Regulation of Globin Gene Expression In Erythroid Differentiation & Development

描述(申请人提供)：珠蛋白基因表达的发育调节为脊椎动物基因控制的机制提供了重要的见解，并与由于珠蛋白基因突变而导致的人类疾病直接相关。表观遗传机制现在被认为是珠蛋白基因调控以及包括癌症在内的常见疾病的基因调控的重要机制。本研究旨在阐明甲基胞嘧啶结合结构域蛋白2(Mbd2)及其辅阻遏物复合体核重塑和去乙酰基酶(NuRD)在抑制出生后红系细胞中胎儿/胚胎β型珠蛋白基因表达中的作用和机制。该项目的长期目标是确定和验证选择性分子靶点，用于安全地激活镰状细胞性贫血和β-地中海贫血中胎儿/胚胎珠蛋白基因的表达。这将通过以下具体目标来实现：1)确定MBD2和MBD2-NuRD复合体成分沉默胎儿γ-珠蛋白基因的作用和机制(S)，以及2)确定在培养的成人表型红系细胞和携带转基因小鼠模型的人β-珠蛋白基因座中干扰MBD2-NuRD复合体的生物效应。实现这些目标的实验方法将包括在培养中对人类β-珠蛋白基因座承载小鼠红系细胞和原代人类红系细胞的遗传、生化和细胞生物学研究，以及转基因小鼠模型，包括β-YAC小鼠和Berkley人源化转基因(BERK)小鼠镰状细胞贫血模型。具体研究将确定培养细胞中Mbd2-NuRD复合体的下游蛋白质和microRNA靶标。将进行生化和遗传学研究，以确定染色质重塑蛋白Mi-2β沉默胎儿/胚胎β型珠蛋白基因表达的机制，而不依赖于它作为Mbd2-NuRD复合体的一部分。遗传转基因小鼠的育种研究和一种来自NuRD复合体p66pha成分的新型共价订书肽将被用于证明Mbd2-NuRD复合体作为治疗镰状细胞性贫血和β-地中海贫血的分子靶标的原则有效性。

DESCRIPTION (provided by applicant): Developmental regulation of globin gene expression has provided significant insight into mechanisms of vertebrate gene control, and is directly relevant tohuman diseases due to mutations of the globin genes. Epigenetic mechanisms are now recognized as important in globin gene regulation as well as regulation of genes in common disorders including cancer. This project is aimed at elucidating the role and mechanism of methylcytosine binding domain protein 2 (MBD2) and components of its associated co-repressor complex Nuclear Remodeling and deacetylase (NuRD) in silencing of fetal/embryonic beta-type globin genes in post-natal erythroid cells. The long term goal of this project is to identify and validate selective molecular targets for safe therapeutic activation of fetal/embryonic globin gene expression in sickle cell anemia and beta-thalassemia. This will be pursued through the following specific aims: 1) To determine the roles and mechanism(s) through which MBD2 and the MBD2-NuRD complex components act to silence the fetal gamma-globin gene, and 2) To determine the biologic effects of disrupting the MBD2-NuRD complex in adult phenotype erythroid cells in culture and in human beta-globin locus bearing transgenic mouse models. The experimental approach to achieve these aims will include genetic, biochemical and cell biologic studies in human beta-globin locus bearing murine erythroid cells and primary human erythroid cells in culture and transgenic mouse models including beta-YAC mice and the Berkley humanized transgenic (BERK) mouse model of sickle cell anemia. Specific studies will characterize the downstream protein and microRNA targets of the MBD2-NuRD complex in cultured cells. Biochemical and genetic studies will be carried out to characterize the mechanism through which the chromatin remodeling protein Mi-2beta silences fetal/embryonic beta-type globin gene expression independently of its role as part of the MBD2-NuRD complex. Both genetic transgenic mouse breeding studies and a novel covalently stapled peptide derived from the p66alpha component of the NuRD complex will be employed to provide proof of principle validation of the MBD2-NuRD complex as a molecular target for therapy of sickle cell anemia and beta-Thalassemia.