdown of combinations of chromatin regulators. KDs were performed in duplicate for each combination for 48 hours.

2011-12-01 | E-GEOD-31008 | biostudies-arrayexpress

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

Project description: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.

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

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Stoichiometry of MBD2 vs. MBD3-NuRD

Project description:To obtain a comprehensive and quantitative view on MBD2 vs MBD3-NuRD complex stoichiometry, we performed biotin co-immunoprecipitations in Mbd3 KO ES cells expressing either biotin-tagged MBD2a or MBD3a and identified known NuRD complex members using label-free mass spectrometry (Supplementary Fig. 8b-c). We then calculated the intensity-based absolute quantification (iBAQ) values of the most predominant and statistically significant MBD-interacting proteins in both cell lines, which can be used to estimate the relative abundance. While we observe very similar complex composition between MBD2a-NuRD and MBD3a-NuRD, peptides shared between SALL1-4 proteins show a preferred interaction with the MBD2a-NuRD complex.

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Project description:Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC.

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