Developmental regulation of globin gene expression has provided significant insight into mechanisms of vertebrate gene control, and is directly relevant tohuman diseases due to mutations of the globin genes. Epigenetic mechanisms are now recognized as important in globin gene regulation as well as regulation of genes in common disorders including cancer. This project is aimed at elucidating the role and mechanism of methylcytosine binding domain protein 2 (MBD2) and components of its associated co-repressor complex Nuclear Remodeling and deacetylase (NuRD) in silencing of fetal/embryonic beta-type globin genes in post-natal erythroid cells. The long term goal of this project is to identify and validate selective molecular targets for safe therapeutic activation of fetal/embryonic globin gene expression in sickle cell anemia and beta-thalassemia. This will be pursued through the following specific aims: 1) To determine the roles and mechanism(s) through which MBD2 and the MBD2-NuRD complex components act to silence the fetal gamma-globin gene, and 2) To determine the biologic effects of disrupting the MBD2-NuRD complex in adult phenotype erythroid cells in culture and in human beta-globin locus bearing transgenic mouse models. The experimental approach to achieve these aims will include genetic, biochemical and cell biologic studies in human beta-globin locus bearing murine erythroid cells and primary human erythroid cells in culture and transgenic mouse models including beta-YAC mice and the Berkley humanized transgenic (BERK) mouse model of sickle cell anemia. Specific studies will characterize the downstream protein and microRNA targets of the MBD2-NuRD complex in cultured cells. Biochemical and genetic studies will be carried out to characterize the mechanism through which the chromatin remodeling protein Mi-2beta silences fetal/embryonic beta-type globin gene expression independently of its role as part of the MBD2-NuRD complex. Both genetic transgenic mouse breeding studies and a novel covalently stapled peptide derived from the p66alpha component of the NuRD complex will be employed to provide proof of principle validation of the MBD2-NuRD complex as a molecular target for therapy of sickle cell anemia and beta-Thalassemia.
The studies in this proposal are focused on defining mechanisms of fetal hemoglobin gene silencing and identifying and validating molecular targets for disrupting this silencing in patients. These studies have direct relevance to the treatment of sickle cell anemia and Cooley's anemia, since increased fetal hemoglobin is known to overcome the pathologic basis for these severe, debilitating diseases which are among the most common, single gene human disorders worldwide and affect thousands of US citizens.
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