Intellectual Merit: Animal, plant and fungal cells alike package their DNA by spooling it in regular intervals about a protein core, forming a jointed chain of DNA spools (nucleosomes). The spooling renders the DNA less accessible and thus interferes with the reading of its genetic information. How then does DNA spooling affect gene activity and regulation? This research addresses this question by investigating the structure of isolated single-gene molecules and gene expression at the single cell level. The essential tools for this research are electron and fluorescence microscopy, combined with mathematical modeling, and yeast genetics. Surprisingly, recent studies have provided evidence that active genes randomly toggle between states during which the gene is either ON or OFF, rather than being on all the time. This random toggling causes the number of gene products produced from a single gene to fluctuate over time. These fluctuations can provide insight into how the gene is activated, but the molecular basis of ON/Off toggling is not understood. One possibility is that random spooling and unspooling of DNA--analogous to the continued opening and closing of a door at randomly chosen time points--may underlie the ON/OFF toggling of active genes. Testing of this hypothesis requires analysis of DNA spooling at the level of single gene molecules. To this end, this research will investigate the structure of specific genes within single cells of baker's yeast by isolation of the gene molecules and subsequent analysis by electron microscopy. The molecules will be isolated from cells that bear mutations in critical biochemical components for DNA spooling, including the proteins that make up the core of the DNA spool. These structural investigations will be combined with analysis of gene expression of single cells by fluorescence microscopy and mathematical modeling to reveal the dynamics and functional significance of DNA spooling for gene expression and regulation.
Broader Impacts: In this project a postdoctoral fellow and one graduate student will be trained. In addition, undergraduate students will be trained through academic-year or summer internships. Student interns will be recruited from underrepresented groups through existing campus programs. These students will be overseen by the PIs, both directly in the lab and in biweekly joint group meetings, and by the post-doctoral fellow and the graduate students of this project. Students will be trained in fluorescence and electron microscopy, yeast genetics and, importantly, in approaching biological problems by quantitative means. Daily interactions with scientists at different stages of their careers will benefit the undergraduates through multiple layers of supervision and will allow the graduate student and post-doctoral trainees to have the opportunity to supervise beginning students. The graduate student and post-doctoral trainee will be given the opportunity to present their work locally and at national meetings.
This project is co-funded by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences and by the Applied Mathematics and Mathematical Biology Programs in the Division of Mathematical Sciences.
