Characterization of the SWR-C Chromatin Remodeling Complex in Yeast Centromeres

Author: Adelle Warford | Major: Biology | Semester: Spring 2023

My sophomore year at the UofA, I was enrolled in Dr. Pinto’s genetics course where I grew an interest in genetics. Shortly after, I joined Dr. Pinto’s lab and began working on a double mutation of the INO80 and SWR1 chromosome remodeling complexes and their role on ploidy. I received two semesters of funding through the honors college to work on my research for my honors thesis. This semester, I chose to take my research further in a new project to identify the characterization of the SWR-C chromatin remodeling complex in yeast centromeres.

Errors in chromosome segregation during cell replication are detrimental to the health of living cells. Chromosome segregation errors can cause aneuploidy: daughter cells with either one or more missing or extra chromosomes, or polyploidy: the complete duplication of a genome. In humans, these segregation errors are responsible for many detrimental diseases and disorders. Down syndrome is a form of aneuploidy caused by the triplication of the 21st chromosome, while certain cancerous tumor cells exhibit aneuploidy and polyploidy, as well as abnormal centrosomes. My research focused on studying the SWR-C chromosome remodeling complex found in the model organism yeast S. cerevisiae known to individually cause an increase in ploidy when removed. The SWR-C chromatin remodeling complex is highly conserved from yeast to humans. The complex contains the SWR1 protein, the catalytic subunit responsible for the exchange of the native H2A histone subunit with the variant H2A.Z subunit. The centromere region of chromosomes is an important structure involved in the division of genetic material during meiosis. If the SWR1 protein is present in the centromere region, it would further advance the understanding of the correlation of SWR1 on polyploidy occurring during cell replication.

I began by creating a yeast strain with an epitope tagging SWR1. I modified the SWR1 gene by adding a short DNA sequence that encodes a specific epitope, cMyc, which is a peptide that is commercially available and not naturally present in yeast cells. This SWR1-cMyc epitope-tagged protein allowed me to identify the presence of the SWR1 protein at the centromere region by using the anti-cMyc antibody to bind to the correlating epitope. Through many trials, the epitope tagging SWR1 was unsuccessful. Dr. Pinto and I redirected our efforts to identify whether the SWR1 protein was active at the centromere region of chromosomes. With chromosome immunoprecipitation, I identified the levels of H2A.Z in centromeres using an antibody against H2A.Z to quantify the levels of this histone within the same centromeric region in a wild type and a mutant swr1D strain. The significantly lower amount of H2A.Z in the mutant strain proved that this modified histone is present and necessary for normal centromeric chromatin.

My research journey would be nothing without the guidance and mentorship of Dr. Pinto. Her knowledge and teaching skills led to my success in the lab and taught me the skills required for successful research. By advising me to apply for a research grant, I not only received funding, but I also learned how to write my findings in a scientific and professional manner. After graduation, I will be attending the University of Alabama Birmingham School of Optometry to pursue a career as an Optometrist. My experience in the lab these past few years prepared me to continue researching throughout my time in my field.