PROJECT SUMMARY/ABSTRACT The regulation of self-renewal and multi-lineage differentiation of hematopoietic stem cells (HSCs) is essential for lifelong maintenance of hematopoiesis, especially under conditions of hematopoietic stress or transplantation. Defects in HSC repopulating activity contribute to immune deficiencies, anemia, and an increased susceptibility to myeloid malignancies. HSCs typically exist in a quiescent state, but to self-renew they must enter the cell cycle. This is associated with an activation of mitochondrial oxidative metabolism. Thus, understanding how HSC quiescence and proliferation are regulated will provide key insights into how HSC function can be maintained in aging, under conditions of hematopoietic stress, or during transplantation. We have demonstrated that MCL-1 is an essential mediator of HSC survival in normal hematopoiesis and during recovery from stress. These functions are associated with MCL-1's pro-survival function on the outer mitochondrial membrane; however, MCL-1 can also be imported into the mitochondrial matrix where it regulates mitochondrial metabolism and fission/fusion dynamics. We have generated a mutant mouse that expresses a truncated MCL-1 protein (MCL-1OMM) which blocks apoptosis, but cannot be imported into the mitochondrial matrix. When subjected to myeloablative stress, Mcl1OMM mice exhibit defective regeneration and most animals die. Furthermore, bone marrow cells from the Mcl1OMM mice exhibit a profound competitive disadvantage in transplantation experiments. Our central hypothesis is that matrix-localized MCL-1 regulates mitochondrial dynamics and respiration in HSCs promoting their ability to self-renew and promote recovery from stress. The following aims will test this hypothesis: Aim 1: Analyze the HSC phenotype/function from mice lacking mitochondrial matrix-MCL-1. Aim 2: Determine how lack of mitochondrial matrix-MCL-1 affects mitochondrial dynamics and oxidative phosphorylation. Aim 3: Interrogate whether enforced expression of matrix-MCL-1 promotes HSC function. My laboratory has made many seminal findings defining the role of MCL-1 in promoting survival during normal hematopoiesis and in leukemia. We are uniquely positioned to successfully perform these studies as we have generated critical, novel model systems that will allow us to functionally dissect MCL-1's functions at the mitochondria. At the end of this study, we will illuminate a previously unrecognized role for MCL-1 in regulating HSC function.