Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Blessing Enya - Savannah State University
Co-Author(s): Tiffany Young and Ann Kirchmaier, Purdue University, West LaFayette, IN
The ‘Central Dogma’ involves replication of DNA, transcription of mRNA, and translation of mRNA into protein. When a gene maintains its sequence, but portrays heritable differences in expression, these differences are often as a result of a process known as epigenetics. Epigenetic regulation of gene expression involves histone modification, chromatin structure, and other factors in all eukaryotes. Using budding yeast (Saccharomyces cerevisiae) as a model to study epigenetics, has provided a strong understanding of how histone modifications and chromatin structures are inherited and maintained throughout the cell cycle. S. cerevisiae has also served as a good model for analyzing genetic interactions. Identifying genetic interaction can be performed by observing differences in growth rate in the presence and absence of DNA damaging agents, of single and double mutants compared to Wild-type. Mutations that occur in many genes responsible for replication, chromatin assembly, or histone modifications cause growth sensitivity in the presence of DNA damage. However, it is not known what role H4 K16 acetylation plays in responses to DNA damage and if this modification genetically interacts with chromatin assembly factor and histone acetyltransferase mutants. We hypothesized that H4K16 acetylation mutants would synthetically interact with defects in chromatin assembly factors and histone modifying-enzymes in the presence of DNA damage. Several genes such as ASF1, CAC1, RTT106, HIF1, etc., were mutated and combined with wild-type histones or H4K16 acetylation mutants. These combinations were assessed in synthetic interaction analyses in yeast, in order to evaluate how this modification influences responses to DNA damage and silencing. Yeast were grown in Yeast Peptone Dextrose media in the absence or presence of different drugs or environmental conditions such as hydroxyurea, methyl methane sulfonate, zeocin, and ultraviolet exposure. Images were taken and compared to see how each drug and mutant combination affect yeast growth. Results show presence of growth defects in single and double mutants in the presence relative to the absence of different DNA damaging agents, highlighting synthetic interaction between the gene mutation and histone mutants in yeast. Silencing was determined based on colony color to indicate expression of HMR:ADE2 in the mutated strains. Red colonies indicate the silencing of ADE2 and loss of gene expression. White colonies indicate fully expressed ADE2 and enhanced gene expression. The results suggest that loss of HMR::ADE2 silencing means defects of silent chromatin formation, and combination of H3K56 and H4K16 acetylation mutants affect yeast growth under different DNA damaging conditions. Combining mutated genes with other altered histone modification, to compare growth defects and DNA damage sensitivity will be conducted to understand genetic pathways regulating DNA damage responses and epigenetic processes.
Funder Acknowledgement(s): National Science Foundation; Purdue University Department of Biochemistry
Faculty Advisor: Ann Kirchmaier, email@example.com
Role: I performed synthetic interaction analysis procedure. This procedure includes plate streaking, making yeast-rich media plate (with presence and absence of DNA Damaging Agents), culture inoculation, taking gel images, comparing yeast cultures based on color, using previous research provided. I also performed yeast strain mutation, bacterial transformation and DNA amplification using Polymerase Chain Reaction (PCR) machine.