Investigating the role of long non-coding RNAs in regulation of DNA methylation

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Project Summary Aberrant DNA methylation patterns are early hallmarks of cancer. In healthy cells, there are “writers” that establish these methylation marks and “erasers” that remove them in specific cell types and at varying points during cell differentiation. Proper regulation of DNA methylation is critical for normal cell function. An important class of writers is the DNA methyl transferases (DNMTs). DNMTs are responsible both for establishing DNA methylation patterns as well as maintaining them during successive rounds of DNA replication. DNMTs are often mutated in cancers and the precise mechanisms for how DNMTs promote gene-specific DNA methylation in healthy cells are not well understood. Recent evidence has suggested that RNA plays important roles in regulating epigenetic marks. For example, several lncRNAs have been shown to interacts with DNMTs, primarily with the maintenance methylase DNMT1. In this extension proposal I aim to investigate the regulation of DNMT1 by RNAs. I will initially aim to elucidate the molecular basis for how RNA interacts with DNMT1 by uncovering core structural features required for DNMT1 binding. I will follow up on the preliminary data I have gathered during my K00 funding which includes uncovering a high affinity of DNMT1 for GU-rich RNA. Additionally, I have found that DNMT1 binds with high affinity to its own mRNA, specifically it’s 3’ UTR, which interestingly is also very GU-rich. I will determine the nature of this specific interaction by performing Cryo-EM on a DNMT1/GU-rich RNA complex together with additional biochemical assays to better understand the binding preference of DNMT1 for various RNAs. Together, this proposal will help elucidate how DNA methylation is misregulated in cancer and will additionally provide information about how RNAs help regulate this important process. Gaining an understanding of the molecular basis for how RNAs interact with DNMT1 may provide structural targets for use in the development of tissue-specific cancer therapeutics.