Sickle cell disease (SCD) is a major hemoglobinopathy caused by a DNA mutation in the ?-globin gene producing abnormal sickle hemoglobin which is associated with severe morbidity and lower than average life expectancy. This group of disorders is often complicated by serious, long-term disability and effects mainly the African American population in the United States. In the immediate future reactivating expression of the fetal specific (-globin genes represents the most attractive prospect for ameliorating disease expression. Fetal hemoglobin (HbF) inhibits polymerization of sickle hemoglobin, the primary pathophysiology of SCD. Therefore, the broad long-term focus of our research effort is to develop drug or gene-based strategies to induce HbF as treatments for hemoglobinopathies. We recently demonstrated that the trans-factors CREB1 and ATF-2 bind a consensus cAMP response element in the G3-globin promoter, to mediate HbF induction by a class of drugs known as the histone deacetylase inhibitors. Our overarching hypothesis is that trans-activating DNA-binding proteins bound to 3-promoter regulatory elements can be used to develop gene-based strategies to increase fetal hemoglobin production. Three interrelated Specific Aims will be completed to test this hypothesis. 1. Determine the role of the cAMP response element in the developmental regulation of 3- globin gene expression during erythroid maturation. Using DNA-binding protein affinity purification techniques we will identify CREB partner proteins for in vivo functional genomics studies using a system created with targeted nucleotide exchange technology. 2. Develop an in vitro model of erythroid differentiation to identify transcription factors involved in (-globin gene regulation. Parallel studies in a primary erythroid culture system will be used to widen our search for potent regulators of 3-globin expression by microarray analysis. 3. Exploit a cell-based, high throughput screening system using transgenic mouse bone marrow cells containing human (-eGFP/?- hcRFP?-YAC dual reporters immortalized by a chemical inducer of dimerization system to identify novel HbF inducers. Novel pharmacological agents will be discovered in this system. This proposal is focused on developing new strategies to treat for the class of blood disorders known as the hemoglobinopathies. This effort will address one aspect of the primary mission of the National Heart Lung and Blood Institute. Employing genome-era technology, we will identify chemical inducers of HbF and explicate molecular and cell-based mechanisms of (-globin gene regulation.
The hemoglobinopathies including sickle cell disease and Cooley's anemia are among the most common genetic disorders in the world. These disorders continue to produce significant morbidity and long-term disability which creates a staggering financial burden in the United States healthcare system. We will exploit the proven efficacy of fetal hemoglobin to ameliorate symptoms in this group as the rationale for developing targeted base-exchange and erythroid liquid culture systems to identify gene-based strategies to induce the fetal globin gene. In a parallel effort, high throughput drug screens will be conducted to discover potent chemical inducers of fetal hemoglobin to improve our chances of success.
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