A GATA switch mechanism in gamma-globin gene re-activation by hydroxyurea

在镰状细胞病（SCD）患者中，羟基脲（HU）通过重新激活成人红细胞中的胎儿y-珠蛋白基因来改善症状。HU在y-珠蛋白基因再激活中的分子机制尚不完全清楚。在本应用中，我们提出验证GATA开关机制是HU对y-珠蛋白基因再激活机制的假设：在GATA-2未检测到且GATA-1高表达的SCD患者成年红系祖细胞中，NF-Y与GATA-1结合形成抑制y-珠蛋白基因的NFY/GATA-1复合物。经HU处理后，GATA-2水平急剧升高，NF-Y通过其对GATA-2较高的亲和力形成NF-Y/GATA-2复合物，激活y-珠蛋白基因。在Aim 1中，我们将通过实时RT-PCR和Western blots检测转基因小鼠和SCD患者红系祖细胞中y-球蛋白基因再激活水平是否与HU诱导的GATA-2水平呈正相关。在Aim 2中，我们将使用染色质免疫沉淀（ChIP）和电泳迁移位移法（EMSA）来确定HU诱导的y-珠蛋白基因再激活水平是否与转基因小鼠和SCD患者红系祖细胞中y-珠蛋白启动子结合的GATA-2水平相关。在Aim 3中，我们将使用转导试验来确定过表达NF-Y和/或GATA-2是否会增强低hu反应的红系祖细胞中y-珠蛋白基因的再激活。预计该项目将为y-珠蛋白基因转录再激活的分子机制提供深入的见解。在翻译水平上，这将通过识别新的和早期的反应标记（GATA-2增加）来改进临床治疗中HU的使用，从而区分反应者和无反应者。由此产生的HU的针对性利用不仅将解决HU机制的重要问题（如最近NIH HU共识会议所确定的），而且还将通过允许在HU和其他抗转换剂之间进行更合理的选择并避免不必要的毒性，从而有助于缓解健康差距。

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.