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系细胞中的胎儿γ-珠蛋白基因来改善症状。HU重新激活γ-珠蛋白基因的分子机制尚不完全清楚。在这一应用中，我们提出了一种假设，即GATA开关机制是HU重新激活γ-珠蛋白基因的机制的基础：在SCD患者的成年红系祖细胞中，GATA-2不可检测到，GATA-1高表达，NF-Y与GATA-1结合，形成抑制γ-珠蛋白基因的NFY/GATA-1复合体。经HU处理后，GATA-2水平显著升高，因此，核因子-Y通过与GATA-2的高亲和力形成激活γ-珠蛋白基因的核因子-Y/GATA-2复合体。在目标1中，我们将通过实时定量RT-PCR和Western blots检测转基因小鼠和SCD患者红系祖细胞中γ-珠蛋白基因的重新激活水平与HU诱导的GATA-2水平是否呈正相关。在目标2中，我们将使用染色质免疫沉淀(ChIP)和凝胶迁移率改变分析(EMSA)来确定HU诱导的转基因小鼠和SCD患者红系祖细胞中γ-珠蛋白基因的重新激活水平是否与与γ-珠蛋白启动子结合的GATA-2水平相关。在目标3中，我们将使用转导实验来确定过表达的NF-Y和/或GATA-2是否增强了低HU反应者红系祖细胞中的γ-珠蛋白基因的重新激活。预计该项目将深入了解γ-珠蛋白基因转录重新激活所涉及的分子机制。在翻译水平上，这将通过识别新的和早期的反应标记物(GATA-2的增加)来改进HU在临床治疗中的使用，从而区分有效者和无应答者。由此产生的HU的有针对性的使用不仅将解决HU机制的一个重要问题(如最近的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理性的选择并避免不必要的毒性来帮助缓解健康差距。

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.