Structural and functional diversity of the methyl-binding domain protein family

甲基结合域蛋白家族的结构和功能多样性

基本信息

批准号：
8451337
负责人：
David Collin Williams
金额：
$ 4.08万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8451337
关键词：
Affect Affinity Base Pair Mismatch Base Pairing Behavior Binding Binding Sites Calorimetry Characteristics Chimera organism Chromatin Communication Complex CpG Islands CpG dinucleotide Cytosine DNA DNA Binding DNA Binding Domain DNA Methylation DNA Sequence Deamination Dependence Development Developmental Gene Dinucleoside Phosphates Family Feasibility Studies Fluorescence Anisotropy Gene Expression Gene Family Gene Silencing Genes Guanine Individual Island Laboratories Length Malignant Neoplasms Mammals Measures Methyl-CpG-Binding Protein 2 Methylation Mismatch Repair Missense Mutation Molecular Mutation NMR Spectroscopy Neurologic NuRD complex Nucleosomes Pathologic Positioning Attribute Property Protein Binding Domain Protein Family Proteins Regulator Genes Relaxation Reporting Role Scanning Site Slide Specificity Structure Surface Plasmon Resonance Technical Expertise Techniques Testing Titrations Tumor Suppressor Genes Work base carcinogenesis chromatin remodeling design ds-DNA experience human disease in vivo in vivo Model inhibitor/antagonist innovation insight member mutant novel preference promoter protein complex protein function research study skills therapeutic gene tissue/cell culture transcription factor

项目摘要

DESCRIPTION (provided by applicant): The long-term objective of this project is to determine the molecular mechanisms by which different MBD proteins target specific methylated DNA sequences to carry out distinct functional roles. In mammals, the MBD protein family consists of at least seven proteins involved in different aspects of normal and pathologic developmental gene silencing, DNA mismatch repair, and aberrant tumor suppressor gene silencing in carcinogenesis. In studying the molecular details of MBD2 bound to methylated DNA, we have shown that the protein can exchange rapidly between binding sites in the same DNA molecular. This observation led us to postulate that the distinct functional roles depend, at least in part, o dynamic and structural DNA binding characteristics of the particular MBD involved. Building on this observation, we plan to investigate the sequence dependent and independent binding of MBDs to DNA in the context of multiple target sites. In particular, we will focus on the structure and binding affinity of MBD2, MBD3, and MBD4. Although MBD2 and MBD3 are highly homologous, MBD2 binds with greater affinity and selectivity for methylated DNA. In contrast, MBD4 recognizes a G-T base pair mismatch arising from hydrolytic deamination of methyl-cytosine. The proposed work will involve NMR spectroscopy, surface plasmon resonance, isothermal titration calorimetry, and fluorescence anisotropy techniques. We will correlate the results of these biophysical and structural analyses with studies in tissue culture cells comparing the function of MBD2 mutants and domain swap chimeras. The latter studies will determine whether functional specificity of different MBD proteins reflects the DNA binding and structural properties of the MBD itself. These experiments will involve co-IP, ChIP, and gene expression studies in tissue culture cells. Ultimately, this work will provide unique insight into the mechanisms of DNA methylation dependent gene silencing and DNA mismatch repair.

描述（由申请人提供）：该项目的长期目标是确定不同MBD蛋白靶向特定甲基化的DNA序列以执行不同功能作用的分子机制。在哺乳动物中，MBD蛋白家族至少由七个参与正常和病理发育基因沉默的不同方面，DNA不匹配修复和异常抑制肿瘤抑制基因沉默的蛋白质。在研究与甲基化DNA结合的MBD2的分子细节时，我们表明该蛋白可以在同一DNA分子中的结合位点之间迅速交换。该观察结果使我们假设不同的功能作用至少部分取决于涉及的特定MBD的动态和结构DNA结合特性。在此观察结果的基础上，我们计划在多个目标位点的背景下研究MBD与DNA的序列依赖性和独立的结合。特别是，我们将重点关注MBD2，MBD3和MBD4的结构和结合亲和力。尽管MBD2和MBD3高度同源，但MBD2的结合具有更大的亲和力和甲基化DNA的选择性。相比之下，MBD4识别甲基 - 胞嘧啶的水解脱氨基引起的G-T碱基对不匹配。提出的工作将涉及NMR光谱，表面等离子体共振，等温滴定量热法和荧光各向异性技术。我们将将这些生物物理和结构分析的结果与组织培养细胞的研究相关联 MBD2突变体和域交换嵌合体的功能。后一项研究将确定不同MBD蛋白的功能特异性是否反映了MBD本身的DNA结合和结构特性。这些实验将涉及组织培养细胞中的Co-IP，CHIP和基因表达研究。最终，这项工作将为DNA甲基化依赖性基因沉默和DNA不匹配修复的机制提供独特的见解。