ABSTRACT The central objective of this proposal is to establish how Tet enzymes regulate the specification of blood cells from endothelial cells during embryonic hematopoiesis, and from pluripotent stem cells in vitro. During embryo- genesis, hematopoietic stem and progenitor cells (HSPCs) emerge from endothelial cells (ECs) in the arterial vasculature. A sub-population of ECs undergo an endothelial-to-hematopoietic transition (EHT) to specify hemogenic endothelial cells (HECs), which then give rise to HSPCs. This specification of ECs to HECs and to HSPCs is guided by timely expression of hematopoietic genes involving the Runx, Gata and Gfi1 family of transcription factors. While the developmental origins of hematopoietic cells are broadly defined, the molecular mechanisms underlying blood formation are poorly understood. A major gap in the field has been how epigenetic paradigms such as DNA methylation/demethylation regulate activation of hematopoietic programs in ECs to allow for specification of HECs and formation of HSPCs. DNA methylation, a major modification that silences genes, is deposited by the DNA methyltransferases and removed by the Tet DNA demethylases. Tet enzymes are dynamically expressed during development and in many hematopoietic cell types. We have developed two mouse models that allow for deletion of Tets temporally (post-gastrulation) as well as spatially (in the endothelial lineage only) during embryogenesis. Using these models, we have recently shown that Tet enzymes are essential for activation of hematopoietic-specific gene expression programs in ECs, and are required for embryonic hematopoiesis in mice; functions that are distinct from the roles of Tet enzymes in adult hematopoiesis. While these findings define a requirement for Tets in emergence of HSPCs from ECs, it is yet to be established (1) how Tets specifically regulate EC-to-HEC commitment, (2) how Tet-mediated DNA demethylation activate hematopoietic programs, (3) How are Tets recruited to hematopoietic enhancers, and (4) whether Tets regulate and enhance derivation of HSPCs from pluripotent stem cells. We hypothesize that Tet enzymes are required for EC-to-HEC transition where Tet-mediated demethylation in ECs is essential for activation of hematopoietic enhancers. We also postulate that Tets regulate derivation of HSPCs from pluripotent stem cells in vitro. We propose two aims. Aim 1 will define the requirements of Tet enzymes in specification of HECs/HSPCs from ECs during embryonic hematopoiesis in vivo, and from mouse embryonic stem cells in vitro. Aim 2 will establish how Tets are recruited to hematopoietic enhancers where they demethylate and activate enhancers to promote transcription factor binding and expression of hemato- poiesis programs. Our findings will establish Tets as major epigenetic regulators of hematopoietic lineage specification in vivo and in vitro. It will inform on epigenetic basis of blood formation with implications in treatment of bl...