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

基本信息

批准号：
8410045
负责人：
DOROTHY Y TUAN
金额：
$ 8.74万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-16 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8410045
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 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

项目摘要

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.

在患有镰状细胞疾病（SCD）的患者中，羟基脲（HU）通过重新激活成人红细胞细胞中的胎儿Y-珠蛋白基因来缓解症状。 HU在重新激活Y-珠蛋白基因中的分子机制尚不完全了解。 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.通过HU的处理大大提高了GATA-2的水平，因此NF-Y通过其对GATA-2的较高亲和力，然后形成激活Y-Globin基因的NF-Y/GATA-2复合物。在AIM 1中，我们将通过实时RT-PCR和Western印迹确定Y-珠蛋白基因的重新激活水平是否与HU在转基因小鼠和SCD患者的红细胞祖细胞中诱导的GATA-2水平呈正相关。在AIM 2中，我们将使用染色质免疫沉淀（芯片）和电泳迁移率转移测定法（EMSA）来确定HU诱导的Y-珠蛋白基因重新激活的水平是否与与Y-Globin启动子的GATA-2绑定的gata-2水平相关。在AIM 3中，我们将使用转导测定法来确定过度表达NF-Y和/或GATA-2是否增强了低HU反应者的红细胞基因祖细胞中的Y-珠蛋白基因的重新激活。预计该项目将洞悉Y-珠蛋白基因转录重新激活的分子机制。在翻译层面上，这将通过识别新颖和早期的反应标记（增加GATA-2），从而使临床疗法中的HU使用细化，从而将反应者与非反应者区分开来。由此产生的针对HU的有针对性利用将不仅要解决HU机制的重要问题（如最近的NIH HU共识会议所述），还将通过允许HU和其他反式转换药物之间的更合理的选择并避免不必要的毒性来帮助减轻健康差异。