REGULATION OF THE HUMAN BETA-GLOBIN LOCUS IN VITRO

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Regulated transcription of the human beta-globin gene family requires the selective expression of individual genes at distinct stages of erythroid development. In vivo studies have shown that the erythroid DNA-binding protein, EKLF, is essential to generate an open chromatin structure in the beta-globin promoter and mediate the fetal-to-adult globin switch during erythropoiesis. In order to understand the mechanisms involved in developmentally-controlled globin gene switching, we have used a biochemical approach to assemble cloned beta-globin genes into specific chromatin structures and then analyze their transcriptional activity in vitro. In the current project, we demonstrate that EKLF-dependent beta-globin activation is achieved on chromatin-repressed templates only in the presence of a newly defined co-activator, E-RC1. We have purified and characterized E-RC1 and shown it to be a distinct member of the mammalian SWI/SNF family of chromatin remodeling complexes. In combination with EKLF, E-RC1 specifically activates adult beta-but not fetal gamma-globin transcription in a chromatin-assembled gamma-beta-globin minilocus containing the two linked genes. In addition, we have isolated another remodeling complex from fetal erythroid cells that activates gamma-globin but not beta- globin transcription in vitro on chromatin templates. Thus, two different remodeling complexes are isolated and shown to recapitulate an important aspect of fetal-to-adult globin gene switching. First, experiments are designed to analyze the ability of E-RC1 to function with a variety of DNA-binding proteins, in addition to EKLF, to remodel chromatin structure and activate transcription in vitro. This will address whether chromatin remodeling complexes display specificity towards particular DNA-binding factors or classes of proteins. Second, the mechanism by which E-RC1 remodels nucleosomal structure and why it is targeted to the beta-globin but not gamma-globin promoter will be analyzed. Third, other remodeling complexes will be examined for their role in globin gene switching. Fourth, the remodeling complex from fetal erythroid cells that specifically activates the gamma-globin gene will be characterized. The beta-globin gene family serves as an important paradigm for the many levels of transcriptional regulation required to achieve tissue-specificity and proper cellular differentiation. The proposed studies should provide insight into these processes and a greater understanding of how changes in gene expression and cellular dedifferentiation occur in many malignancies.