Screen identifies demethylation regulator
DNA methylation was one of the first epigenetic mechanisms discovered, but there is a limited understanding of its regulation and dysregulation in the context of development and disease. Dixon et al. performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to identify DNA methylation regulators (see the Perspective by Gu and Goodell). A top screen hit, QSER1, proved to be essential for maintaining low methylation at DNA methylation valleys, which overlap with developmental genes and broad H3K27me3 and EZH2 peaks. Mechanistic examination revealed that QSER1 and the demethylating enzyme TET1 cooperate to safeguard developmental programs from de novo methylation by the enzyme DNMT3.
DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions, including immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) and microcephalic dwarfism. The DNMT and TET enzymes are responsible for the addition and removal of DNA methylation, but how they coordinate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells (hESCs) to discover DNA methylation regulators.
We focused on bivalent promoters, defined by the presence of both active (H3K4me3) and repressive (H3K27me3) histone marks and typically occupied by Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2). In stem or progenitor cells, bivalent promoters are believed to maintain developmental regulators in a “poised state” ready for activation upon differentiation, and they are sensitive to DNA hypermethylation in dysfunctional cellular contexts such as cancer or aging. Creation of a knockin DNA methylation reporter line provided a singular opportunity to visualize epigenetic alterations otherwise “invisible” in terms of gene expression changes in the stem cell state. Using the bivalent PAX6 P0 promoter as a representative locus, we aimed to discover mechanisms that regulate DNA methylation at regions with similar chromatin features, which would inform not only gene regulation during development but also epigenetic dysregulation in disease.
Our screen successfully identified not only known methylation regulators such as TET1, TDG, and KDM2B but also functionally uncharacterized genes, including QSER1. Like the TET proteins, QSER1 safeguards bivalent promoters and poised enhancers (marked by H3K4me1 but not H3K27ac) against hypermethylation. However, distinct from the more general protective effect of the TET proteins on regulatory regions, QSER1 preferentially protects broad PRC2-bound and H3K27me3-marked regions and DNA methylation valleys (DMVs). Also known as DNA methylation canyons, DMVs identify large (≥5 kb) hypomethylated regions present in cells of many lineages and conserved across vertebrates. They are enriched with bivalent promoters, developmental genes, and transcription factors, including PAX6 and the HOX genes. QSER1 and TET1 showed high correlation in genomic occupancy measured by chromatin immunoprecipitation sequencing (ChIP-seq) and both were high at DMVs, whereas the binding of de novo methyltransferases DNMT3A and DNMT3B was excluded in DMVs and relatively enriched in the flanking regions. Further proteomic and genomic analyses revealed that QSER1 and TET1 share many common interacting proteins, depend on each other for efficient recruitment to DNA, and cooperate to limit the encroachment of DNMT3A and DNMT3B in DMVs. In addition, deleting DNMT3B reversed the hypermethylation in QSER1 knockout (KO) hESCs. Furthermore, combined KO of QSER1 and TET1 had a stronger impact on DNA methylation and gene expression than either single KO and resulted in a failure of hESC differentiation to PDX1+NKX6.1+ pancreatic progenitors.
We show that QSER1 cooperates with TET1 to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation at important genomic loci, especially in DMVs and bivalent promoters where hypermethylation has been linked to developmental disorders and cancers. Our work provides mechanistic insight into the region-specific regulation of the TET pathway and highlights the utility of unbiased genome-wide screens and locus-specific epigenetic measurements, including, but not limited to, DNA methylation for probing the epigenetic regulatory mechanisms relevant to human health.
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 regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 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 genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.