Transcriptional profiling of Mbd3 knockout and wild-type embryonic stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Paul Bertone
PROVIDER: E-MTAB-997 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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. 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.
2011-12-01 | E-GEOD-31690 | biostudies-arrayexpress
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.
2023-06-30 | PXD042407 | Pride
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.
2011-12-01 | GSE31690 | GEO
Project description:Maintenance of chromatin structure is essential to eukaryotic life; dysregulation is known to be causal for aberrant development and disease. The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. We identified the localization of MBD3, a component of Mi-2/NuRD complex, in two breast cancer cell lines (MCF7 and MDA-MB-231) using ChIP-Seq. MBD3 showed cell-type specific localization with overlap across cell lines being less than 50%. MBD3 localized across gene bodies, peaking around the transcription start site (TSS). Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have roles in fine tuning expression for active genes. These data represent an important first step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and gene expression. Identification of MBD3 localization in human breast cancer cell lines
2013-10-31 | E-GEOD-44737 | biostudies-arrayexpress
Project description:Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Samples include Mbd3+/+, Mbd3flox/- and Mbd3-/- cells from mouse ES cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors). Two histone modifications are given: H3K4me3, H3K27me3. In addition binding data of Mbd3 and Mi2B in various stages.
2013-09-12 | E-GEOD-49766 | biostudies-arrayexpress
Project description:The heterogeneous collection of NuRD complexes can be grouped into the MBD2 or MBD3 containing complexes MBD2-NuRD and MBD3-NuRD. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here we show a strong enrichment for MBD2 at methylated CpG sequences, whereas CpGs bound by MBD3 are devoid of methylation. Gene activity of MBD2 bound genes is four fold lower as compared to genes bound by MBD3. When depleting cells for MBD2, the MBD2 bound genes increase their activity, whereas MBD2 plus MBD3 bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes. This suggests a functional connection between MBD2 binding to chromatin and splicing. V5 ChIP followed by high throughput sequencing in HeLa cells transiently transfected with either V5-MBD2b, V5-MBD3 or V5.
2013-02-08 | E-GEOD-41006 | biostudies-arrayexpress
Project description:We and others have identified that MBD3/NuRD localizes at active promoters and enhancers, suggesting an active role of NuRD at open chromatin region. Because NuRD includes nucleosome remodelers, CHD3 and CHD4, we hypothesized that NuRD regulates nucleosome organization at open chromatin region. To test this idea, we performed micrococcal nuclease digestion followed by massively parallel sequencing (MNase-seq) in MBD3 knockdowned MCF-7 cells. We observed the decrease of nucleosome occupancy at promoters and enhancers in MBD3 knockdowned cells. Our results suggest a regulatory role of MBD3/NuRD at open chromatin region. Mapped nucleosome positioning in control (shLuc) and MBD3 knockdowned MCF-7 cells, in duplicate.
2013-10-31 | E-GEOD-51097 | biostudies-arrayexpress
Project description:Maintenance of chromatin structure is essential to eukaryotic life; dysregulation is known to be causal for aberrant development and disease. The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. We identified the localization of MBD3, a component of Mi-2/NuRD complex, in two breast cancer cell lines (MCF7 and MDA-MB-231) using ChIP-Seq. MBD3 showed cell-type specific localization with overlap across cell lines being less than 50%. MBD3 localized across gene bodies, peaking around the transcription start site (TSS). Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have roles in fine tuning expression for active genes. These data represent an important first step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and gene expression. DamID experiment was performed in human breast cancer cell lines (MCF-7 and MDA-MB-231) in duplicate. Samples were hybridized to Nimblegen 2.1M Deluxe promoter array. MBD3-Dam material was hybridized over Dam-only control.
2013-10-31 | E-GEOD-44773 | biostudies-arrayexpress
Project description:Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Reduced representation bisulfite sequencing (RRBS) was applied to mouse iPS cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors) from randomly selected Mbd3+/+ and Mbd3flox/- clonal cell line series. Polyclonal donor cell cultures were harvested at days 0,4 and 8 after DOX reprogramming without selection or sorting for any marker or passaging, and mapped for similarity to subcloned iPSC lines.
2014-12-13 | E-GEOD-64115 | biostudies-arrayexpress
Project description:Maintenance of chromatin structure is essential to eukaryotic life; dysregulation is known to be causal for aberrant development and disease. The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. We identified the localization of MBD3, a component of Mi-2/NuRD complex, in two breast cancer cell lines (MCF7 and MDA-MB-231) using ChIP-Seq. MBD3 showed cell-type specific localization with overlap across cell lines being less than 50%. MBD3 localized across gene bodies, peaking around the transcription start site (TSS). Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have roles in fine tuning expression for active genes. These data represent an important first step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and gene expression. DamID experiment was performed in human breast cancer cell lines (MCF-7 and MDA-MB-231) in triplicate. Samples were hybridized to NimbleGen Human Whole-Genome Tiling Arrays (0701_HG18_TILE_05_HX1 and 100718_HG18_TILE_05_HX1). MBD3-Dam material was hybridized over Dam-only control.
2013-10-31 | E-GEOD-44774 | biostudies-arrayexpress