Role for DNA methylation in response to Gata4 activation in embryonic stem cell-derived mesoderm
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ABSTRACT: During embryogenesis, many key transcription factors are used repeatedly, achieving different outcomes depending on cell type and developmental stage. The epigenetic modification of the genome functions as a memory of a cell’s developmental history, and it has been proposed that such modification shapes the cellular response to transcription factors. To investigate the role of DNA methylation in the response to transcription factor Gata4, we examined expression profiles of Dnmt3a-/-Dnmt3b-/- ES cell-derived mesoderm cells cultured for 4 days with or without Gata4 activation, as well as the wild-type counterparts, using Affymetrix microarrays. Wild-type and Dnmt3a-/-Dnmt3b-/- (DKO) ES cell clones stably expressing dexamethasone (Dex)-inducible Gata4 were generated by introducing an expression plasmid for Gata4 fused with the ligand-binding domain of the human glucocorticoid receptor (Gata4GR) driven by the CAG promoter, by electroporation. For mesoderm differentiation, 1 x 10^5 ES cells were plated on a type IV collagen-coated 10-cm dish and cultured for 4 days. Cultured cells were harvested and collected as single-cell suspensions using 0.25% trypsin-EDTA, followed by incubation in differentiation medium at 37˚C to allow cell-surface markers to recover. Flk1(+)/PDGFRalpha(+) double-positive (DP) cells, which are considered equivalent to the lateral plate mesoderm, were sorted by FACS-Aria (BD Biosciences). Sorted DP cells were further cultured on type IV collagen-coated culture dishes in the absence or presence of 100 nM Dex for 4 days. For reference data, primitive endoderm (PE) cells were directly differentiated from undifferentiated ES cells by addition 100 nM Dex in ES cell-maintenance medium.
Project description:DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm in part. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells. To understand how mouse cells differentially respond to Gata4 in cell type- and DNA methylation-dependent manners and to examine what genes are differentially regulated, we analyzed temporal change of gene expression profile in response to Gata4 in ES or Flk1(+) mesoderm cells in WT and Dnmt3a-/-Dnmt3b-/- (DKO) background, in total 108 samples.
Project description:Screening for genes regulated by Etv2 within Flk-1+/PDGFRa+ ES derived mesoderm.Microarray analysis performed to screen for the candidate genes regulated by Etv2. TT2 ES cells differentiated on OP9 feeder cells were sorted using Flk-1 and PDGFRa antibodies.Gene expressions from these two populations were compared. Extract RNA from sorted Flk-1+/PDGFRa+ populations from Etv2Het vs KO cells. To obtain primitive mesoderm cells TT2 ES cells of corresponding genotypes were differentiated on OP9 cells for 4 days. Flk-1+/PDGFRa+ populations were sorted from Etv2 Het vs. KO cells for RNA extraction.
Project description:DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm in part. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.
Project description:During embryogenesis, many key transcription factors are used repeatedly, achieving different outcomes depending on cell type and developmental stage. The epigenetic modification of the genome functions as a memory of a cell’s developmental history, and it has been proposed that such modification shapes the cellular response to transcription factors. To investigate the role of DNA methylation in the response to transcription factor Gata4, we examined expression profiles of Dnmt3a-/-Dnmt3b-/- ES cell-derived mesoderm cells cultured for 4 days with or without Gata4 activation, as well as the wild-type counterparts, using Affymetrix microarrays.
Project description:We found that mouse ES cell-derived Flk1+ cells could be subdivided into three population by the expression of PDGFRa and CAR (Flk1+PDGFRa-CAR-, Flk1+PDGFRa-CAR+, and Flk1+PDGFRa+CAR+). Therefore, global gene expression analysis was perfomed by microarray to characterize these mesodermal subsets. RNA isolated from five separate experiments was pooled and used for comparison
Project description:We have determined the genome-wide binding profile of the transcription factor Sp5. Flag-tagged Sp5 was targeted to the HPRT locus in A2Lox.cre ES cells to allow for Doxycycline inducible expression. ES cells were cultured as embryoid bodies for 2 days to prime them for germ layer differentiation. Cells were then treated with Doxycycline for 24 hrs. We found that Flag-Sp5 prefentially bound to promoters associatd with several growth factor signalling pathways, but most prominently with the Wnt/beta-catenin pathway to selectively promote mesoderm differentiation over neural. These data implicate Sp5 as new regulator of Wnt/beta-catenin target expression to promotes ES cell differentation into the mesoderm lineage. Examination of Sp5 transcription factor binding in differentiating embryonic stem cells.
Project description:During embryogenesis, many key transcription factors are used repeatedly, achieving different outcomes depending on cell type and developmental stage. The epigenetic modification of the genome functions as a memory of a cell’s developmental history, and it has been proposed that such modification shapes the cellular response to transcription factors. To investigate the role of DNA methylation in the response to transcription factor Gata4, we have tried to identify GATA4-binding associated genes of WT-type and Dnmt3a-/-Dnmt3b-/-(DKO) in Flk1-based mesoderm progenitor by Gata4 ChIP-analysis.
Project description:Pluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage specific behavior of selected factors. In addition to the orchestrated remodeling of the chromatin landscape, we find that the binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer and in many cases a reciprocal gain in the other layers. Taken together, our work shows context-dependent rewiring of transcription factor binding, downstream signaling effectors, and the epigenome during human embryonic stem cell differentiation. 200 ChIP-seq experiments profiling 38 transcription factors (TFs) and several chromatin marks in 5 cell types--male human ES cell line HUES64 and directed differentiation of HUES64 towards mesendoderm (dMS, 12 hours), endoderm (dEN, 120 hours), mesoderm (dME, 120 hours), and ectoderm (dEC, 120 hours). In addition, three ES cell lines were derived with shRNA mediated knockdown of GATA4 and differention toward endoderm (dEN_shGATA4) and mesoderm (dME_shGATA4). These cell lines were used for MNChIP-seq of GATA4, SMAD1, and H3K27Ac and for 4 RRBS experiments in GATA4 knockdown and control cell lines.
Project description:Scl/Tal1 confers hemogenic competence and prevents cardiomyogenesis in embryonic endothelium. Here we show that Scl both directly activates a broad gene regulatory network required for hematopoietic stem/progenitor cell (HS/PC) development, and represses transcriptional regulators required for cardiogenesis. Cardiac repression occurs during a short developmental window through Scl binding to distant cardiac enhancers that harbor H3K4me1 at this stage. Scl binding to hematopoietic regulators extends throughout HS/PC and erythroid development and spreads from distant enhancers to promoters. Surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic versus cardiac specification and Scl binding to the majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors to facilitate HS/PC emergence from hemogenic endothelium. These results uncover a dual function for Scl in dictating hematopoietic versus cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates. Examination of Scl target genes in ES cell derived day4 EB (embryoid body) Flk+ mesodermal cells
Project description:In this study, the composition of ES of male and female L4 stage Heligmosomoides polygyrus bakeri in the presence (cultured together) or absence (cultured alone) of the opposite sex was examined using mass spectrometry.