Transcription factor GATA-1 is the central nuclear regulatory for erythroid development. Without GATA-1 erythroid precursors arrest at the pro-erythroblast stage and undergo apoptosis. During the formation of embryonic red blood cells in the developing yolk sac blood islands, GATA-1's function overlaps with that of GATA-2. Thus, a GATA-factor, either of the hematopoietic system. Uncovering how GATA-1 functions in transcriptional control and the nature of its target genes are both fundamental to our understanding of the molecular mechanisms responsible for hematopoietic development, erythropoiesis, and more generally lineage-specific differentiation. In the proposed studiers, aspects of GATA-1 structure will be related to in vivo function through gene targeting in the mouse. The contribution of GATA-1's N-finger to DNA-binding and function in vivo will be addressed by modification of amino acid residues that are involved in DNA contacts but not binding of the protein cofactor FOG-1. The potential roles of the putative N-terminal activation domain of GATA-1 and phosphorylation of specific serine residues will be examined by targeted knockin mutations and creation of mice. In the latter experiments, collaboration with the Lodish laboratory will reveal effects of mutation of specific serines on response to erythropoietin, cell survival, and STAT signaling. In a second area of studies, we will determine the in vivo role of a new GATA-1 target gene, ABC-me, in heme biosynthesis and erythropoiesis. The ABC-me gene encodes a mitochondrial transporter, believed to participate in heme biosynthesis. Through targeted mutation in the mouse and in culture mouse erythroleukemia cells, the requirement for ABC-me will be tested. Moreover, to pursue the genetics of ABC-me more broadly, we will isolate and study the zebrafish homologue in collaboration with the Zon laboratory. This work should provide a comprehensive assessment of the biology of this new GATA-1 regulated transporter.
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