Date of Graduation

5-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Pinto, Ines

Committee Member

Durdik, Jeannine M.

Second Committee Member

Stites, Wesley E.

Third Committee Member

Korth, Kenneth L.

Keywords

Acetylation; Chromosome segregation; Gene dosage; Histones

Abstract

Chromatin plays a role in all cellular functions that involve DNA. These include, but are not limited to replication, recombination, transcription, and chromosome segregation. Chromosome segregation is an extremely well conserved cellular process and is essential for maintaining the genetic integrity of a cell. There is very strong evidence indicating that chromatin structure is critical for maintaining the fidelity of chromosome transmission, but its specific role(s) in this process remains unclear. Chromatin is comprised of arrays of nucleosomes that serve to compact DNA. These nucleosomes consist of 146 bp of DNA that is wrapped around a histone octamer; two each of histones H2A, H2B, H3, and H4. The overall goal of this project has been to elucidate and understand the function of histones during chromosome segregation. Previous work has shown that a mutation in histone H2A, hta1-300 can cause both increase in ploidy and increase in chromosome loss, and that these defects correlate with an altered chromatin structure at the centromere.1 Suppressor analysis of this allele has identified a mutation in one of the two genes that encode histone H3 (hht1) is able to suppress the increase in ploidy phenotype.2 This suppression has been confirmed by deletion of the hht1 allele, and it has also been found that deleting the accompanying histone H4 allele (hhf1) suppresses the increase in ploidy caused by hta1-300. A new phenotype for the hta1-300 allele has been identified through mass spectrometry and western blotting; there is a marked increase in acetylation of lysine 12 of histone H4 (H4K12) in strains carrying the hta1-300 allele. Interestingly, the hht1Δ allele has a decrease in acetylation on H4K12. To further characterize these mutations at the centromere in order to understand their function in chromosome segregation, chromatin immunoprecipitation was done using an antibody against H4 acetylated at lysine 12. The increase in acetylation caused by hta1-300 was observed around the centromere, but not the decrease in acetylation caused by the hht1Δ allele. In contrast to these data, increasing the expression of HHT1, HHF1, or the gene pair results in severe growth phenotypes. Overexpression of the single genes in the presence of hta1-300 leads to a synthetic sickness, whereas overexpression of both leads to cell death. Previous work described an increased rate of chromosome loss as a result of high copy H3-H4 in a WT background,3 suggesting an additive effect of chromosome instability as a cause for the inviability of the H2A mutant strain. Taken together, these results stress the sensitivity of the Saccharomyces cerevisiae cell to histone gene dosage and histone pair stoichiometry. The data presented here suggest that histone modifications are altered in the H2A mutant and deletion of either H3 or H4 genes suppresses by restoring a balance in histone modifications. Also, these data support hypotheses that for proper cell function, histone genes must be stoichiometrically balanced as well as stoichiometrically balanced in their modifications across chromatin and that histone gene ratio has a function in the maintenance of histone post-translational modifications.

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