The goal of this project is to discover fundamental epigenomic regulatory mechanisms that commit cells to defined fates during early stages of embryogenesis. Early in development, commitment of the epiblast to germ layers is followed by activation of key regulatory genes that drive lineage fate. These genes control normal development and underlie the genetic basis for a broad range of human structural birth defects. We have studied members of the TET family of hydroxylation enzymes, which regulate the demethylation of DNA, or block active methylation of DNA, to control gene expression. We discovered requirements for TET enzymes during early development in the zebrafish model, and for progenitor specification from human embryonic stem cells (hESCs). Major gaps in understanding include: i) whether common or distinct mechanisms control demethylation for progenitors from different germ layers, ii) whether different TET family members distinguish developmental programs, and iii) how TETs are targeted to regulatory regions such as bivalent promoters. Suspecting that additional proteins beyond TETs are needed to target DNA demethylation, we carried out a genome-wide CRISPR screen and discovered QSER1, a previously uncharacterized chromatin-binding protein. We showed that QSER1 is a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys, broadly across different cell fates. We found biochemical and genetic interactions between QSER1 and TETs, suggesting that they cooperate to safeguard transcriptional and developmental programs from methylation. QSER1 variant alleles were recently linked to coronary artery disease, while haploinsufficiency of a QSER1 paralog, PRR12, is associated with multi-organ developmental birth defect syndromes. We propose to fully explore the genetic relationships and downstream networks of TET/QSER1 (TQ) family members, including how they function to control methylation and impact chromatin structure in the context of two complementary model systems, zebrafish and hESCs. The zebrafish model allows full genetic analysis of potentially compensatory or cooperating family members (including tet1, tet2, tet3, qser1, and prr12), in an animal model with highly conserved developmental programs. The hESC model provides outstanding biochemical and “omics” capacity, and validation in developing human progenitor and differentiated cells. The multi-PI project represents a continued collaboration among investigators with complementary and overlapping expertise, with a strong record of productivity. Specific Aims are proposed to determine the relative contribution of these genes for directing early progenitor fate, discover the regulatory networks in which they function, and to test interacting factors as candidates for linking TQ methylation control to chromatin modification. Because regulation of methylation is a fundamental step of progenitor fate determination, our...