TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

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. We generated TET single-, double-, and triple-knockout hESCs and differentiated them towards the pancreatic endocrine fate. Two replicates were used for sequencing. We performed RNA-seq for WT and TET triple deficient lines (clone 2 and clone 6) of hESC, definitive endoderm (DE) and pancreatic progenitor (PP), as well as TET1 single-deficient line of PP. We performed WGBS (one replicate), CMS-IP, ATAC, H3K4me1, H3K27ac ChIP-seq data for WT and TET triple deficient lines. One replicate of WT TET1 ChIP-seq were also sequenced.