Project description:Current knowledge about the role of epigenetic modifiers in pancreas development has been exponentially increased. However, the precise function of TET dioxygenases in pancreas specification remains poorly understood. Using a stepwise human embryonic stem cell (hESC) differentiation system, TET1/TET2/TET3 triple-knockout (TKO) cells displayed severe defects in pancreatic differentiation. Whole-genome analysis revealed TET depletion led to aberrant DNA methylation and chromatin remodeling. In comparison with methylome and hydroxymethylome datasets previously generated from hESCs, we identified unique pancreas-specific hyper-methylated and hypo-hydroxymethylated regions in TKO cells, where binding of pioneer transcription factor FOXA2 was remarkably enriched. Interestingly, transduction of full-length TET1 in TKO cells effectively rescued pancreatic differentiation and the expression of PAX4, a key determinant for -cell specification. Taking these findings together with genome-wide mapping of TET1 in pancreatic progenitors, we uncovered that TET1 co-occupied at a specific subset of FOXA2-bound loci featuring high levels of active chromatin. Locus-specific DNA methylation analysis revealed significant increases of 5-methylcytosine at the PAX4 enhancer in a TET1-dependent manner, consistent with defective generation of functional beta-cells from TET1-knockout hESCs. Thus, our study not only highlights the importance of TET-dependent epigenetic regulation in pancreas development but also unveils an essential role of TET1 in establishing beta-cell identity.
Project description:Tet enzymes (Tet1/2/3) catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC) and are dynamically expressed in various embryonic and adult cell types. While loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development has not been established. To define the role of Tet enzymes and 5hmC in development we have generated Tet1, Tet2 and Tet3 triple knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential in vitro and in vivo. Combined deficiency of all three Tet enzymes led to complete depletion of 5hmC and impaired ESC differentiation as seen in poorly differentiated TKO embryoid bodies and teratomas. Consistent with impaired differentiation, TKO ES cells exhibited limited contribution to the chimeric embryos and could not support embryonic development in tetraploid complementation assays. Gene expression profiles and genome wide methylome analyses of TKO embryoid bodies revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet and 5hmC-mediated DNA demethylation in proper regulation of gene expression during differentiation of embryonic stem cells and development. Methylation patterns in tissue samples from a series of wt and Tet1/Tet2 DKO embryos, neonates and adults were generated using ethylated DNA immunoprecipitation with antibodies against 5mC (MeDIP) and 5hmC (hMeDIP) followed by deep sequencing.