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

羟基脲重新激活γ-珠蛋白基因的GATA开关机制

• 批准号: 7684411
• 负责人: DOROTHY Y TUAN
• 金额: $ 22.97万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-28 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7684411
• 关键词: Address; Adult; Adverse effects; Affect; Affinity; Binding; Biological Assay; Blood; Blood specimen; Cells; Chemicals; Clinic; Clinical; Complex; Consensus; Data; Development; Electrophoretic Mobility Shift Assay; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; GATA2 transcription factor; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Globin; Human; K562 Cells; Laboratories; Lead; Lentivirus Vector; Mediating; Messenger RNA; Molecular; Mus; National Center on Minority Health and Health Disparities; Patients; Pharmaceutical Preparations; Primer Extension; Proteins; Recruitment Activity; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Sickle Cell; Sickle Cell Anemia; Stem cells; Symptoms; TATA Box; Testing; Therapeutic; Time; Toxic effect; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; Western Blotting; chromatin immunoprecipitation; cytotoxic; fetal; gamma Globin; health disparity; human GATA1 protein; hydroxyurea; insight; novel; outreach; overexpression; peripheral blood; promoter; response marker; sickling; symposium; transcription factor

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.

在镰状细胞病 (SCO) 患者中，羟基脲 (HU) 通过重新激活细胞来改善症状 成人红细胞中的胎儿v-珠蛋白基因。 HU重新激活v-珠蛋白基因的分子机制是 没有完全理解。在此应用中，我们建议测试 GATA 切换机制的假设 HU 重新激活 y 球蛋白基因的机制的基础：在 SCO 的成年红系祖细胞中 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 y 珠蛋白基因的 NFY/GATA-1 复合物。 HU 的治疗大大增加了 GATA-2 水平，因此 NF-Y 通过其对 GATA-2 更高的亲和力形成 NF-Y/GATA-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 和蛋白质印迹确定是否 y-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 (ChIP) 和电泳迁移率变动测定 (EMSA) 来确定水平是否 HU 诱导的 Y 珠蛋白基因重新激活的程度与与 v 珠蛋白结合的 GATA-2 水平相关 转基因小鼠和 SCO 患者的红系祖细胞中的启动子。在目标 3 中，我们将使用 转导测定以确定过度表达 NF-Y 和/或 GATA-2 是否增强 v-珠蛋白基因 低 HU 反应者的红系祖细胞重新激活。预计该项目将 深入了解 v 珠蛋白转录再激活所涉及的分子机制 基因。在转化层面，这将通过以下方式改进 HU 在临床治疗中的使用： 识别新的和早期的反应标记（GATA-2 的增加），从而将反应者与 无反应者。由此产生的对 HU 的有针对性的利用不仅将解决一个重要问题 HU 机制（如最近的 NIH HU 共识会议所确定），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来消除健康差异 避免不必要的毒性。