investigator s abstract) In a variety of species, the activity of the b-globin locus is regulated by a locus control region (LCR). The human and murine LCRs are comprised of five DNase I hypersensitive sites (5' HS 1-5) located 5' prime of the embryonic beta-like globin genes. Studies conducted by the P.I. indicate that naturally occurring deletions of the human LCR results in a failure to activate the beta-globin locus at the levels of chromatin transcription and replication timing. In addition, this group has shown that the LCR is essential for the function of the origin of replication of the locus located 50 kb upstream of the LCR, between the delta-and the beta-globin genes. In order to examine how the components of the LCR interact to regulate the endogenous beta-globin locus, homologous recombination (HR) strategies to mutate the endogenous human beta-globin LCR in situ, in somatic cell lines and in murine beta-globin LCR embryonic stem (ES) cells have been developed. Initial analysis of mice derived from the ES cells containing HR-generated mutations of the 5' HS-2 and 5' HS-3 have revealed that the beta-globin locus functions almost normally without these sites, results that would have not been predicted from results of transfection and transgenic assays. Accordingly, the goal of the current proposal is to use the newly developed HR strategies to elucidate how the beta-globin LCR and its component hypersensitive sites regulate the chromium structure, replication timing, and origin use, and transcription of the beta-globin locus. Specifically, this proposal will : 1) Determine the sequence of the human beta-globin LCR that are necessary and sufficient for the formation of the active chromatin structure, transcription, and replication timing and origin use characteristic of the beta-globin locus in the erythroid cells. 2) Determine the sequences necessary and sufficient for replication initiation between the human delta- and beta-globin genes, and determine the mechanism by which the LCR controls origin choice. 3) Determine the consequence of HR-generated LCR mutations on transcription, chromatin structure, and replication of the murine beta-globin locus. These studies will complement and extend previous analysis of the human beta-globin LCR, by determining the consequences of LCR mutations after passage through the germ line and at all stages of erythropoiesis. 4) Optimize the HR analysis of the murine beta-globin LCR by developing strategies to (a) increase the efficacy of generating HRs, and (b) allow rapid analysis of mutation in vitro in chimeric embryonic and adult mice.
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