Role for DNA methylation in Gata4-binding in embryonic stem cells-derived mesoderm

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. Overall design: Wild-type and 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, 5.4 x 10^5 ES for WT, 6.5 x 10^5 ES cells for DKO, were plated onto OP9 stroma-culture in 15-cm dish. Day. 4.5-differentiated cells were treated with 100 nM Dex for 3 hours and then 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(+) cells were sorted by AutoMACS (Miltenyi Biotec) and crosslinked with 1% formaldehyde for Gata4 ChIP-seq analysis.