Ubiquitin-mediated degradation down-regulates hematopoiesis regulators, including AML1, p130, p27Kip, and JAK2. AML1 and JAK2 both play roles in causing leukemia, so determining mechanisms for how ubiquitination regulates hematopoiesis is critical for devising methods to prevent or treat leukemia. This pathway's substrate specificity is largely determined by E3 ubiquitin ligase complexes. Little is known about which ligases target these regulators for destruction. The CUL-4A cullin is a core E3 component, and this gene affects hematopoiesis. CUL-4A -/- embryos die in utero. Half of CUL-4A +/- mice die in utero and half display hematopoietic dysregulation. CUL-4A overexpression in cell lines interferes with cell cycle exit and granulocyte and erythrocyte terminal differentiation. These results lead to the hypothesis that a Cul-4A E3 liqase targets for degradation regulators that inhibit proliferation and promote differentiation, and this function is required to establish progenitor and stem cell homeostasis. Proposed studies: Identify Cul-4A E3 targets by (a) identifying targets by co-purification with Cul-4A, and (b) testing if known regulators that inhibit proliferation and promote differentiation are degraded by Cul-4A-mediated ubiquitination (Aim1). Evaluate the effect of CUL-4A haploinsufficiency on the differentiation, proliferation, and survival of hematopoietic progenitors and stem cells (Aim2). Analyze hematopoietic cells from CUL-4A +/- mice by competitive repopulation assay and multilineage analysis (2a). Affected lineages identified in 2a will be evaluated in clonogenic assays for progenitors (2b). Proliferation and apoptosis will be evaluated in affected progenitors and stem cells by clonogenic assays and analyses of phenotypically defined populations of cells (2c & d). Future in vivo and in vitro studies will focus on the functions of identified Cul-4A E3 targets, their regulation by Cul-4A, and how these targets regulate processes (apoptosis/proliferation) in progenitors and more primitive cells found to be affected by CUL-4A haploinsufficiency.
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