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

Texas A&M University, College Station TX

Investigators

Abstract

DNA is methylated – chemically modified by a methyl group – at specific positions throughout the genome. Establishing and maintaining proper patterns of methylation is a highly choreographed process involving both the addition (methylation) and removal (demethylation) of methyl groups. This project will investigate how some enzymes responsible for demethylating DNA are targeted to specific sites in the genome. This is an important question because accurate demethylation is critical for cellular homeostasis, and dysregulation of this process can result in abnormal development of living organisms as well as diseases such as cancer. The knowledge gained from this research will advance our understanding of how genome methylation landscapes are established and maintained under physiological or pathological states, and may prove useful for future biotechnology applications. Additionally, this project will offer research training opportunities for a diverse group of graduate and undergraduate students, enable development of a new educational curriculum, and provide research opportunities to K-12 students to increase participation in STEM fields. Enzymatic reversal of 5-methylcytosine (5mC) back into cytosine (referred to as “active” DNA demethylation) is an essential pathway in mammals that impacts gene expression and organismal development. As the only known enzyme capable of removing DNA demethylation intermediates 5-formylcytosine and 5-carboxycytosine from DNA in mammals, thymine DNA glycosylase (TDG) has an essential role in the demethylation pathway. Yet, how TDG is targeted to specific genomic loci and how its activities are regulated at these sites remains largely unknown. This project will employ a series of in vitro and mammalian cell studies aimed at uncovering the molecular actions of TDG and how its interactions with chromatin shape genomic methylation landscapes. In vitro studies using chemically defined nucleosome arrays will provide molecular-level details regarding the relationship between TDG activity and chromatin structure. Building on in vitro observations, the biological relevance of TDG-chromatin interactions will be explored in the context of estrogen-responsive gene promoters in cells. The outcomes will advance mechanistic understanding of how chromatin structure impacts TDG activity and thus contributes to methylation dynamics at important gene regulatory elements. 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.