A chemical crosslinking strategy to determine DNA methylating protein complexes

描述（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，重复的DNA是高甲基化和转录沉默，而转录的基因启动子是甲基化不足，并与开放的染色质。癌细胞的特征在于异常的DNA甲基化：重复DNA序列和一些基因启动子是低甲基化和转录活性的，而许多肿瘤抑制基因启动子是高甲基化和转录失活的。虽然许多研究都集中在分类哪些基因改变了癌细胞中的DNA甲基化模式，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的方法，以确定参与乳腺癌启动子高甲基化的因素。我们的新策略是基于在He实验室内合成的新型化合物的开发，并且与其他方法相比具有显着的优势，因为它直接在特定的生物相关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，我们建议集中精力确定介导BRCA 1（一种经典的肿瘤抑制基因）和CDH 1（编码一种对细胞粘附重要的蛋白质）启动子超甲基化的蛋白质复合物，使用两个特定目的：（1）将新的化学交联化合物掺入对应于BRCA 1和CDH 1启动子的寡核苷酸中，对乳腺癌细胞的蛋白质提取物进行交联，并通过质谱鉴定交联的蛋白质;和（2）使用我们的技术定量比较人乳腺肿瘤与正常组织中的DNA甲基化复合物。 在未来，对特定基因启动子处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靶向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，新的化学允许以高效率形成DNMT-DNA复合物，这可以促进人DNMT-DNA复合物的结构表征。

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.