This proposal addresses the role played by coactivator and corepressor complexes in myelopoiesis and their role in abnormal differentiation patterns in AML. Specifically, this application outlines a series of experiments that focus on the transcriptional co-factor SHARP (SMRT/HDAC Associated Repressor Protein) and a newly identified splice variant, which I call SHARP2. SHARP, a co-repressor protein that has SMRT/HDAC, retinoic acid receptor (RAR), and DNA binding domains, represses transcription by regulation of HDAC activities. SHARP2, which is absent from stem cells and is rapidly upregulated in all differentiated hematopoietic cells in both murine and human bone marrow, is identical to the 3' end of SHARP, which contains the repressive SMRT/HDAC binding domain. However, it lacks the more 5' RAR and DNA interaction domains. Based on its differential expression pattern and its regions of homology to SHARP, I hypothesize that a normal function of SHARP is to block differentiation by negative regulation of lineage specific genes, and that SHARP2 antagonizes gene inhibition mediated by SHARP, thereby permitting the activation of differentiation specific genes. I propose to test thishypothesis first by using hematopoietic cell lines, in order to focus on how SHARP2 acts as a myeloid co-factor to promote hematopoietic differentiation. Because SHARP has been implicated as playing a regulatory role in receptor tyrosine kinase signaling (RTK) and SHARP and SHARP2 are reciprocally expressed during hematopoiesis, we will determine whether SHARP and SHARP2 are differentially regulated in different forms of human AML, in which RTK signaling is dysregulated. Because SHARP2 likely regulates gene transcription by modifying the co-repressor complex, we will also assess the relative affinities of SHARP and SHARP2 for co-repressor versus co-activator complexes, and determine how this correlates with myeloid gene expression. In addition, we will determine the contribution of SHARP2 to myelopoiesis by creating mice that express SHARP2 on a tet-inducible promoter. SHARP is highly homologous to the gene on chromosome 1 that has been identified from chromosomal translocation t(1;22) in acute myeloid leukemia (AML), FAB M7. Understanding the mechanisms by which SHARP and SHARP2 mediate development of normal blood cells and leukemia will help us to gain more information about leukemogenesis and could lead us to possible therapeutic approaches for leukemia patients.
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