Transcription factor GATA-1 is critical for the development of multiple blood lineages, including erythroid cells, megakaryocytes, mast cells and eosinophils. Mutations in the Gata1 gene underlie a variety of congenital and acquired hematological disorders. GATA-1 can activate and repress gene expression to promote cell differentiation, cell cycle arrest and cell survival. In previous work we have shown that GATA-1 is acetylated at conserved lysine-rich motifs near the zinc finger regions, and that acetylation is essential for its function. Recently, we found that acetylation is required for the stable association of GATA-1 with chromatin in vivo but not for binding to naked DNA in vitro. In new studies, we discovered that GATA-1 via its acetylated region interacts with the bromodomain protein Brd3, and that Brd3 is recruited to GATA-1-dependent sites in erythroid cells vivo. Brd family proteins are of great interest since they can interact with components of the basal transcriptional machinery to govern initiation and elongation. Moreover, some Brd proteins have been shown to remain bound to chromatin during mitosis whereas the great majority of nuclear factors are dispersed into non-chromatin space. This might provide an epigenetic memory function to re-assemble transcription factor complexes at the appropriate sites upon entry into the G1 phase of the cell cycle.
In Aim 1, we will characterize the GATA-1- Brd3 interaction using biochemical and structural tools. We will determine the acetylation sites of GATA-1 in vivo and examine the function and regulation of GATA-1 acetylation during the cell cycle and under varying conditions of cell growth and differentiation.
In Aim 2 we will examine the Brd3 genomic occupancy patterns. We will define the mechanisms by which Brd3 regulates GATA-1 in cell based assays focusing on chromatin occupancy during mitosis and interphase as well as a role during transcription elongation. Experiments proposed in Aim 3 will investigate the in vivo function of Brd3 during hematopoietic development and GATA-1-regulated gene expression in zebrafish and mice. Together these studies are aimed at elucidating a novel transcriptional circuitry that might underlie epigenetic maintenance of gene expression patterns throughout the cell cycle and elucidate novel functions for transcription factor acetylation.
The proposed studies are aimed to better understand the mechanisms underlying the formation of the blood lineages and their associated disorders. Our work focuses on how nuclear factors and their chemical modifications control the genes that govern the specification and differentiation of blood cells. Progress is this area promises novel approaches to treat blood-related disorders including leukemias and anemias.
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