Bacterial Rogue Methyltransferases Inducing Human Epimutations

点击翻译按钮获取中文摘要

PROJECT SUMMARY: DNA methylation is dysregulated in every form of cancer. While there is substantial knowledge regarding the location and function of abnormal DNA methylation across tumor types, we are severely lacking in understanding how such abnormal DNA methylation is established during cancer formation. Colorectal cancer (CRC), the third most common cancer type worldwide, has been heavily associated with alterations in DNA methylation, however, mutations in DNA methylation/demethylation pathway genes are rare and cannot account for the exaggerated DNA hypermethylation. Roughly 1.5 kg of bacteria reside in our gut and have been shown to play a substantial role in the development of CRC. Recent evidence suggests that these bacteria can alter the colon cell epigenome by inducing aberrant DNA hypermethylation, although the mechanism promoting this is still not completely understood. One such microorganism, Fusobacterium nucleatum, is able to invade into colon epithelial cells and has been heavily associated with CRC development, as well as hypermethylation of tumor suppressor genes. Roughly 90% of bacteria, including Fusobacterium nucleatum, contain DNA methyltransferase enzymes as a component of their restriction-modification system; a defense mechanism aimed at protecting their genome against invading viruses by methylating specific motifs within their DNA as to distinguish between their own and invading viral DNA. To date, >3500 different R-M motifs have been identified, thus our gut microbiome is filled with microbial genome-modifying enzymes, however their potential to modify human DNA has not been evaluated. The long-term objective of this proposal is to evaluate whether F. nucleatum DNA methyltransferase enzymes are able to access and aberrantly methylate colon cell DNA, potentially giving rise to the epimutations observed in CRC development. This will be tested with three specific aims. The first aim will experimentally evaluate F. nucleatum methyltransferase activity in vitro, by ectopically expressing F. nucleatum DNA methyltransferase enzymes in CRC cell lines and evaluating their ability to enter the nucleus, methylate DNA, as well as determine the locations of DNA methylation. The second aim will take advantage of the microbiome core facilities at Lerner Research Institute to preform bacterial co-culture experiments using F. nucleatum that have been genetically modified to contain tagged-DNA methyltransferase enzyme, to test whether F. nucleatum DNA methyltransferase can access and methylate colon cell DNA. The third aim is to detect F. nucleatum activity in vivo, by searching for F. nucleatum R-M motif enrichment at CRC hypermethylated loci, using our labs published MBD-seq data and the TCGA CRC cohort. Results could reveal a novel paradigm in host-microbiome interactions, as direct epigenetic crosstalk between microbial and host cells has not been fully explored. However, if true, this new paradigm will transform studies of bacterial-associated cancers and open new therapeutic avenues for treatment and risk-management.