A chemical crosslinking strategy to determine DNA methylating protein complexes

描述(申请人提供)：表观遗传改变改变染色质结构，从而调节基因转录。在正常细胞中，重复的DNA高甲基化，转录沉默，而转录的基因启动子甲基化不足，并与开放染色质相关。肿瘤细胞的特征是DNA甲基化异常：重复的DNA序列和一些基因启动子低甲基化并转录活性，而许多肿瘤抑制基因启动子高甲基化而转录不活性。尽管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，但调节这些变化的DNA甲基化机制的确切组成部分还没有确定。 我们建议开发一种独特的化学交联和蛋白质复合体鉴定方法来鉴定乳腺癌中涉及启动子超甲基化的因素。我们的新策略是基于在实验室内合成的新型化合物的开发，与其他方法相比具有显著的优势，因为它直接在特定的生物相关DNA上组装蛋白质复合体。我们设想应用这一策略来确定癌细胞中DNA甲基化复合体的定量分子特征。 为了发展我们的新技术，我们建议专注于确定介导经典抑癌基因BRCA1和CDH1启动子超甲基化的蛋白质复合体，使用两个特定目标：(1)将新型化学交联化合物结合到与BRCA1和CDH1启动子对应的寡核苷酸中，与乳腺癌细胞的蛋白质提取物进行交联，并通过质谱分析鉴定交联的蛋白质；以及(2)使用我们的技术对人类乳腺肿瘤与正常组织中的DNA甲基化复合体进行定量比较。 在未来，对DNMT/其他蛋白质在特定基因启动子上的接触的详细了解可能会导致以序列特异的方式针对这些启动子的低甲基化药物的开发，从而避免全基因组低甲基化的后果。此外，这种新的化学方法可以高效地形成DNMT-DNA复合体，这有助于人类DNMT-DNA复合体的结构表征。

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.