In patients with sickle cell disease (SCD), hydroxyurea (HU) ameliorates the symptoms by re-activating the fetal y-globin gene in adult erythroid cells. The molecular mechanism of HU in reactivating y-globin gene is not fully understood. In this application, we propose to test the hypothesis that a GATA switch mechanism underlies the mechanism of y-globin gene re-activation by HU: In adult erythroid progenitor cells of SCD patients, in which GATA-2 is non-detectable and GATA-1 is highly expressed, NF-Y binds to GATA-1 in forming a NFY/GATA-1 complex that represses y-globin gene. Treatment by HU drastically increases the level of GATA-2, so NF-Y through its higher affinity for GATA-2 then forms the NF-Y/GATA-2 complex that activates y-globin gene.
In Aim 1, we will determine by real-time RT-PCR and Western blots whether the level of y-globin gene re-activation is positively correlated with the level of GATA-2 inducible by HU in the erythroid progenitor cells of transgenic mice and SCD patients.
In Aim 2, we will use chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) to determine whether the level of y-globin gene re-activation induced by HU is correlated with the level of GATA-2 bound to the y-globin promoter in the erythroid progenitor cells of transgenic mice and SCD patients.
In Aim 3, we will use transduction assays to determine whether over-expressing NF-Y and/or GATA-2 enhances y-globin gene re-activation in the erythroid progenitor cells of low HU-responders. It is anticipated that this project will provide insight into the molecular mechanisms involved in the transcriptional reactivation of the y-globin gene. At the translational level, this will lead to a refinement of HU usage in clinical therapeutics by identifying novel and early markers of response (increase in GATA-2) and thus differentiate responders from non-responders. The resultant targeted utilization of HU will not only address an important question of the mechanism of HU (as identified in the recent NIH HU consensus conference) but will also help alleviate a health disparity by allowing a more rational choice between HU and other anti-switching agents and avoiding unnecessary toxicities.
Understanding the activities of the NF-Y/GATA complexes assembled on the y-globin promoter may lead to development of a new generation of small chemical drugs that can specifically activate the y-globin gene without affecting other genes in adult erythroid cells of SCD patients. Such globin gene-specific drugs may avoid producing the wide-ranging, cytotoxic side effects of HU.
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