The eukaryotic genome has to be organized to fit into the cell and this is achieved by packing of DNA into chromatin. The basic repeating structural unit of chromatin is the nucleosome, which consists of DNA wrapped around histone proteins. Histones are subjected to multiple covalent posttranslational modifications including, acetylation, methylation, phosphorylation, and ubiquitination. These modifications take part in gene regulation by changing the structure of chromatin and by recruiting gene regulatory proteins. Histone acetylation can be removed by histone deacetylases (HDACs), which are highly conserved enzymes that regulate a diverse number of biological processes including gene expression and chromosome segregation, and have shown to be closely linked to major diseases like cancer. This thesis described the genome-wide role of HDACs and transcription in S. pombe. We studied the genome wide binding targets and enzymatic specificity of different S. pombe HDACs and uncovered different roles for the enzymes at silent regions and in repression and activation of gene expression. We proposed that independent of gene length, a typical fission yeast gene shows a 5 to 3 polarity, i.e., the histone acetylation levels peak near the ATG and gradually decrease in the coding regions. We also observed that different HDACs are responsible for different position within the ORF regions. Our genome-wide study of two different Mediator complexes reviled that they displayed similar binding patterns, and interactions with promoters and upstream activating sequences correlated with increased transcription activity. We also found that Mediator associates with the downstream coding region of many genes. We finally developed a method, E-map, which made it possible to systematically construct haploid double mutants. This method was used for constructing genome-wide genetic interaction maps of HDACs in S. pombe. From our preliminary results we discovered a new link between the Class III HDACs and a biosynthesis protein. Our data also suggest that different HDACs are involved in distinct biological processes.

DNA is wrapped almost two times around a group of proteins called histones to form a chromosomal structure known as the nucleosome. Both DNA and histones can be modified with different chemical tags by several enzymes to activate or suppress a particular gene or group of genes. Histones can be covalently modified at several places. Among many different types of post-translational histone modifications, histone acetylation and methylation are two important modification types that are associated with transcriptional activation and repression. Histone acetylation and methylation can be added by histone acetyltransferases (HATs) and histone methyletransferases (HMTs), whereas these modifications can be removed by histone deacetylases (HDACs) and histone demethylases (HDMs). Histone modifications are not only involved in the regulation of gene expression, but also in DNA-based processes, such as replication, repair, and the formation and maintenance of heterochromatin. Combinations of modified and unmodified states of histones can form distinct histone modification patterns. In many different genome-wide studies, it was observed that a distinctive pattern of histone modification in various organisms is important for gene regulation, DNA replication, chromosome segregation and heterochromatin-mediated silencing. In this thesis, we have conducted several genome-wide investigations to uncover different histone modification patterns and their roles in transcriptional control in fission yeast. Our analysis of six different HDACs in fission yeast showed that Clr6 and Clr3 are mainly involved in keeping repressed genes silent; Sir2 and Hst2 repress non-expressed genes, and Hst4 acts globally to reduce gene expression, whereas Hos2 is required for the activation of gene expression. By investigating the influence of each HDAC on nucleosome density, we found that all sirtuins and Hos2 enzymes are required to maintain normal nucleosome density and distribution in the S. pombe genome. We have reported that histone acetylation patterns show a 5` to 3` polarity, i.e., the modification levels peak near the ATG and gradually decrease in the coding regions. We also found that histone acetylation patterns depend on gene expression but are independent of gene length. Comparing our data with other published datasets, we observed that different HDAC mutants affect acetylation in different parts of open reading frames (ORFs). We have demonstrated that histone H4 acetylation proceeds in the direction from K16 to K5, consistent with a `zip` model that may be involved in transcriptional control. Our analysis revealed antagonistic crosstalk between H3K36me2/me3 and H3K27ac at promoter regions. We observed that histone H3 K18, K27 and K9 acetylation positively correlate with gene expression, and a conserved pattern was also reported in other organisms. Finally, we report that histone H4K20me1 is strongly linked to active genes, whereas H4K20me3 is associated with weakly expressed genes. Our analysis further shows that H4K20me1 modification levels peak at 3‟UTR regions in active genes. Thus, our analysis revealed many different aspects of histone modification patterns and their roles in transcriptional control in fission yeast.

