Project Summary Infections are the number one cause of morbidity and mortality in hematopoietic stem cell transplant (HSCT) patients. Infections contribute to delayed or failed engraftment of hematopoietic stem and progenitor cells (HSPCs). To study the mechanism underlying infection-related impaired HSPC function, our lab has utilized a Mycobacterium avium infection model and discovered that chronic infection depletes HSCs by impairing self- renewal and promoting myeloid differentiation – through increased activation and transcription of myeloid differentiation genes such as Batf2, Fosb, and Jun– via an interferon-gamma (IFNγ)-dependent mechanism. Further, the lab showed that inflammation-induced myeloid differentiation is epigenetically driven, as the knockout of DNA methyltransferase DNMT3A led to suppression of the myeloid differentiation response. Based on these data from our group’s previous studies, I hypothesize that HSPCs undergo a malleable epigenetic reprogramming in response to IFNγ that promotes myeloid differentiation and affects downstream immune responses. By defining the extent of epigenetic reprogramming, I seek to identify strategies to preserve HSC function despite infectious stress and thereby improve HSCT outcomes. Whereas our lab showed that IFNγ-dependent changes in DNA methylation contribute to HSC differentiation and exhaustion during chronic infection, the impact of IFNγ stimulation on histone modifications, another key mechanism of epigenetic regulation, has not been studied in HSPCs. Therefore, the first objective is to determine whether IFNγ induces histone modifications in HSPCs to promote myeloid differentiation. Specifically, I will use epigenomic sequencing techniques CUT&RUN-seq and ATAC-seq to determine the changes in histone modifications and chromatin accessibility in HSPCs under inflammatory stress. Next, I will identify which pre-stimulated HSPC subpopulations are functionally reprogrammed by transplanting M. avium- stimulated HSPC subpopulations into naïve recipients and challenging the recipients 3 months later with M. avium. Finally, I will investigate the malleability of these epigenetic modifications by exposing M. avium- stimulated HSPCs to subsequent high-dose LPS. I will perform RNA-seq, CUT&RUN-seq, and ATAC-seq, and immune challenge experiments post-transplant to determine whether HSPCs exposed to LPS after M. avium stimulation show altered differentiation responses compared to those exposed to M. avium alone. Overall, the work in this proposal will uncover the mechanisms by which IFNγ promotes myeloid differentiation and HSC exhaustion during chronic inflammation and enable the development of therapeutic approaches to prevent graft loss in the early post-transplant period.