Understanding the mechanisms by which globin genes are regulated in vivo is relevant to the design of novel approaches to management of thalassemia and hemoglobinopathies by drug or somatic gene therapy. Moreover, elucidating the basis of erythroid expression will lead to fundamental insights into the development of hematopoietic cells. Recent studies have implicated an abundant DNA-binding protein, GATA-1, as a central regulator of gene expression in erythroid cells. Gene targeting in mouse embryonic stem (ES) has established GATA-1 as an essential protein for erythroid cells to accomplish its proposed roles. This research plan is directed toward resolution of these outstanding issues. First, two approaches will be undertaken to define the cis-regulatory elements of the mouse GATA-1 gene: (i) GATA-1/lacZ gene constructs will be used to delineate sequences required for gen activation in transgenic mice; (ii) a two-step procedure involving gene targeting and Cre-mediated site-specific excision in ES cells will be used to test the role of upstream and intronic regions. By these approaches, the critical cis- regulatory sequences of the GATA-1 gene will be mapped. A long-range goal is identification of the regulatory proteins involved in turning-on GATA-1 in progenitors and maintaining its expression. Study of the structure and function of GATA-1 will include (i) x-ray crystallography of the novel two-finger DNA-binding domain complexed to DNA; (ii) analysis of the role of phosphorylation at multiple ser residues in the protein in DNA-binding, cellular localization, and its capacity to direct differentiation either in the 416B megakaryocytic differentiation assay or in rescue of GATA-1 minus ES cells; (iii) detection of protein-protein interactions of GATA-1 with other transcription factors (SP1, EKLF) and with itself in vitro in a GST-pull down assay and in """"""""two-hybrid"""""""" assays in mammalian cells and yeast; and (iv) the search for novel erythroid proteins that interact wit GATA-1 or components of the basal transcription complex (TFIID and TFIIB) by cloning of cDNAs in yeast using GATA-1 as the tethered protein and in vitro using a protein interaction screen of gammagt11 MEL cDNAs. A long-range goal is the discovery of novel proteins that are critical to the establishment of an erythroid transcriptional complex, but which do not themselves directly contact DNA.
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