The DNA in every eukaryotic cell is wrapped around eight core histones to form the nucleosome. Therefore all events that involve DNA must also involve chromatin and nucleosomes. By regulating chromatin structure the cell can regulate the reactivity of the DNA. One of the most common ways of altering nucleosomes is the acetylation of lysine residues. Two enzymes are required to maintain the correct equilibrium for optimal cell growth: histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs). In general, histone hypoacetylation is correlated with transcriptional inactivation, while hyperacetylation is correlated with active gene transcription. In Schizosaccharomyces pombe, mating type loci are silenced. Deletion of HDAC Hos2 had previously been shown to slightly increase silencing at the mating type locus. To assess whether any other HDAC was necessary for mating type silencing, cells were treated with HDAC poison Trichostatin A (TSA). TSA was found to cause a mild derepression of the mating type locus, indicating that another HDAC was responsible for silencing in this region. The RNA interference nuclease Dcr1 was later identified, and showed to degrade double stranded RNA into small nucleotide fragments. Deletion of dcr1 caused chromosome segregation defects and derepression of centromeric silencing. Rpd3 in S. cerevisiae is recruited to genomic targets by interacting with co-regulator Sin3. S. pombe has three Sin3 homologs. Pst1 interacts with the HDAC Clr6, and like Clr6 is an essential gene, mutants of which display chromosome mis-segregation and derepression of centromeric silencing. Pst1 was required for centromeric cohesion, and localized to centromeres in late S phase. Thus a co-repressor paradigm could be applied to centromere silencing as well. A comparative characterization of HDACs in S. pombe showed that the HDACs had different localizations and histone specificities. The comparison of HDACs was taken further with a genome wide expression analysis and histone density study of mutants. Results indicated that Clr6 was most often involved in promoter initiated gene repression, whereas Hos2 promoted the high expression of growth related genes by deacetylating H4K16ac in their coding regions. A class II HDAC, Clr3, was found to act cooperatively with Sir2 throughout the genome. Using a genomic approach to analyze Pst3, it was established that Clr6 and Pst3 could cooperate to negatively regulate genes by binding to their promoter regions. On the other hand, Pst3 was also involved in the up-regulation of ribosome biosynthesis genes, and could bind to the rDNA.

Regulation of chromatin structure is essential in a wide variety of processes including transcriptional regulation, recombination, replication, chromosome segregation, development and differentiation. The enzymes that are central in regulating chromatin structure can be classified into two major groups. The first group of proteins consists of the histone modifying enzymes that catalyse the addition or removal of posttranslational modifications of histones. The second group of proteins is the highly conserved ATP-dependent remodeling factors that modify the nucleosome structure. Evidence is emerging that these two groups of proteins are intimately linked in chromatin function. This thesis describes the roles of the S. pombe Hrp1 and Hrp3 CHD remodeling factors in chromatin regulation, which have been shown to be important in centromere function and transcriptional regulation. The Hrp remodeling factors are functionally linked to the histone chaperone Nap1 as well as acetylation and methylation activities. We have demonstrated that Hrp1 has both independent and overlapping roles with Hrp3 in regulating centromere assembly and function. Both hrp1 and hrp3 deficient cells are disrupted in centromere silencing and display various chromosome segregation defects indicative of functions at both the outer repeats and the central core of the centromere. These phenotypes are likely to originate from the requirement of Hrp1 in keeping the centromeres hypoacetylated and for maintaining the histone H3 variant CENP-A at the central core of the centromere. Genetic interactions combined with chromatin immunoprecipitation and fluorescent in situ hybridisation indicate that Hrp1 stimulates CENP-A assembly during DNA replication. In addition to their centromere functions, the Hrp remodeling factors contribute to transcriptional regulation by promoting histone removal. Biochemical purifications identified a physical interaction between Hrp1 and Hrp3 and with the histone chaperone Nap1. Consistent with the physical interaction data, genome wide analysis showed that the CHD remodeling factors together with Nap1 have a common function in removing histones particularly at promoter regions. Interestingly, we found that histone disassembly in coding regions by both Hrp1 and Hrp3 promote transcriptional activation. Cell synchronisation studies revealed that the Hrp1 dependent histone disassembly occurs in a DNA replication independent manner. A functional interaction between acetylation and remodeling activity was established based on the high degree of overlap between the Hrp ATPases, regions affected by Nap1 histone density, and corresponding histone deacetylase and histone acetylase targets. Finally, we discovered that regions with upregulated genes and altered levels of histone modifications in the HDAC clr6-1 mutant were significantly similar to equivalent lists for the histone demethyl transferase swm1 mutant. In addition, the same regions with upregulated genes and effects on histone modification levels in the swm1 and clr6 mutant overlapped with Hrp1 and Hrp3 binding targets. Thus, it is likely that Swm1 act in concert with Clr6 and Hrp1 to mediate transcriptional silencing. Thus, HDACs, HATs, and HMTs are intimately linked in vivo to CHD nucleosome remodeling factors as well as histone chaperones in centromere assembly and transcriptional regulation.