ChIP-seq of QSER1-FLAG, TET1-V5, DNMT3A, DNMT3B, EZH2, BCOR, H3K4me3, H3K27me3, H3K36me2, H3K36me3, H3K27ac, and H3K4me1.

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. Overall design: ChIP-seq was performed for QSER1-FLAG (H1 WT and H1 TET1 KO hESCs), TET1-V5 (H1 WT and H1 QSER1 KO hESCs), DNMT3A (H1 WT, H1 QSER1 KO, H1 TET1 KO, and H1 QSER1/TET1 DKO hESCs), DNMT3B (H1 WT, H1 QSER1 KO, H1 TET1 KO, and H1 QSER1/TET1 DKO hESCs), EZH2 (H1 WT and H1 QSER1 KO hESCs), BCOR (H1 WT and H1 QSER1 KO hESCs), H3K4me3 (H1 WT and H1 QSER1 KO hESCs), H3K27me3 (H1 WT and H1 QSER1 KO hESCs), H3K36me2 (H1 WT and H1 QSER1 KO hESCs), H3K36me3 (H1 WT and H1 QSER1 KO hESCs), H3K27ac (HUES8 WT hESCs), and H3K4me1 (HUES8 WT hESCs).