Globin Gene Expression during Erythroid Differentiation

描述（申请人提供）：脊椎动物红系分化和发育过程中珠蛋白基因的调控是高等真核生物基因调控的信息模型。越来越多的证据支持表观遗传机制在控制脊椎动物珠蛋白基因表达中的重要作用。本项目旨在阐明DMA甲基化在胚胎发育调控中的作用和机制，以及成年红细胞中胎儿珠蛋白基因的表达。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，以确定安全治疗性激活胎儿/胚胎珠蛋白基因表达的选择性分子靶点。地中海贫血和镰状细胞性贫血。这一目标将通过以下具体目标来实现：1)表征优先结合甲基化胚胎珠蛋白基因DMA序列的红细胞甲基化胞嘧啶蛋白结合复合体（MeCPC）的蛋白质成分，并验证关键成分的功能重要性；2)确定甲基化胞嘧啶结合结构域蛋白2 （MBD2）使人类发育沉默的机制。-珠蛋白基因在转基因小鼠模型中的表达，并在CD34+人造血祖细胞衍生的红细胞中检测其功能。实现这些特定目标的实验计划将包括转基因小鼠模型系统，其中包含单拷贝酵母人工染色体（YAC）和细菌人工染色体（BAG），其中包含人类？球蛋白轨迹;染色质免疫沉淀测定；生化净化方法；SDS凝胶分离和离子喷雾质谱法鉴定蛋白质；小抑制RNA （SiRNA）在人原代红细胞和培养细胞系中敲除基因。在大多数实验中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起作用的机制。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疾病之一，造成严重痛苦和残疾。已知导致胎儿血红蛋白激活的自然突变可以克服？地中海贫血和镰状细胞性贫血。了解DMA甲基化介导的胎儿/胚胎珠蛋白基因沉默机制可能导致更有效的治疗这些疾病，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主要作用。

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.