RUI: Connecting Threonine Metabolism to Chromatin Silencing and Genome Stability

Traditionally, proteins have been understood to have only one primary function. However, recent research has brought attention to a new class of multi-functional proteins known as “moonlighters.” Hundreds of moonlighters have now been characterized, revealing unexpected connections between important biological processes that were previously thought to be unrelated. This project seeks to identify and characterize new moonlighters that operate at the intersection of amino acid synthesis and DNA accessibility. Because these two pathways are essential for proper cellular function, this work has the potential to provide new insights into processes that contribute to aging and disease. The project will train undergraduate researchers in a diverse array of molecular biology and genetic techniques in the budding yeast Saccharomyces cerevisiae that are relevant to biotechnology. Students will also play central roles in experimental design, data interpretation, and scientific communication and will have multiple opportunities to engage with the broader scientific community at the local and national levels. A growing class of multifunctional enzymes known as moonlighters challenges the traditional “one protein, one function” paradigm that has shaped our understanding of cellular pathways. In Saccharomyces cerevisiae, the ribosomal DNA (rDNA) is a silenced locus characterized by a delicate balance of epigenetic modifications that regulate transcriptional levels in response to cellular cues. Disruption of this silencing can significantly affect key cellular processes, including genomic stability and cellular lifespan. In this project, undergraduate researchers will employ molecular biology and genetic techniques to determine how aspartate kinase (Hom3) and aspartic beta semi-aldehyde dehydrogenase (Hom2), two enzymes canonically associated with threonine biosynthetic functions but now implicated in chromatin-based roles, contribute to rDNA silencing. CRISPR-based mutagenesis will be used to investigate the dependence of rDNA silencing on threonine metabolic activity. Flow cytometry will be used to quantify silencing dynamics, and double-mutant and overexpression studies will shed light on functional genetic interactions with established rDNA epigenetic modifiers. Because amino acid metabolism and chromatin silencing are well-conserved and ancient processes, this work may provide insight into how distinct cellular pathways evolve functional connections. Additionally, since the threonine biosynthetic pathway is absent in humans, these studies may also provide insight into biotechnological applications such as the design of antifungal therapies. The project will provide undergraduate students with genuine discovery-driven research experiences, enhance their intellectual, analytical, and technical skills, and prepare them for future careers in science and technology. 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. NSF Award ID: 2548778 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Jennifer Chik | Institution: California Polytechnic State University Foundation, SAN LUIS OBISPO, CA | Award Amount: $310,099 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2548778 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2548778.html