The budding yeast, Saccharomyces cerevisiae, serves as an excellent model for identifying fundamental mechanisms of DNA repair. A Local Coherence Detection (LCD) algorithm that uses biclustering to assign genes to multiple functional sub-groups was applied on the chromosome E-MAP containing genetic interactions among genes involved in nuclear processes. Using this method, we found that Asf1 and Rtt109, genes that are together required for histone H3K56 acetylation, cluster together with Ctf4, Ctf18, Ctf8 and Dcc1, genes important for efficient sister chromatid cohesion. It is known that H3K56 acetylation is required for post-repair chromatin reassembly at sites of DNA double-strand breaks (DSBs). The cohesion genes were previously implicated in the repair of some DNA DSBs, but the nature of their involvement has not been reported. The experimental data in my thesis work suggest that Ctf4, Ctf8, Ctf18 and Dcc1 function in the post-repair chromatin reassembly pathway.

Chromatin is the combination of DNA and proteins in the nucleus that is used to aid in the compaction of DNA. Histones are a group of proteins used to condense DNA by forming a complex (nucleosome) around which DNA wraps around; there are two of each type of histone in a nucleosome: H2A, H2B, H3 and H4. Once the DNA is wrapped around the histones, the genome is further compacted. A shortened, "clipped" version of histone H3 has been found in some organisms including yeasts, flies, mammalian stem cells, and the ciliated protozoan, Tetrahymena thermophila. In each organism, clipping occurs at a different site on the N-terminus, usually before an alanine residue. Clipping is important as it may affect other epigenetic modifications and gene regulation in cell differentiation, but the regulation of this histone proteolysis has remained largely unstudied. In Tetrahymena thermophila, approximately half of the histone H3 molecules are clipped between residues 6 and 7 on histone H3, solely in the transcriptionally silent micronucleus. The histones in the micronuclei are deacetylated, while histones in the macronuclei can be acetylated or deacetylated. It is hypothesized that the post-translational acetylation modification to the histone tails may inhibit histone H3 clipping. Immunoblot analyses were carried out with acetylated and deacetylated micronuclei, demonstrating an increase of clipping when acetylated. Additionally, mutations were created at lysine 9 upstream of the clip site on the histone H3 tails to mimic acetylation and deacetylation to study whether the modification of that site has a regulatory effect.

No description available.

Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.

No description available.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Histone H2A.Z is a highly conserved replication-independent histone variant that is essential for survival in diverse organisms including Tetrahymena thermophila, Drosophila melanogaster, Xenopus laevis, and Mus musculus. H2A.Z has been shown to play a role in many cellular processes including, but not limited to, gene expression, chromosome segregation, cell cycle progression, heterochromatin maintenance and epigenetic transcriptional memory. However, the mechanism by which H2A.Z and its post-translationally modified forms participate in these diverse cellular events and their subsequent effects on chromatin structure and function are not entirely clear. A thorough review of H2A.Z is provided in Chapter 1. We have isolated native non-acetylated and acetylated forms of H2A.Z and characterized nucleosome core particles (NCPs) reconstituted with these proteins using the analytical ultracentrifuge (Chapter 2). We report that NCPs reconstituted with native non-acetylated H2A.Z exhibit a slightly more compact conformation compared to those reconstituted with H2A. Furthermore, we show that acetylation of H2A.Z in conjunction with acetylation of the histone complement, results in NCPs that are less compact and less stable than H2A.Z-containing NCPs reconstituted with non-acetylated histones. Acetylated H2A.Z NCPs are nevertheless more compact and stable than acetylated H2A-containing NCPs. We have also identified the presence of two H2A.Z protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, and characterized the sites and abundances of their N-terminal peptide acetylation. Further characterization of the human H2A.Z isoforms is presented in Chapter 3 and indicates that they are expressed across a broad range of human tissues, and that they exhibit a similar but non-identical distribution within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1, and the phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution. Overall, these data suggest that the two isoforms of H2A.Z present in vertebrates may have acquired a degree of functional independence. In Chapter 4, we show that H2A.Z and an N-terminally acetylated form of H2A.Z associate with the prostate specific antigen (PSA) gene promoter and the levels of these proteins are reduced upon induction of the gene with androgen. Furthermore, H2A.Z protein levels increase in response to treatment with androgen which correlates with an increase in the mRNA expression levels of the H2A.Z-1 gene. Preliminary Western Blot and quantitative PCR analysis of H2A.Z (-1 and -2) levels in a tumor progression model of prostate cancer indicate that increased H2A.Z expression may be involved in the development of androgen independent prostate cancer. Collectively, our results contribute to our understanding of H2A.Z biology in vertebrates and support a role for this protein and its acetylated forms in poising promoter chromatin for subsequent gene transcription.

