5-hydroxymethylcytosine (5hmC) profiling of H1 WT hESCs. 5-hydroxymethylcytosine (5hmC) profiling of H1 WT hESCs

DNA methylation is essential to mammalian development, and aberrant regulation can lead to disease. The enzymes responsible for deposition and removal of DNA methylation have been identified, but how tightly regulated DNA methylation patterns are achieved globally remains a central question. To discover regulators of DNA methylation in human Embryonic Stem Cells (hESCs), we engineered a DNA methylation reporter line and performed a genome-wide CRISPR/Cas screen. Through our screen, we identified the functionally uncharacterized gene QSER1, which proved to be essential for protection from DNA hypermethylation at bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA Methylation Valleys (DMVs). Further mechanistic enquiry revealed that QSER1 protein depends on TET1 for efficient recruitment to DNA, and QSER1 occupancy inhibits the binding of DNMT3B. Our discovery of QSER1 is a major advance in understanding TET-dependent protection from DNA hypermethylation and provides mechanistic insight into how epigenetic factors can cooperate to target locus-specific regulation. Overall design: For 5hmC profiling (5hmC-Seal), genomic DNA was isolated from H1 WT hESCs using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. Two replicates were performed for each sample.