Molecular Mechanisms of Globin Gene Expression

St. Jude Children'S Research Hospital, Memphis TN

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Abstract

The long term objectives of this project are to determine the mechanisms by which erythroid Kriippel-like factor[unreadable] (EKLF) contributes specifically to the developmental control of 13-globin gene expression and more generally to[unreadable] erythropoiesis in vivo. Utilizing an EKLF-dependent erythroblast model, studies of the structural determinants[unreadable] of EKLF function have identified separable chromatin remodeling and transactivation domains. Moreover, these[unreadable] experiments demonstrate that additional sequences outside the previously defined in vitro remodeling domain[unreadable] are required for modulation of [3-globin promoter structure. In contrast to studies utilizing transient reporter[unreadable] assays, a novel internal activation domain, which is sufficient for induction of endogenous 13-globin gene[unreadable] expression to wild type levels was observed. To extend these observations, the first specific aim will assess the[unreadable] ability of the defined domains to modulate local and regional chromatin remodeling, transcription and globin[unreadable] gene switching in the context of an intact animal. This will be accomplished by deriving knock-in mouse strains[unreadable] that express various EK.LF domains. Two of these mouse lines will test the hypothesis that an EK_LFdomain[unreadable] which can mediated chromatin remodeling but lacks transactivation potential, is sufficient to recruit the distal[unreadable] locus control region enhancer to the 13-globin promoter in definitive erythroid cells. In complementary[unreadable] experiments, a similarly derived knock-in EKLF mutant encoding the novel transactivation domain but lacking a[unreadable] second previously described amino terminal transactivation region will be tested for its ability to rescue normal[unreadable] erythropoiesis. The determination that additional polypeptide sequences are required for remodeling of the[unreadable] endogenous 13-globin promoter has resulted in a working hypothesis that additional as yet unidentified factors[unreadable] are necessary for this process. Studies in the second specific aim focus on the identification and characterization[unreadable] of these factors. Biochemical approaches utilizing reagents already in hand will be exploited to identify the[unreadable] components of this complex. Long-term, the genes identified will be studied by deriving mice in which the[unreadable] corresponding genomic loci are targeted. Together, the studies will provide important insights into the critical[unreadable] functions of EKLF that are essential for erythropoiesis. This fundamental knowledge is likely to expand our[unreadable] understanding of the molecular mechanisms regulating the 7- to [3-switch in globin gene expression, potentially[unreadable] identifying therapeutic targets for the treatment of sickle cell disease.