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:RNA Sequencing of H1 WT hESCs, H1 QSER1 KO hESCs, H1 TET1 KO hESCs, H1 QSER1/TET1 DKO hESCs, WT Day10 embryoid bodies (EBs), QSER1 KO Day10 EBs, TET1 KO Day10 EBs, QSER1/TET1 DKO Day10 EBs, WT pancreatic progenitors (PP1), QSER1 KO PP1, TET1 KO PP1, and QSER1/TET1 DKO PP1. DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to tightly regulate the methylation landscape remains a central question. Utilizing a knockin DNA methylation reporter, we performed a genome-wide CRISPR/Cas screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate cooperation of QSER1 and TET1 through genetic and biochemical interactions to inhibit DNMT3-mediated de novo methylation and safeguard developmental programs.
Project description:Tet enzymes (Tet1/2/3) convert 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 is not established. We have generated Tet1/2/3 triple knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO-ESCs contributed poorly to chimeric embryos and could not support embryonic development. Global gene expression and methylome analyses of TKO-EBs 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 ESCs and development. To quantify global gene expression in differentiating embryoid bodies (EBs) derived from wild type (WT) and Tet triple knockout (TKO), TKO and WT mouse embryonic stem cells (ESCs) were differentiated in vitro to EBs and cultured for 10 days. RNA was extracted using Qiagen RNeasy kit and subjected to microarray analysis. Global gene expression profile of two technical replicas of WT embryoid bodies (2 samples in total) was compared to two technical replicas of two independent TKO embryoid bodies (4 TKO samples in total).
Project description:Purpose: To investigate the effect of Tet1 depletion on global DNA methylation, we performed whole-genome bisulfite sequencing (WGBS). Methods: Starting with as little as 1400-5251 manually micro-dissected PGCs, we used an ultra-low input method, Tn5mC-seq. Results: We generated 945 million reads for Tet1Gt/Gt PGCs and 302 million reads for wild-type PGCs. We obtained 14-16 million CpG sites per genotype at 1.76-2.66x genome coverage, which enables a comprehensive view of genome-wide DNA methylation patterns in E13.5 PGCs. PGCs are almost completely unmethylated genome-wide. Loss of Tet1 led to a subtle increase of methylation level in various genomic elements including promoters, exons, introns and repetitive elements in Tet1Gt/Gt PGCs (p<0.01). Local analysis identified 4,337 differentially methylated regions (DMRs) between Tet1-/- PGCs and wild-type cells. These DMRs are associated with 5,261 genes, among which 271 genes also exhibited differential gene expression and enriched for the cell cycle pathway (FDR=0.02). Conclusions: This result revealed that demethylation of certain set of cell cycle genes is largely abolished in the Tet1-/- PGCs. Genome-wide methylation profiles of primordial germ cells derived from the wild type (WT) and Tet1-null female embryos at E13.5 were generated by whole genome bisulfite sequencing using Illumina Hiseq.