Unknown,Transcriptomics,Genomics,Proteomics

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Role for DNA methylation in response to Gata4 activation in embryonic stem cell-derived mesoderm

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.

ORGANISM(S): Mus musculus

SUBMITTER: Masaki Okano

PROVIDER: E-GEOD-36814 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.