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 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。在本申请中，我们提出测试假设，即加塔开关机制 在SCO的成人红系祖细胞中，HU是γ-珠蛋白基因再激活的机制之一： 在加塔-2不可检测而加塔-1高表达的患者中，NF-Y与加塔-1结合， 形成抑制γ-珠蛋白基因的NFY/加塔-1复合物。通过HU的治疗大大增加了 水平的加塔-2，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， 激活V-珠蛋白基因。在目标1中，我们将通过实时RT-PCR和蛋白质印迹来确定 γ-珠蛋白基因再激活水平与HU诱导的加塔-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（ChIP）和电泳迁移率变动分析（EMSA），以确定是否水平 HU诱导的γ-珠蛋白基因再激活与γ-珠蛋白结合的加塔-2水平相关 在转基因小鼠和SCO患者的红系祖细胞中，在目标3中，我们将使用 转导测定以确定过表达NF-Y和/或加塔-2是否增强V-珠蛋白基因 低HU应答者的红系祖细胞中的再活化。预计该项目将 提供了对v-珠蛋白转录再激活的分子机制的深入了解 基因在翻译水平上，这将导致临床治疗中HU使用的细化， 鉴定新的和早期的应答标志物（加塔-2增加），从而区分应答者和 无应答者。因此，有针对性地利用HU不仅将解决一个重要问题， HU的机制（如最近NIH HU共识会议所确定的），但也将有助于缓解 通过允许在HU和其他抗转换剂之间进行更合理的选择， 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.