The covalent modifications to the histone 2A, 2B, 3, and 4 N-terminal tails that affect gene expression have been deemed the “Histone Code.” Mis-regulation of these signalling pathways is of great interest as are important in human disease. A variety of peptides containing post-translationally modified histone 3 and 4 sequences were read using a supramolecular sensor array approach, where two or three sensors gave a unique response for each analyte when compared to others. These sequences were chosen to determine what type of modifications could be read (phosphorylation, acetylation, methylation) and if this type of array would be suitable for reading analytes on which antibodies—the leading technology—typically perform poorly. It was found that three sensors, which operate in neutral aqueous solution, were able to discriminate 16 different histone analytes. Additionally, it was shown that this array could report simultaneously on both concentration and the identities of histone analytes. / Graduate

Histone phosphorylation is often a direct outcome of activated intracellular signaling pathways, and functions to translate extracellular signals into appropriate biological outputs such as changes in gene expression. Growth factors and cellular stress trigger rapid and transient expression of immediate-early genes (such as c-fos, c-jun) in mammalian cells, and their induction strongly correlates with a transient phosphorylation of S10 and S28 on histone H3. While many signaling cascades that lead to H3 phosphorylation have been mapped out, mechanistic details of the downstream events and how H3 phosphorylation contributes to transcriptional activation are still poorly defined. To investigate the direct effects of H3 phosphorylation on transcription, we targeted the H3 kinase MSK1 to endogenous c-fos promoter, and found that this is sufficient to activate its expression. Moreover, targeting MSK1 to the tissue-specific -globin gene induces H3S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Mechanistically, H3S28 phosphorylation not only disrupts binding of polycomb repressive complexes, but also induces a methyl-acetylation switch of the adjacent K27 residue. This provides the first indication that H3 phosphorylation is involved in antagonizing polycomb silencing. To further identify post-translational modifications (PTMs) that function together with MSK1-mediated H3 phosphorylation, I developed a novel nucleosome purification approach called Biotinylation-assisted Isolation of CO-modified Nucleosomes (BICON). This technique combines in vivo biotinylation by BirA and H3 phosphorylation by MSK1, allowing enrichment of phosphorylated nucleosomes using streptavidin. I found that MSK1-phosphorylated nucleosomes are hyper-acetylated on H3 and H4, and importantly, I identified a trans-tail crosstalk between H3 phosphorylation and H4 acetylation on K12. This proof-of-principle study demonstrates that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes. Taken together, work described in this thesis shows that histone H3 phosphorylation can initiate additional PTM changes on other residues within the nucleosome, and such crosstalk plays an important role in regulating gene expression.

Histone phosphorylation is often a direct outcome of activated intracellular signaling pathways, and functions to translate extracellular signals into appropriate biological outputs such as changes in gene expression. Growth factors and cellular stress trigger rapid and transient expression of immediate-early genes (such as c-fos, c-jun) in mammalian cells, and their induction strongly correlates with a transient phosphorylation of S10 and S28 on histone H3. While many signaling cascades that lead to H3 phosphorylation have been mapped out, mechanistic details of the downstream events and how H3 phosphorylation contributes to transcriptional activation are still poorly defined. To investigate the direct effects of H3 phosphorylation on transcription, we targeted the H3 kinase MSK1 to endogenous c-fos promoter, and found that this is sufficient to activate its expression. Moreover, targeting MSK1 to the tissue-specific -globin gene induces H3S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Mechanistically, H3S28 phosphorylation not only disrupts binding of polycomb repressive complexes, but also induces a methyl-acetylation switch of the adjacent K27 residue. This provides the first indication that H3 phosphorylation is involved in antagonizing polycomb silencing. To further identify post-translational modifications (PTMs) that function together with MSK1-mediated H3 phosphorylation, I developed a novel nucleosome purification approach called Biotinylation-assisted Isolation of CO-modified Nucleosomes (BICON). This technique combines in vivo biotinylation by BirA and H3 phosphorylation by MSK1, allowing enrichment of phosphorylated nucleosomes using streptavidin. I found that MSK1-phosphorylated nucleosomes are hyper-acetylated on H3 and H4, and importantly, I identified a trans-tail crosstalk between H3 phosphorylation and H4 acetylation on K12. This proof-of-principle study demonstrates that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes. Taken together, work described in this thesis shows that histone H3 phosphorylation can initiate additional PTM changes on other residues within the nucleosome, and such crosstalk plays an important role in regulating gene expression.