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.
The DNA within a cell can be modified by methylation to alter its structure and affect gene expression. DNA methylation is involved in many normal cellular processes and is abnormally distributed in cancer cells, leading to some of the phenotypes of cancer cells. Promoter hypermethylation represses the expression of a diverse set of genes, although the machinery that produces the increased level of DNA methylation is not known. We hypothesize that novel chemical probes can be used to trap and identify the protein complexes that regulate the DNA methylation of these sequences thereby determining a quantitative molecular signature for DNA methylating complexes in cancer cells. We will focus on the protein complexes that regulate DNA methylation of the BRCA1 and CDH1 genes in breast cancer. If successful, our strategy could be applied to other genes whose promoters are hypermethylated in breast cancer cells, thereby establishing a proteomic framework in which to understand the complex epigenetic changes seen in breast cancer. The knowledge gained from the proposed experiments could provide a basis for novel diagnostic and therapeutic strategies for patients with breast cancer, and more broadly, could reveal paradigms common to other processes that involve DNA methylation, such as mammalian embryonic development, X-chromosome inactivation, genomic imprinting, and aging.
