Methyl capture sequencing (Truseq Epic from Illumina) of H1 WT, H1 QSER1 KO, H1 TET1 KO, H1 DNMT3B KO, H1 QSER1 KOpm (passaged matched with DKOs), H1 QSER1/TET1 DKO, and H1 QSER1/DNMT3B DKO hESCs

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. For methyl capture sequencing, genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. Two replicates were done for each genotype.