Analysis of gene regulation in the beta-globin loci has been instructive in establishing a number of the founding principles for the differential, tissue-specific transcriptional control of vertebrate genes. The PI proposes to continue analysis of the cis-regulatory mechanisms and trans-acting factors involved in regulating the chicken and human beta-globin gene clusters, in particular to address the underlying mechanisms responsible for erythroid tissue- and developmental stage-specific transcriptional control (""""""""hemoglobin switching""""""""). This application proposes three lines of study to investigate cis- regulatory mechanisms that are responsible for globin gene switching using different experimental approaches: stable or transient transfection of mutated chicken beta-globin gene-bearing constructs into definitive or primitive chicken erythroid cells, and transgeneic mouse experiments in which a variety of mutated yeast artificial chromosomes (YACs) bearing the human beta-globin locus have been incorporated into the murine germ line. Both strategies focus on elucidating the underlying mechanisms controlling the differential expression of beta- globin locus genes during development, with particular emphasis on whether or, and if so how, regulatory sequence competition among distal and gene proximal transcriptional control elements of locus dictate developmental stage-specific expression. The proposal will also examine the question of whether or not developmental stage-specific transcription factor binding to the beta/epsilon-globin gene enhancer differentially contributes to chicken beta-globin gene switching.
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