Uncovering the molecular mechanisms of DNMT1-mediated inheritance of gene silencing

Every cell in the body harbors essentially identical DNA sequences, so how are different cell types established and maintained? Different cells express a distinct subset of DNA sequences, i.e., they make only the RNA and proteins characteristic of that particular cell type. This project focuses on the study of one of the most critical proteins in this process, DNA methyltransferase 1 (DNMT1). This enzyme is responsible for silencing the subset of DNA sequences that need not be expressed for the cell to function. This project will utilize structural biology, biochemistry, and cell biology methods to understand DNMT1 regulation in cells and to discover the mechanism cells utilize to ensure DNMT1 only acts on a specific subset of DNA sequences. This will provide insight into gene regulation, and by extension dysregulation that may result in a loss of function and disease states. The principal investigator will develop a practical learning module integrating teaching and research to advance critical thinking for undergraduate students, with the goal to inculcate the ability to utilize computational methods for biological data handling and visualization. The practical learning module will be adopted as part of classroom (undergraduate or graduate level courses in Biochemistry), and non-classroom teaching (undergraduate research experience through programs at CU Boulder). It will also be implemented in outreach (summer research exposure for community college students) and made publicly available on GitHub. DNA methlyation patterns define the distinct cell types that arise from a single genome, and these patterns must be accurately passed onto daughter cells by the enzyme DNMT1, which requires specific activation cues. Several studies have established that DNA hemi-methylation, and the modification of the histone proteins which package DNA impact DNMT1 activity. More recent studies have shown that DNMT1 is also regulated by interactions with G-quadruplex RNA. Precisely how the RNA and the different activation partners interact with DNMT1 to regulate its activity remain unknown. This project will combine RNA and chromatin structural biology to visualize DNMT1 interaction with RNA and chromatin. The tight integration of structural and functional studies promises to reveal the essential elements of DNMT1 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.