Intellectual Merit: Every time the genome is replicated, the epigenetic state, which is controlled by chromatin organization, is either maintained to retain cell identity or reset to establish a new cell identify. The pathways regulating replication-coupled packaging of DNA into chromatin are largely unknown. This research is aimed at understanding how proteins involved in this process interact with each other during DNA replication to ensure proper maintenance of epigenetic states and maintenance of genome integrity. Genetic, biochemical and single-molecule strategies will be applied to define the relationships among members of this evolutionarily conserved replication-coupled chromatin assembly network in the budding yeast Saccharomyces cerevisiae. These results will be important for uncovering how this network is organized at the levels of protein-protein interactions, the sub-cellular location and the timing of these interactions, and connections between multiple pathways within this network. Mechanisms revealed by the research will contribute to understanding how the chromatin assembly network enables downstream genome-dependent processes, including: formation of silent chromatin, which inactivates gene expression; maintenance of appropriate chemical modifications on histones; and resistance to DNA damage. In addition, the research will produce broadly applicable strategies for measuring protein-protein interactions in single, living or fixed cells or in cell extracts at the single-molecule level.
Broader Impacts: The research will continue to encourage entry and promote retention of students in science. This research will be conducted primarily by graduate and undergraduate students and will provide an excellent opportunity for cross-disciplinary training in the fields of Biochemistry, Genetics and Biophysics. Graduate trainees will receive mentoring on career development, scientific writing, presentation and Problem Based Learning teaching strategies while conducting the proposed research. Trainees will share their findings with the broader scientific community by presenting their results at departmental, university-wide and international meetings, as well as in written publications.
Within the nucleus of the cell, DNA that makes up genes in chromosomes is organized by cellular proteins into chromatin. The kinds of protein complexes formed at any given site on the chromosome will determine whether genes are expressed (turned "on") or silenced (turned "off") and the expression state of every gene is reset or maintained every time the genome is replicated to change or maintain cell identity. The process of maintaining expression states, termed epigenetic gene regulation, plays critical roles in biology by ensuring stable patterns of gene expression during normal growth and differentiation. Improper formation of epigenetic processes results in the wrong genes being turned on or off and, thus, can lead to developmental defects and other catastrophic disorders. Understanding how proteins involved in assembling DNA into chromatin interact is important because these interactions are required to ensure proper maintenance of epigenetic states and are critical for genome integrity. Using the yeast Saccharomyces cerevisiae as a model, this research has explored how evolutionarily conserved proteins interact with each other to package DNA into chromatin during DNA replication. This research demonstrates how proteins involved in replication-coupled chromatin assembly interact at the single cell level to enable downstream genome-dependent processes. This work has helped to uncover how proteins are organized at the levels of protein-protein interactions, and the regulation of timing of these interactions. Significantly, this research describes how defects in these interactions perturb responses to DNA damage and epigenetic processes. These proteins serve as potential targets for environmental factors that adversely affect DNA damage responses or epigenetic processes in multiple organisms. This NSF support provided an opportunity for five undergraduate students (three female, one minority students) to gain an introduction to their discipline plus "one-on-one" training and "hands on" research experience as well as advanced training, including cross-disciplinary training in Biochemistry and Biophysics, for graduate students in designing, conducting and overseeing scientific research. Students received assistance in identifying professional development opportunities based on their career goals and gained experience in scientific writing, outreach, presentation and teaching.