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

项目总结 珠蛋白基因表达的发育调控研究提供了重要的机制洞察力 转化为正常的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控的中心，以及白血病和其他疾病中基因的失调 癌症。这个项目的目的是阐明关键的蛋白质之间的相互作用的组件 成人胎儿β-型珠蛋白基因沉默背景下的MBD2-NuRD染色质重塑复合体 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-地中海贫血患者的血红蛋白表达这一目标将通过 以下协作目标：1)从功能上确定Mbd2和NuRD综合体特定区域的作用 Mbd2-nuRD沉默成人红系细胞中胎儿γ-珠蛋白基因能力的组成部分和 2)从生物物理和结构上表征对MBD-NuRD复合体形成至关重要的界面以及 稳定性。该实验方法采用了基因组编辑与密钥之间的实时迭代反馈 成人红系细胞中的蛋白质相互作用及关键蛋白-蛋白质结构研究 用标准核磁共振方法研究Mbd2、GATAD2a和CHD4NuRD组分的相互作用 结晶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)。直接被激活的红系基因 Mbd2-nuRD的破坏和反过来缓解γ-珠蛋白基因沉默将是两个rna-seq的特征 以及芯片序列分析和生物信息学分析以及芯片分析验证。这些实验将 确定并验证主肽和小分子靶点的证据，为未来治疗的发展提供依据 镰状细胞性贫血和β-地中海贫血症。

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.