Mechanisms of DNA Demethylation: The Molecular Interplay Between Thymine DNA Glycosylase and RNA

About This Grant

DNA methylation is a biological process that involves the attachment of methyl groups to the building blocks of DNA. Establishing and maintaining proper patterns of DNA methylation throughout the genome is a highly choreographed process involving not only the addition of methyl groups, but also their removal (termed DNA demethylation). This project will investigate how the enzyme thymine DNA glycosylase, a key player in the DNA demethylation pathway, is targeted to specific methyl groups throughout the genome. This is an important question because accurate removal of methyl groups from DNA is critical for normal cellular function, and dysregulation of this pathway can result in abnormal human development and disease, including cancer. Thus, the scientific outcomes will lead to an improved understanding of how DNA methylation landscapes are established and maintained under both physiological and pathological states and may provide impetus for future clinical applications. Additionally, this project will promote research training of graduate and undergraduate students and will provide research opportunities to K-12 students to encourage participation in STEM fields. Thymine DNA glycosylase (TDG) plays important roles in maintaining appropriate genetic and epigenetic states, yet it remains unclear how this multifaceted enzyme is targeted and regulated at the genome level. While most studies aimed at answering this question have focused on protein interactions, emerging evidence now indicates a regulatory relationship between TDG and RNA. Indeed, TDG was recently shown to bind tightly to RNA. Moreover, RNA competes with DNA for binding to TDG and inhibits TDG-mediated excision, supporting a model wherein TDG-mediated processes, such as DNA demethylation, are regulated through the direct interactions of TDG with RNA. Yet, several critical questions remain: How does TDG bind to RNA? To what extent does TDG interact with RNA in cells and what is the functional relevance of those interactions? To answer these questions, this project will employ a series of biochemical, genetic, and genome-wide approaches to uncover the molecular basis of RNA recognition by TDG and to establish how these interactions contribute to TDG chromatin occupancy and function in vivo. The findings will have broad implications for all TDG-associated biological pathways and will provide new insights into the role of RNA in gene regulation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.