Type of Document	Dissertation
Author	Liang, Dun
Author's Email Address	dun.liang@gmail.com
URN	etd-07252011-230244
Title	Role of Histone Gene Dosage in DNA Damage Repair
Degree	Doctor of Philosophy
Department	Biomedical Sciences, Department of
Advisory Committee	Advisor Name Title Akash Gunjan Committee Chair Johanna Paik Committee Member Myra Hurt Committee Member Yanchang Wang Committee Member David Gilbert University Representative
Keywords	Double Strand Break Epigenetics Homologous Recombination Budding Yeast Genomic Stability
Date of Defense	2011-07-01
Availability	unrestricted
Abstract In eukaryotes, each individual chromosome is one large DNA molecule packed by histone proteins into a compact nucleoprotein filament. Two molecules each of core histone proteins H2A, H2B, H3 and H4 assemble to form an octamer protein core around which 147 base pairs of DNA is wrapped to form the nucleosome core particle and this structure is repeated to form chromatin. Histones are essential proteins as they package the genomic DNA to fit it inside the relatively tiny nucleus and regulate DNA accessibility. However, when present in excess, the positively charged histones can bind non-specifically to negatively charged DNA and affect all forms of DNA metabolism such as transcription, replication, repair and recombination. The DNA of all organisms is under constant threat of damage from both exogenous and endogenous agents that can contribute to genomic instability, which is characterized by the increased rate of acquisition of alterations in the genome and is a hallmark of cancer cells. Hence, cells have evolved multiple mechanisms to ensure genomic stability. Since DNA damage and repair occurs in a chromatin context in eukaryotes, chromatin structure and histones may affect genomic stability. Not surprisingly, scarcity of histones during DNA replication results in spontaneous DNA damage. On the other hand, accumulation of excess histones leads to genomic instability in the form of excessive chromosome loss, enhanced sensitivity to DNA damaging agents and cytotoxicity. Therefore, histone synthesis is tightly regulated at transcriptional, posttranscriptional, as well as posttranslational levels. Here, we have investigated the mechanism/s via which excess histones exert their deleterious effects in vivo in the budding yeast. We find that the presence of excess histones saturates certain histone modifying enzymes, potentially interfering with their activities. Additionally, excess histones appear to bind non-specifically to DNA as well as RNA, which can adversely affect their metabolism. Microarray analysis revealed that upon overexpression of the histone H3 and H4 gene pair or all four core histones but not individual histones, about 240 genes were either up or downregulated by 2-fold or more. Interestingly, histone overexpression does not affect the bulk chromatin structure, but alters the fine structure of chromatin. Overall, we present evidence that excess histones are likely to mediate their cytotoxic effects via multiple mechanisms that are primarily dependent on inappropriate electrostatic interactions between the positively charged histones and diverse negatively charged molecules in the cell. We have also investigated how changes in histone gene dosage affects the DNA damage sensitivity of budding yeast cells that have two copies of each histone gene when only one copy is needed for survival. We found that overexpression of histones led to an increase in DNA damage sensitivity. Next, we deleted the second copy of the gene pair (HHT2-HHF2) encoding histones H3 and H4 that contributes 6-8 fold more histone mRNA than the first gene pair (HHT1-HHF1), to create an experimental system to study the effects of reduced histone levels in vivo . A reduction in the dosage of histone H3-H4 resulted in a significant decrease in DNA damage sensitivity. By taking the advantage of a HO endonuclease induced DNA double stand break (DSB) at the budding yeast mating type (MAT) locus, we were able to study the DSB repair process in detail in strains with a reduced histone gene dosage. We found that the efficiency of Homologous Recombination (HR) at a DSB, as well as genome wide HR, was elevated in hht2-hhf2 deletion strain, while Non-Homologous End Joining remained unchanged. These effects were not associated with global changes in the expression of DNA repair genes or DNA damage checkpoint responses. We also found that there was no alteration of gross chromatin structure in response to changes in histone gene dosage. One mechanism by which reduced histone dosage leads to elevated HR mediated repair of a DSB at the MAT locus is through enhanced recruitment of the HR factors, as determined by the Chromatin Immunoprecipitation (ChIP) assay. Concomitant with this, cells experience a greater histone loss around this DSB upon a reduction in histone gene dosage. We propose that high levels of endogenous histones generated by multiple genes in eukaryotes compete with HR factors, thereby reducing HR efficiency and may normally function to restrain potentially excessive HR activities during S-phase. Overall, our findings help to explain the basis for the existence of multiple mechanisms that regulate histone levels and highlight their role in maintaining genomic stability and cell viability. Our findings could have major implications for DNA repair, genomic stability, carcinogenesis and aging in human cells that have dozens of histone genes.
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