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）：红细胞分化和发育过程中脊椎动物球蛋白基因的调节是较高真核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控制脊椎动物球蛋白基因表达中的重要作用。该项目旨在阐明DMA甲基化在成年红细胞细胞中胚胎和胎儿球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性球蛋白基因调节的机理成分，以鉴定选择性分子靶标，以确定胎儿/胚胎球蛋白基因在？ - thalassalassia and ackle cans贫血和镰状细胞贫血中的安全治疗激活。该目标将通过以下特定目的来实现：1）表征红斑细胞甲基化的胞嘧啶蛋白蛋白结合复合物（MECPC）的蛋白质成分，该蛋白质蛋白结合复合物（MECPC）优先结合甲基化的胚胎球蛋白基因DMA序列并验证关键成分的功能重要性； 2）为了确定甲基化的胞嘧啶结合结构域蛋白2（MBD2）在转基因小鼠模型中人类？ - 格珠蛋白基因并测试其在CD34+人类造血祖细胞中得出的红斑细胞的功能。实现这些特定目标的实验计划将包括包含单拷贝酵母人工染色体（YAC）和细菌人造染色体（BAG）的转基因小鼠模型系统（携带人造人造染色体）？染色质免疫沉淀测定；生化纯化方法；通过SDS凝胶分离和离子喷雾质谱法鉴定蛋白质；在原代人红细胞和培养的细胞系中，小抑制性RNA（siRNA）敲低基因。对于大多数提出的分析，用于选择转基因小鼠模型和原代红细胞细胞的基本原理是确定DMA甲基化和其他表观遗传对照在体内与正常红细胞动物细胞中的模型中的作用。镰状细胞性贫血和？ - 地中海贫血是人类中最常见的单基因介导的疾病之一，并造成严重的痛苦和残疾。已知导致胎儿血红蛋白激活的自然突变可以克服thalassyal和镰状细胞贫血中的分子缺陷。了解胎儿/胚胎球蛋白基因沉默的DMA甲基化介导的机制可能会导致对这些疾病的更有效治疗，并可以促进其他常见疾病（包括癌症）的治疗策略，其中DNA甲基化介导的基因沉默被认为起着主要作用。