Hematopoietic cell development is programmed by nuclear regulatory factors (transcription factors), many of which are targeted by chromosomal translocation or somatic mutation in leukemias. The transcription factor GATA-1, originally isolated as an erythroid enriched factor, has served as a paradigm for dissecting how hematopoietic development is controlled in vertebrates. Prior work has shown that GATA-1 can dominantly reprogram hematopoietic lineages and is also required for proper erythroid, megakaryocytic, mast, and eosinophil cell differentiation. GATA-1's activity in erythroid and megakaryocytic, but not mast or eosinophil, lineages is dependent on a zinc-finger cofactor known as FOG-1. The function of FOG-1 appears to be context-dependent in various developmental contexts. How the transcriptional activities of GATA-1 are affected by interaction with FOG-1 is poorly understood. Without insights into these mechanisms, it is not possible to account for how GATA-1 acts to direct hematopoietic differentiation in erythroid and megakaryocytic lineages, or explain how its functions may differ in mast and eosinophil lineages, where FOG-1 is not expressed. The principal goal of the proposed research is an improved understanding of how FOG-1 acts to modulate GATA-1-dependent gene regulation. The following specific aims are proposed: 1. Targeted mutation of the FOG-1 gene to assess the role of its N-terminus in vivo; 2. Affinity-tagging of FOG- 1 protein within cells to facilitate purification of FOG-1 associated proteins and chromatin fragments; 3. Testing the in vivo roles of FOG-1 associated proteins, including a protein isolated by yeast two-hybrid screening (TACC3) and any proteins that are identified by mass spectrometric sequencing of protein complexes; 4. Determining the chromatin occupancy by FOG-1, either in the presence or absence of associated GATA-1, and an unbiased search for FOG-1 associated chromatin; 5. Assess the capacity of FOG-1 to reprogram the fate of mast cells, and their committed precursors, to erythroid/megakaryocytic pathways. Addressing these specific aims should define how FOG-1 acts to control GATA-1-dependent transcription in hematopoietic cells and also distinguish target genes depend on GATA-1 + FOG-1 and GATA- 1 alone. These findings will be of general relevance to hematopoietic development and to leukemogenesis, particularly megakaryoblastic leukemia associated with somatic mutation of GATA-1.
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