PROJECT SUMMARY The goal of this proposal is to understand how KMT2C/MLL3 regulates hematopoietic stem cell (HSC) self- renewal and why KMT2C mutations convey a selective advantage that can lead to leukemia. KMT2C encodes MLL3, a COMPASS family histone methyltransferase that binds enhancer elements and promotes transcription. KMT2C is mutated in human leukemias, both as part of large deletions of chromosome 7q and at specific domains such as the PHD zinc finger domains (which bind chromatin) or the SET methyltransferase domain (which can prime enhancer elements for activation). Prior murine studies have established that Kmt2c deletions enhance HSC self-renewal and promote leukemogenesis, but the mechanism is not clear. To better understand how Kmt2c regulates HSC self-renewal, we generated germline and conditional loss-of-function mice. Kmt2c deletions enhanced HSC self-renewal, consistent with prior observations, but the mutations did not alter cell cycle kinetics by themselves. Instead, Kmt2c deletions allowed serially transplanted or chemotherapy treated HSCs to retain self-renewal capacity after multiple division cycles. This allowed the mutant HSCs to outcompete wild type HSCs during marrow recovery. In the absence of stress, Kmt2c deletions did not convey a selective advantage. Altogether, our data suggest that Kmt2c mutations mitigate a phenomenon, called HSC exhaustion, in which HSCs lose self-renewal capacity after several cumulative divisions. Our mechanistic data suggest that MLL3 primes HSCs to differentiate in response to IL-1, and possibly other inflammatory cytokines, by either enhancing IL-1 signal transduction or by facilitating IL-1 target gene expression. The aims of this proposal are designed to extend these observations. Aim 1 will test whether Kmt2c/MLL3 deficiency conveys a selective advantage to dividing HSCs by reducing sensitivity to IL-1 and other inflammatory cytokines. Aim 2 will characterize the structure, regulation and IL-1 responsiveness of MLL3 target enhancers in HSCs with short and extensive division histories. Changes in enhancer priming may allow HSCs to archive their division histories and favor commitment, rather than self-renewal, after multiple division cycles. Aim 3 will test whether MLL3 requires functional SET or PHD domains to restrict HSC self-renewal capacity. This structure-function analysis will help us better understand how specific KMT2C mutations might convey a self-renewal advantage. If we can understand how HSCs change as they undergo cumulative self-renewing divisions, and how Kmt2c deletions convey a selective self-renewal advantage, we may ultimately be able to preserve HSC function through periods of stress, such as post-chemotherapy periods, without increasing leukemia risk.