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：
7628907
负责人：
LUCY Ann GODLEY
金额：
$ 19.92万
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/7628907
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）：表观遗传变化改变了染色质结构，从而调节基因转录。在正常细胞中，重复的DNA是高甲基化的，并在转录上保持沉默，而转录的基因启动子则是甲基化的，并与开放的染色质相关。癌细胞的特征是异常的DNA甲基化：重复的DNA序列，一些基因启动子是甲基化的，并且具有转录活性，而许多肿瘤抑制基因启动子是高甲基化的，并且具有转录性不活性。尽管许多研究集中于分类哪些基因改变了癌细胞中DNA甲基化模式，但尚未确定介导这些变化的DNA甲基化机制的确切成分。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，以识别乳腺癌中启动子高甲基化的因素。我们的新策略基于HE实验室中合成的新型化学化合物的发展，并且与其他方法具有显着优势，因为它直接在特定的生物学相关的DNA上组装了蛋白质复合物。我们设想采用该策略来确定癌细胞中DNA甲基化复合物的定量分子特征。 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从乳腺癌细胞中提取物，并通过质谱鉴定交联的蛋白质；（2）使用我们的技术在人类乳腺肿瘤中定量地比较DNA甲基化复合物。将来，对特定基因启动子的DNMT/其他蛋白质接触的详细理解可能会导致以序列特异性方式靶向这些启动子的低甲基化剂的发展，从而避免全基因组低甲基化的后果。此外，新的化学物质允许在高效率下形成DNMT-DNA复合物，这可以促进人DNMT-DNA复合物的结构表征。