The symptoms of Sickle Cell Disease and Cooley's Anemia can be ameliorated by reactivating the fetal globin genes. Although fetal globin is not normally expressed at high level in adults, significant expression is observed when erythroid cells or patients are treated with certain chemicals, which are believed to act by inhibiting transcriptional repression. Fetal globin expression also occurs when the fetal globin promoters are altered by point mutations, as seen in some cases of heriditary persistence of fetal hemoglobin (HPFH). We have focused on point mutations in the fetal globin promoters that are associated with HPFH and examined the roles of DNA-binding proteins that recognize the elements implicated by these mutations. We have shown that Basic Kruppel-like Factor (BKLF) binds together with GATA-1 over one site and the orphan receptor COUP-TF2 recognizes a separate site. In the case of BKLF experiments involving breeding BKLF knockout mice to transgenic mice carrying the entire human beta globin locus have confirmed that BKLF is required for fetal globin silencing in vivo. In vitro experiments also argue in favor of a role for BKLF. We now wish to carry out further in vivo and in vitro work to verify that BKLF is directly involved, to identify other components of the silencing complex and to investigate the mechanism of silencing. We will use chromatin immunoprecipitation assays to confirm the presence of the silencing complex, biochemical fractionation to identify additional components, and experiments with cells immortalized from BKLF knockout mice to test the direct functional relevance of these proteins. We will also use a dominant negative strategy in cell lines and primary cells to assess the role of COUP-TF2. It has previously been reported that Ikaros plays a role in fetal globin silencing and we will also analyze a novel Ikaros mutant mouse bred to globin transgenic mice to test the effect of Ikaros in vivo. These experiments are aimed at confirming the in vivo relevance of BKLF, GATA-1, COUP-TF2 and Ikaros and may ultimately lead to the development of inhibitors that mimic the effect of specific HPHF point mutations and allow reactivation of fetal globin gene expression as a treatment for Sickle Cell Disease and Cooley's anemia.
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