Histone proteins wrap DNA around themselves to form filaments called chromosomes, thereby regulating all aspects of DNA function, including DNA repair. Chromosome structure in turn is regulated by posttranslational modifications (PTMs) on the histones and the use of primary sequence variants of the canonical histones. Unlike its canonical counterparts, the histone variant H3.3 is deposited throughout the cell cycle in transcriptionally active regions, where it forms unstable nucleosomes. The research will examine a new potential role for H3.3 in DNA repair. The research will benefit society by promoting science education and training future scientists, while contributing to a better understanding of how histone proteins promote DNA repair. This research will be carried out with the involvement of K-12, undergraduate, and graduate students as well as postdoctoral scholars and will have a broad impact on science education and the training of future scientists. Early exposure to the joys of science encourages students to pursue future careers in science. Through a college-based summer outreach program, rural and minority K-12 students will be exposed to research concepts in the PIs lab. Genetic model systems such as the budding yeast and fruit flies are ideal for educating K-12 (and undergraduate) students in the methods of scientific research and explaining genetic principles. The PI will also continue to host high school juniors in his lab each summer under the Young Scholars Program (YSP) as they work on mini-projects. These mini-projects usually perform very well at science competitions, which helps them secure admission and scholarships at top universities.
While the transcriptional roles of histone H3.3 are well known, recent evidence also suggests critical transcription independent roles of H3.3. Consistent with this evidence, preliminary data pointing to a crucial role for H3.3 in homologous recombination (HR)-mediated DNA repair has been obtained, and this project will investigate the molecular mechanisms involved. The specific hypothesis to be tested is that histone H3.3 and its PTMs modulate chromatin structure at DNA damage sites to help recruit HR repair factors, thereby contributing to genomic integrity. The hypothesis will be tested by (i) determining the role of H3.3 in HR mediated DNA repair in distinct chromatin structures, (ii) identifying its DNA repair specific chaperones, and (iii) defining the role of H3.3 modifications in DNA repair. The research will use genome sequencing based biochemical assays, live cell microscopy, and mass spectrometry (MS) to determine the role of H3.3 and its modifications in HR-mediated DNA repair. The analyses will be carried out primarily in normal and H3.3 deficient cultured human cells, as well as in patient derived tumor cell lines carrying H3.3 mutations. A potentially useful byproduct of the MS data generated during the course of this research is the identification of novel DNA damage inducible PTMs on histones other than H3.3. Hence, the findings from this research will establish the role for H3.3 in DNA repair and will also lay the groundwork for the functional analysis of other histone variants implicated in DNA repair. Furthermore, due to the evolutionary conservation of the major processes involving chromatin and DNA repair, the research will yield useful information on mechanisms that contribute to genomic stability across eukaryotic species.
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.
