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

在镰状细胞病(SCO)患者中，羟基脲(HU)通过重新激活细胞内的 成人红系细胞中的胎儿v-珠蛋白基因。HU激活v-珠蛋白基因的分子机制是 没有完全理解。在这个应用程序中，我们建议测试GATA开关机制的假设 人脐血造血祖细胞γ-珠蛋白基因再激活机制的研究 在GATA-2检测不到而GATA-1高表达的患者中，NF-Y与GATA-1结合 形成抑制γ-珠蛋白基因的NFY/GATA-1复合体。胡的治疗极大地增加了 GATA-2的水平，所以NF-Y通过其对GATA-2的较高亲和力而形成NF-Y/GATA-2复合体 激活v-珠蛋白基因。在目标1中，我们将通过实时RT-PCR和Western blots来确定 γ-珠蛋白基因再激活水平与HU诱导的GATA-2水平呈正相关 转基因小鼠和SCO患者的红系祖细胞。在目标2中，我们将使用染色质 免疫沉淀法(CHIP)和凝胶电泳法(EMSA)测定 HU诱导的Y-珠蛋白基因再激活与v-珠蛋白结合的GATA-2水平相关 转基因小鼠和SCO患者红系祖细胞中的启动子。在目标3中，我们将使用 转导试验确定过表达的NF-Y和/或GATA-2是否增强v-珠蛋白基因 低HU应答者红系祖细胞的再激活。预计该项目将 深入了解v-珠蛋白转录重新激活的分子机制 吉恩。在翻译层面上，这将通过以下方式改进HU在临床治疗中的使用 识别新的和早期的应答标记(GATA-2的增加)，从而将应答者与 无回应者。由此产生的对HU的有针对性的利用不仅将解决 HU机制(如最近的NIH HU共识会议所确定的)，但也将有助于缓解 通过允许在HU和其他抗转换剂和 避免不必要的毒性。

In patients with sickle cell disease (SCO), hydroxyurea (HU) ameliorates the symptoms by re-activating the fetal v-globin gene in adult erythroid cells. The molecular mechanism of HU in reactivating v-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 SCO 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 v-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 SCO 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 v-globin promoter in the erythroid progenitor cells of transgenic mice and SCO patients. In Aim 3, we will use transduction assays to determine whether over-expressing NF-Y and/or GATA-2 enhances v-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 v-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.