Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program
Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Gene Expression; Nucleosomes; Histones; Dissertations, UMMS
Genetics and Genomics | Life Sciences | Medicine and Health Sciences
There are two copies of each core histone in a nucleosome, however, it is unclear whether post-translational modifications on each molecule function redundantly or if symmetrical modifications are required to properly regulate gene expression. We tried to address this question by breaking nucleosomal symmetry and measuring its impact on gene expression. Our strategy includes re-engineering specific residues at the H3-H3 interface, generating pairs of mutant proteins, which were predicted by computational methods to form obligate heterodimers. Using S. cerevisiae as a model system, we tested the viability of strains with mutant histones, and analyzed the interaction between by co-immunoprecipitation from mononucleosome preparations. We also measured the changes of gene expression in the strains bearing single-tailed or tailless H3 heterodimers. The data suggested that the best computationally-derived H3 pair was frequently, but not exclusively heterodimeric in vivo. In order to obtain a more stringent H3 heterodimer, random mutagenesis was performed on four codons in the original computational design, and then genetic screening of the mutant libraries was performed.
Chen, Yuanyuan, "Generating Nucleosomal Asymmetry in Saccharomyces cerevisiae: A Masters Thesis" (2010). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 500.