This application is aimed at identifying inducible transcription factors that activate fetal hemoglobin (HbF) expression, for the development of novel therapies for Sickle cell disease and Cooley's anemia. The rationale for this strategy is based on clinical, genetic and biochemical evidence illustrating the beneficial effect of HbF in both genetic disorders. Histone deacetylase inhibitors (HDAIs) are validated HbF inducers but available analogues are not widely used due to anti-proliferative effects and poor bioavailability. HDAIs alter DNA-protein interactions in the fetal globin gene promoter, which indicates initial activation of hitherto unknown transactivator molecules, which in turn activate fetal globin. Based on this deduction, we initiated studies with two HDAIs sodium butyrate and trichosatin A and found both to activate p38 mitogen activating protein kinase (p38 MAPK) and activating transcription factor 2 (ATF-2). We have confirmed this mechanism of g globin activation in experiments using a specific activator (anisomycin) and inhibitor (SB203580) of p38 MAPK. From these findings we have developed a hypothesis to be tested in four major specific aims, 1) Determine the mechanism for p38 MAPK activation by histone deacetylase inhibitors. We will establish whether HDAI activation of p38 MAPK is direct or through reactive oxygen species in the mitochondria and test novel HDAIs currently in Phase I clinical trials for g globin inducibility. 2) Define the cis-regulatory element(s) required for p38 MAPK dependent g gene induction. These studies will include functional analysis of the g promoter using reporter and expression vectors. 3) Identify transcription factor(s) required for p38 MAPK dependent g gene activation. Cognate binding transcription factors for the elements identified in Aim 2 will be characterized by gel shift analysis and immunoprecipitation studies. Experiments in this Aim will include validation that the identified transactivation or co-activator proteins activate endogenous g globin. 4) Determine the ability of novel HDAIs to induce g gene activity in vivo in transgenic lines. Novel HDAIs found to be effective in Aim 1 will be tested in sickle transgenic mice. Successful completion of the experiments described in the Specific Aims will provide the basis for developing effective drug or gene therapy protocols for Sickle cell disease and Cooley's anemia.
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