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

DNA在整个基因组的特定位置上被甲基化--由甲基进行化学修饰。建立和维持适当的甲基化模式是一个高度精心设计的过程，包括甲基的添加(甲基化)和去除(去甲基化)。这个项目将调查一些负责脱甲基化DNA的酶是如何针对基因组中的特定位置的。这是一个重要的问题，因为准确的去甲基化对细胞内稳态至关重要，而这一过程的失调可能会导致生物的异常发育以及癌症等疾病。从这项研究中获得的知识将促进我们对基因组甲基化图景如何在生理或病理状态下建立和维持的理解，并可能被证明对未来的生物技术应用有用。此外，这个项目将为不同的研究生和本科生群体提供研究培训机会，使新的教育课程得以开发，并为K-12学生提供研究机会，以增加对STEM领域的参与。5-甲基胞嘧啶(5mC)的酶促逆转(称为“主动”DNA去甲基化)是哺乳动物中影响基因表达和器官发育的基本途径。胸腺嘧啶DNA糖基酶(TDG)是目前已知的唯一能从哺乳动物DNA中去除DNA去甲基化中间体5-甲酰胞嘧啶和5-羧基胞嘧啶的酶，在DNA去甲基化途径中起着至关重要的作用。然而，TDG如何被靶向于特定的基因组位点，以及它的活动如何在这些位点上受到调控，在很大程度上仍然是未知的。该项目将采用一系列体外和哺乳动物细胞研究，旨在揭示TDG的分子作用及其与染色质的相互作用如何形成基因组甲基化格局。使用化学定义的核小体阵列的体外研究将提供关于TDG活性和染色质结构之间关系的分子水平的细节。在体外观察的基础上，将在细胞中雌激素反应基因启动子的背景下探索TDG-染色质相互作用的生物学相关性。这一结果将促进对染色质结构如何影响TDG活性的机械性理解，从而有助于重要基因调控元素的甲基化动力学。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.