Project Summary Chronic infections affect over 2 billion people worldwide and can impair the function and differentiation of hematopoietic stem cells (HSCs), which produce all circulating blood cells. Patients chronically infected with Mycobacterium tuberculosis often have bone marrow suppression and pancytopenia, resulting in the depletion of platelets, erythrocytes, and leukocytes. However, the molecular mechanism of Mycobacterium-induced bone marrow suppression is not fully elucidated. Our lab has established a mouse model of Mycobacterium avium (M. avium) infection to study the influence of chronic inflammation on HSCs. Using this model, we have shown that chronic infections significantly reduce the number of hematopoietic stem and progenitor cells (HSPCs). We have also demonstrated that basic leucine zipper ATF-like transcription factor 2 (Batf2), an interferon-activated immune response regulator, mediates the terminal differentiation of HSCs in the context of infection. Specifically, overexpression of Batf2 in mice promotes myeloid differentiation of HSCs. Conversely, the depletion of BATF2 in human HSCs reduces interferon gamma-dependent myeloid differentiation. Our preliminary data further show that Batf2 is not efficiently induced in mice lacking DNA methyltransferase 3a (Dnmt3a), suggesting that expression of Batf2 may be epigenetically regulated by Dnmt3a. However, the mechanisms underlying how Batf2 drives HSCs differentiation in response to infection remain poorly understood. We hypothesize Batf2 expression is epigenetically regulated through DNA methylation and Batf2 interacts with transcription factors to drive HSC myeloid differentiation during chronic infection. Based on data from our group’s previous studies, the first object of this project is to determine whether Batf2 is necessary to promote HSC myeloid differentiation and impair self-renewal in chronic infection. Specifically, we will characterize the HSPC populations in Batf2-deficient mice in the setting of chronical M. avium infection using flow cytometry and bone marrow transplant. Next, we will identify Batf2-interacting partners in HSPCs during chronic infection by performing co-IP and CUT&RUN to identify the proteins and DNA sequences that bind to Batf2 in HSPCs during infection. We also will use ATAC-seq and RNA-seq to study the potential genes that are regulated by Batf2 in HSPCs from M. avium-infected mice. Finally, we will investigate how epigenetic modification affects Batf2 expression in HSPCs during infection. We will study the methylation status of Batf2 and previously described binding partners in HSCs from Dnmt3a-deficient (Dnmt3a KO) and control mice using whole-genome bisulfite sequencing. We will overexpress Batf2 in HSPCs from these mice to address the dependency of Dnmt3a-regulated HSC self-renewal on Batf2. Overall, the work in this proposal will uncover a novel mechanism by which Batf2 promotes HSCs differentiation during chronic infection and may e...