Epigenetic Regulation of Embryonic and Intestinal Stem Cells by DNA Hydroxymethylation

DNA methylation at the 5 position of cytosine is a well-characterized epigenetic modification that is important for essential cellular processes including stem cell proliferation and differentiation. 5-methylcytosine (5mC) can be oxidized by the Ten Eleven Translocation (TET) family of enzymes to 5-hydroxymethylcytosine (5hmC), which has been suggested as either an intermediate in DNA demethylation or as an independent epigenetic modification that directly regulates gene expression. Gene silencing and activation are important processes in embryonic stem cells (ESCs) and intestinal stem cells (ISCs) for their self-renewal and differentiation, but our understanding of the potential role of 5hmC and TET hydroxylases in these processes is still limited. Here, I generate genome-wide maps of the 5hmC mark in ISCs and their differentiated progeny and utilize Tet1-/- mice model to investigate the role of TET1 in the intestinal epithelium. Genes with high levels of hydroxymethylation in ISCs are strongly enriched for developmental regulation functions. The Tet1-deficient postnatal intestine shows significantly reduced numbers of proliferative cells and decreased Wnt target genes expression, which correlates with lower 5hmC levels at their promoters. These data demonstrate that Tet1-mediated DNA hydroxymethylation is important for the self-renewal of the intestinal epithelium. To determine a novel role of 5hmC, we perform integrative analyses of various epigenomic sequencing datasets, and identify a group of distal transcription factor binding sites as putative silenced enhancers, which are lacking active histone marks and nascent transcription, but are nevertheless highly enriched for the 5hmC modification in ESCs. During lineage specification of ESCs, these silenced elements lose 5hmC and acquire the H3K4me1/2 histone marks and become active. I demonstrate that these elements function as enhancers when the 5hmC mark is removed by a reporter assays. These data suggest that 5hmC suppresses the activity of a specific subset of enhancers in ESCs. In summary, DNA hydroxymethylation contributes to both transcriptional activation and repression for epigenetic gene regulation in stem cells.