Globin Gene Expression during Erythroid Differentiation

红系分化过程中的球蛋白基因表达

• 批准号: 7901949
• 负责人: GORDON D GINDER
• 金额: $ 10万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-21 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7901949
• 关键词: Adult; Bacterial Artificial Chromosomes; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Birds; CD34 gene; Cell Culture Techniques; Cell Line; Cells; Chromosomes, Artificial, Yeast; Complex; Cytosine; DNA Methylation; Data; Development; Disease; Embryo; Epigenetic Process; Erythroid; Erythroid Cells; Evolution; Fetal Hemoglobin; Funding; Gel; Gene Cluster; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Globin; Goals; Hematopoietic; Histones; Human; Ions; Laboratories; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Methylation; Modeling; Modification; Molecular; Molecular Target; Mus; Mutation; Play; Protein Binding; Protein Binding Domain; Proteins; RNA; Regulation; Role; Sickle Cell Anemia; Staging; Testing; Thalassemia; Therapeutic; Tissues; Transgenic Mice; Transgenic Model; Transgenic Organisms; beta Globin; chromatin immunoprecipitation; disability; effective therapy; erythroid differentiation; fetal; fetal globin; in vivo; insight; knock-down; knockout gene; mouse model; progenitor; treatment strategy

DESCRIPTION (provided by applicant): Regulation of vertebrate globin genes during erythroid differentiation and development is an informative model of higher eukaryotic gene control. An increasingly large body of evidence supports an important role for epigenetic mechanisms in the control of vertebrate globin gene expression. This project is aimed at elucidating the role and mechanism of DMA methylation in the developmental regulation of embryonic and fetal globin gene expression in adult erythroid cells. The long-term goal of this project is to characterize mechanistic components of developmental globin gene regulation in order to identify selective molecular targets for safe therapeutic activation of fetal/embryonic globin gene expression in ?-thalassemia and sickle cell anemia. This objective will be pursued through the following specific aims: 1) To characterize the protein components of erythroid cell methylated cytosine protein binding complex (MeCPC) that binds preferentially to methylated embryonic globin gene DMA sequences and verify the functional importance of key components; and 2) To determine the mechanism(s) through which methylated cytosine-binding domain protein 2 (MBD2) developmentally silences the human ?-globin gene in a transgenic mouse model and test its function in erythroid cells derived from CD34+ human hematopoietic progenitors. The experimental plan to achieve these specific aims will include transgenic mouse model systems containing single-copy yeast artificial chromosome (YAC) and bacterial artificial chromosomes (BAG) harboring the human ?-globin locus; chromatin immunoprecipitation assays; biochemical purification methods; protein identification by SDS gel separation and ion spray mass spectroscopy; and gene knock-down by small inhibitory RNA (SiRNA) in primary human erythroid cells and cultured cell lines. The rationale for selection of transgenic mouse models and primary erythroid cells for most of the proposed assays is to identify mechanisms through which DMA methylation and other epigenetic controls operate in models that closely resemble those in normal erythroid cells in vivo. Sickle cell anemia and ?-thalassemia are among the most common single-gene-mediated diseases in humans and inflict severe suffering and disability. Natural mutations that result in activation of fetal hemoglobin are known to overcome the molecular deficits in ?-thalassemia and sickle cell anemia. Understanding the DMA methylation-mediated mechanisms of fetal/embryonic globin gene silencing could lead to more effective treatment of these diseases and could facilitate treatment strategies for other common diseases, including cancer, in which DNA methylation-mediated gene silencing is believed to play a major role.

描述（由申请人提供）：在红细胞分化和发育过程中脊椎动物珠蛋白基因的调节是高等真核生物基因控制的信息模型。越来越多的证据支持表观遗传机制在脊椎动物珠蛋白基因表达控制中的重要作用。本项目旨在阐明DNA甲基化在成人红系细胞中胚胎和胎儿珠蛋白基因表达的发育调控中的作用和机制。该项目的长期目标是表征发育珠蛋白基因调控的机制成分，以确定选择性分子靶点，用于安全治疗激活？地中海贫血和镰状细胞性贫血。1）鉴定红系细胞甲基化胞嘧啶蛋白结合复合物（MeCPC）中与甲基化胚胎珠蛋白基因DNA序列优先结合的蛋白组分，并验证关键组分的功能重要性;和2）为了确定甲基化胞嘧啶结合结构域蛋白2（MBD 2）通过其使人类？在转基因小鼠模型中检测珠蛋白基因的表达，并检测其在来源于CD 34+人造血祖细胞的红系细胞中的功能。实现这些特定目标的实验计划将包括含有单拷贝酵母人工染色体（YAC）和细菌人工染色体（BAG）的转基因小鼠模型系统，这些系统携带人类？珠蛋白基因座;染色质免疫沉淀分析;生化净化方法;通过SDS凝胶分离和离子喷雾质谱进行蛋白质鉴定;以及通过小抑制RNA（SiRNA）在原代人红系细胞和培养细胞系中进行基因敲低。大多数拟定试验选择转基因小鼠模型和原代红系细胞的基本原理是确定DMA甲基化和其他表观遗传控制在与体内正常红系细胞非常相似的模型中发挥作用的机制。镰状细胞性贫血和？-地中海贫血是人类最常见的单基因介导的疾病之一，造成严重的痛苦和残疾。已知导致胎儿血红蛋白活化的自然突变可以克服？地中海贫血和镰状细胞性贫血。了解DNA甲基化介导的胎儿/胚胎珠蛋白基因沉默机制可能会导致这些疾病的更有效的治疗，并可能促进其他常见疾病的治疗策略，包括癌症，其中DNA甲基化介导的基因沉默被认为发挥了重要作用。