The role of the MBD2-NuRD complex in gamma-globin gene silencing

项目摘要 珠蛋白基因表达的发育调控研究提供了重要的机制见解 正常哺乳动物基因控制和疾病中的异常基因表达。表观遗传机制是 现在被认为是珠蛋白基因调节以及白血病和其他疾病中基因失调的中心。 癌的该项目旨在阐明蛋白质-蛋白质相互作用的关键组成部分， MBD 2-NuRD染色质重塑复合物在成人胎儿β型珠蛋白基因沉默中的作用 红系细胞长期目标是确定和验证安全治疗激活胎儿的靶点， 镰状细胞性贫血和β-地中海贫血血红蛋白表达这一目标将通过 以下合作目标：1）功能上定义MBD 2和NuRD复合物特定区域的作用 MBD 2-NuRD沉默成人红系细胞中胎儿γ-珠蛋白基因的能力中的组分， 2)从生物药理学和结构上表征MBD-NuRD复合物形成的关键界面， 稳定该实验方法采用了关键基因组编辑之间的实时迭代反馈， 成人红系细胞中蛋白质相互作用及关键蛋白质-蛋白质结构研究 MBD 2、GATAD 2A和CHD 4 NuRD组分的相互作用， 晶体学技术以及最先进的顺磁弛豫增强（PRE）和 生物发光共振能量转移（BRET）。直接被激活的红细胞基因 MBD 2-NuRD的破坏并进而减轻γ-珠蛋白基因沉默将通过RNA-seq 和ChIP-seq测定和生物信息学分析以及ChIP测定验证。这些实验将 鉴定和验证用于未来治疗发展的主要肽和小分子靶点的证据 镰状细胞性贫血和β-地中海贫血。

PROJECT SUMMARY Studies of developmental regulation of globin gene expression have provided important mechanistic insight into normal mammalian gene control and abnormal gene expression in diseases. Epigenetic mechanisms are now recognized as central to globin gene regulation as well as dysregulation of genes in leukemia and other cancers. This project is aimed at elucidating the key protein-protein interactions among components of the MBD2-NuRD chromatin remodeling complex in the context of fetal β-type globin gene silencing in adult human erythroid cells. The long term goal is to identify and validate targets for safe therapeutic activation of fetal hemoglobin expression in sickle cell anemia and β-Thalassemia. This goal will be pursued through the following collaborative aims: 1) To functionally define the roles of specific regions of MBD2 and NuRD complex components in the ability of MBD2-NuRD to silence the fetal γ-globin gene in adult human erythroid cells and 2) to biophysically and structurally characterize interfaces critical for MBD-NuRD complex formation and stability. The experimental approach employs a real-time iterative feedback between genome editing of key protein interactions in adult erythroid cells in conjunction with structural studies of key protein-protein interactions of MBD2, GATAD2A and CHD4 NuRD components using both standard NMR, and crystallographic techniques as well as state-of-the-art paramagnetic relaxation enhancement (PRE) and bioluminescence resonance energy transfer (BRET). The erythroid genes that are directly activated by disruption of MBD2-NuRD and in turn relieve γ-globin gene silencing will be characterized by both RNA-seq and ChIP-seq assays and bioinformatics analyses as well as ChIP assay validation. These experiments will identify and validate proof of principle peptide and small molecule targets for future development of therapy of sickle cell anemia and β-Thalessemia.