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A chemical crosslinking strategy to determine DNA methylating protein complexes

确定 DNA 甲基化蛋白复合物的化学交联策略

基本信息

批准号：
7798219
负责人：
LUCY Ann GODLEY
金额：
$ 16.48万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7798219
关键词：
Affect Aging BRCA1 gene Breast Cancer Cell CDH1 gene Cell Adhesion Cell physiology Cells Characteristics Chemicals Chemistry Chromatin Chromatin Structure Complex DNA DNA Methylation DNA Methyltransferase DNA Modification Methylases DNA Sequence Development Diagnosis E-Cadherin Embryonic Development Enzymes Epigenetic Process Estrogen Receptors Failure Future Gene Expression Genes Genetic Transcription Genomic Imprinting Glycoproteins Hematopoietic Neoplasms Hereditary Breast Carcinoma Histone Deacetylase Inhibitor Hormonal Human Hypermethylation Knowledge Laboratories Lead Malignant Neoplasms Mammary Neoplasms Mass Spectrum Analysis Mediating Methodology Methods Methylation Molecular Molecular Profiling Mutate Neoplasm Metastasis Normal Cell Normal tissue morphology Oligonucleotides Oncogenes Oncogenic Patients Pattern Phenotype Process Progesterone Receptors Proteins Proteomics Reaction Refractory Signal Transduction Solid Neoplasm Structure Techniques Therapeutic Tumor Suppressor Genes X Inactivation base cancer cell crosslink genome-wide hormone therapy human DNA link protein malignant breast neoplasm new technology novel novel diagnostics promoter protein complex research study tumor

项目摘要

DESCRIPTION (provided by applicant): Epigenetic changes alter chromatin structure, thereby regulating gene transcription. In normal cells, repetitive DNA is hypermethylated and transcriptionally silent, whereas transcribed gene promoters are undermethylated and associated with open chromatin. Cancer cells are characterized by abnormal DNA methylation: repetitive DNA sequences and some gene promoters are hypomethylated and transcriptionally active, whereas many tumor suppressor gene promoters are hypermethylated and transcriptionally inactive. Although many studies have focused on categorizing which genes have altered DNA methylation patterns in cancer cells, the precise components of the DNA methylation machinery mediating those changes have not been established. We propose to develop a unique method of chemical cross-linking and protein complex identification to identify the factors involved in promoter hypermethylation in breast cancer. Our new strategy is based on the development of novel chemical compounds synthesized within the He Laboratory and has significant advantages over other approaches in that it assembles a protein complex directly on a specific biologically relevant DNA. We envision applying this strategy to determine a quantitative molecular signature for DNA methylating complexes in cancer cells. To develop our new technology, we propose focusing on determining the protein complexes that mediate the hypermethylation of the promoters of BRCA1, a classic tumor suppressor gene, and CDH1, which encodes a protein important for cell adhesion, using two Specific Aims: (1) To incorporate novel chemical cross-linking compounds into oligonucleotides corresponding to the BRCA1 and CDH1 promoters, perform cross-linking to protein extracts from breast cancer cells, and identify the cross-linked proteins by mass spectroscopy; and (2) To compare the DNA methylating complexes quantitatively in human breast tumors versus normal tissue using our technique. In the future, a detailed understanding of the DNMT/other protein contacts at particular gene promoters could lead to the development of hypomethylating agents targeted to these promoters in a sequence-specific manner, thereby avoiding the consequences of genome-wide hypomethylation. In addition, the new chemistry allows formation of DNMT-DNA complexes at high efficiency, which could facilitate the structural characterization of human DNMT-DNA complexes.

描述（由申请人提供）：表观遗传变化改变染色质结构，从而调节基因转录。在正常细胞中，重复 DNA 高度甲基化且转录沉默，而转录基因启动子甲基化不足且与开放染色质相关。癌细胞的特点是DNA甲基化异常：重复的DNA序列和一些基因启动子甲基化程度低且转录活性高，而许多抑癌基因启动子甲基化程度高且转录活性低。尽管许多研究都集中于对哪些基因改变了癌细胞中的 DNA 甲基化模式进行分类，但介导这些变化的 DNA 甲基化机制的精确组成部分尚未确定。我们建议开发一种独特的化学交联和蛋白质复合物鉴定方法，以鉴定与乳腺癌启动子高甲基化有关的因素。我们的新策略基于 He 实验室合成的新型化合物的开发，与其他方法相比具有显着优势，因为它直接在特定的生物学相关 DNA 上组装蛋白质复合物。我们设想应用这种策略来确定癌细胞中 DNA 甲基化复合物的定量分子特征。为了开发我们的新技术，我们建议重点确定介导 BRCA1（一种经典肿瘤抑制基因）和 CDH1（编码对细胞粘附重要的蛋白质）启动子高甲基化的蛋白质复合物，使用两个具体目标：（1）将新型化学交联化合物纳入与 BRCA1 和 CDH1 启动子相对应的寡核苷酸中，与蛋白质进行交联从乳腺癌细胞中提取，并通过质谱鉴定交联蛋白； (2) 使用我们的技术定量比较人类乳腺肿瘤与正常组织中的 DNA 甲基化复合物。将来，对特定基因启动子处的 DNMT/其他蛋白质接触的详细了解可能会导致以序列特异性方式针对这些启动子的低甲基化剂的开发，从而避免全基因组低甲基化的后果。此外，新的化学方法可以高效形成 DNMT-DNA 复合物，这有助于人类 DNMT-DNA 复合物的结构表征。