PROJECT SUMMARY Clonal hematopoiesis of indeterminate potential (CHIP) refers to the presence of expanded blood cell clones with one or more somatic mutations without other hematologic abnormalities. CHIP is common in the elderly, affecting more than 10% of individuals over 65 years. CHIP is associated with a 10-fold increase risk of hematologic malignancies and a doubled risk of atherosclerotic cardiovascular disease, contributing to an increase in all-cause mortality. The two most commonly mutated genes in CHIP are DNMT3A and TET2, both of which encode epigenetic modifiers. Recent studies have found evidence of increased inflammation and worsened atherosclerosis when the murine ortholog of TET2 was perturbed in myeloid cells in vivo. However, the specific epigenetic mechanism underlying this phenotype and whether DNMT3A and TET2 mutations target the same biological pathway are unclear. Furthermore, it remains unexplained why loss-of-function mutations in DNMT3A and TET2 have shared a clinical outcome despite the two genes encode enzymes with opposite biochemical functions (DNA methylation vs. de-methylation). To address this knowledge gap, we developed human pluripotent stem cell (hPSC)-derived macrophage models of DNMT3A- and TET2- haploinsufficiency. hPSC-derived macrophages are inexhaustible, scalable, and amenable to genetic manipulation, offering a powerful in vitro model well-suited for mechanistic studies. In this proposal, we propose the use of this model to (1) define unique and shared epigenetic features of DNMT3A and TET2 haploinsufficiency, and (2) identify transcription factors driving altered immune gene expression in DNMT3A- and TET2-haploinsufficient immune cells. This project is built on our previous work, and we anticipate that findings from this study will guide future studies on targeting an epigenetic vulnerability shared between DNMT3A- and TET2-mutated cells as well as mechanistic studies on other drivers of CHIP.