Abstract During the previous funding period, we made important strides in answering the fundamental mechanistic question of how TET2, DNMT3A and ASXL1 mutations give rise to clonal hematopoiesis (CH), cancer and inflammation. We performed biochemical and genomic analyses that linked TET deficiency to an unexpected loss of DNA methylation in heterochromatin. Given that DNMT3A and TET proteins have opposite biochemical functions, our finding of decreased DNA methylation in heterochromatin of both Dnmt3a KO and Tet2 KO cells is the only common feature that explains the unexpectedly greater disease severity of Dnmt3a KO, Tet2 KO (DKO) mice compared to mice with individual Dnmt3a or Tet2 gene disruptions. We also showed that CH- and cancer-associated mutations in ASXL1 result in reduced histone 3 lysine methylation (H3K9me2/me3) in hetero- chromatin, thus linking all three of the top proteins mutated in clonal hematopoiesis – TET2, DNMT3A and ASXL1 – to the fundamental process of impaired heterochromatin integrity due to reduced DNA or H3K9 methylation in heterochromatin. Interference with DNA or H3K9 methylation in heterochromatin has been known for decades to result in increased expression of transposable elements, genome instability and inflammation, all common features of cancer, inflammation and aging. Finally, we showed that OGT normally restrains TET activity in mESC, and that consequently, OGT deficiency increases TET activity genome-wide and results in genome-wide DNA demethylation. In this renewal application, we will ask how the TET-OGT interaction regulates DNA methylation, focusing on the protein-protein interactions and regulatory mechanisms that operate within TET- OGT complexes in mESC and hematopoietic-lineage cells. We will define how DNMTs and TETs cooperate to reduce DNA methylation in heterochromatin by examining DNMT3A redistribution and DNMT1/UHRF1 stability. We will explore the mechanism of how CH-associated mutations in ASXL1 lead to reduced H3K9 methylation in heterochromatin, again focusing on protein-protein interactions and regulatory mechanisms that operate within two distinct ASXL1-associated protein complexes that we have recently defined. We will identify individual members of TE families that are uniquely upregulated in TET2, DNMT3A and ASXL1-mutant cells, so as to determine how increased TE expression can have potentially indirect and stochastic effects on the expression of nearby genes by acting as promoters, enhancers or both. Lastly, we will perform CRISPR/Cas9 screens to elucidate how TET deficiency and ASXL1 mutations are linked to inflammation. Our studies will illuminate the mechanistically elusive connections between DNA and H3K9 methylation, clonal hematopoiesis, cancer, inflammation and aging, and improve our understanding of heterochromatin, a compartment of the genome that is very poorly understood.