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激活γ-珠蛋白基因的分子机制尚不完全清楚。在本申请中，我们提出检验加塔转换机制是HU再激活γ-珠蛋白基因机制的基础的假设：在SCD患者的成人红系祖细胞中，其中加塔-2不可检测，而加塔-1高度表达，NF-Y结合加塔-1形成抑制γ-珠蛋白基因的NFY/加塔-1复合物。HU处理显著增加了加塔-2的水平，因此NF-Y通过其对加塔-2的更高亲和力形成NF-Y/加塔-2复合物，从而激活γ-珠蛋白基因。在目的1中，我们将通过实时RT-PCR和蛋白质印迹确定在转基因小鼠和SCD患者的红系祖细胞中，γ-珠蛋白基因再激活的水平是否与HU诱导的加塔-2的水平正相关。在目的2中，我们将使用染色质免疫沉淀（ChIP）和电泳迁移率变动分析（EMSA）来确定转基因小鼠和SCD患者的红系祖细胞中由HU诱导的γ-珠蛋白基因再激活的水平是否与结合至γ-珠蛋白启动子的加塔-2的水平相关。在目的3中，我们将使用转导测定来确定过表达NF-γ和/或加塔-2是否增强低HU应答者的红系祖细胞中的γ-珠蛋白基因再活化。预计该项目将提供深入了解的Y-珠蛋白基因的转录激活的分子机制。在翻译水平上，这将通过鉴定新的和早期的应答标志物（加塔-2的增加）导致HU在临床治疗中的使用的改进，从而区分应答者和非应答者。因此，有针对性地利用HU不仅可以解决HU机制的重要问题（如最近的NIH HU共识会议所确定的），而且还可以通过在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来帮助缓解健康差异。

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.