The underlying goal of this proposal is to experimentally determine the mechanisms and molecules employed in the process of """"""""hemoglobin switching"""""""" in chickens, and to extend these studies into the homologous processes employed in globin gene regulation in mice and humans. During the past five years, many of the cis-regulatory sequences required for erythroid- specific transcriptional regulation of globin genes in several vertebrate organisms have been determined. Additionally, the identities of several of the protein factors used to accomplish these molecular switches have been revealed. During the next five years, we plan to define more precisely the cellular and molecular mechanisms which vertebrate organisms employ to elicit these erythroid cell lineage-specific changes in gene expression. We propose to test a number of central questions in erythropoiesis and in generation of the erythroid cell lineage. 1) Is the developmental process of hemoglobin switching homologous in chickens, mice and humans (are conceptually comparable mechanisms and trans-acting factors involved)? 2) Biochemically and mechanistically, how does the murine GATA-1 transcription factor activate erythroid-specific genes? 3) Do GATA transcription factor family members other than GATA-1 (all of which are found in erythroid cells) functionally contribute to erythroid cell differentiation and to hemoglobin switching? 4) Is hemoglobin switching determined in a single kind of progenitor cell, or do multiple erythroid progenitors exist during embryonic development? 5) How does transcription factor NF-E2 regulate cell lineage-specific transcriptional activation in chicken and mammalian erythroid differentiation? 6) Is NF-E4 a central factor involved in effecting the embryonic to adult beta-globin gene switch in chickens, and is the homologous factor playing a similar role in human globin gene switching? 7) Do the chicken and human beta-globin gene enhancers display a developmental stage-specific preference for the gene to be activated? 8) Is the sequential transcriptional activation and silencing of the human embryonic and fetal beta-globin genes due to developmentally regulated alternative utilization of globin gene distal and proximal regulatory elements during development? The answers to these questions, should these experiments prove to be successful, will provide unprecedented insight into this most fundamental developmental regulatory process, and coherent strategies for direct molecular intervention into altering human hemoglobin disease states could then be proposed.
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