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.

Epistasis analysis, which reports on the extent to which the function of one gene depends on the presence of a second, is a powerful tool for studying the functional organization of the cell. Systematic genome-wide studies of epistasis, however, have been limited, with the majority of data being collected in the budding yeast, Saccharomyces cerevisiae. Here we present two 'pombe epistasis mapper' strategies, PEM-1 and PEM-2, which allow for high-throughput double mutant generation in the fission yeast, S. pombe. These approaches take advantage of a previously undescribed, recessive, cycloheximide-resistance mutation. Both systems can be used for genome-wide screens or for the generation of high-density, quantitative epistatic miniarray profiles (E-MAPs). Since S. cerevisiae and S. pombe are evolutionary distant, this methodology will provide insight into conserved biological pathways that are present in S. pombe, but not S. cerevisiae, and will enable a comprehensive analysis of the conservation of genetic interaction networks.

Mediator exists in a free form containing the Med12, Med13, CDK8, and CycC subunits (the Srb8-11 module) and a smaller form, which lacks these four subunits and associates with RNA polymerase II (Pol II), forming a holoenzyme. We use chromatin immunoprecipitation (ChIP) and DNA microarrays to investigate genome-wide localization of Mediator and the Srb8-11 module in fission yeast. Mediator and the Srb8-11 module display similar binding patterns, and interactions with promoters and upstream activating sequences correlate with increased transcription activity. Unexpectedly, Mediator also interacts with the downstream coding region of many genes. These interactions display a negative bias for positions closer to the 5' ends of open reading frames (ORFs) and appear functionally important, because downregulation of transcription in a temperature-sensitive med17 mutant strain correlates with increased Mediator occupancy in the coding region. We propose that Mediator coordinates transcription initiation with transcriptional events in the coding region of eukaryotic genes.

We have used oligonucleotide tiling arrays to construct genome-wide high-resolution histone acetylation maps for fission yeast. The maps are corrected for nucleosome density and reveal surprisingly uniform patterns of modifications for five different histone acetylation sites. We found that histone acetylation and methylation patterns are generally polar, i.e. they change as a function of distance from the ATG codon. A typical fission yeast gene shows a distinct peak of histone acetylation around the ATG and gradually decreased acetylation levels in the coding region. The patterns are independent of gene length but dependent on the gene expression levels. H3K9Ac shows a stronger peak near the ATG and is more reduced in the coding regions of genes with high expression compared with genes with low expression levels. H4K16Ac is strongly reduced in coding regions of highly expressed genes. A second microarray platform was used to confirm the 5' to 3' polarity effects observed with tiling microarrays. By comparing coding region histone acetylation data in HDAC mutants and wild type, we found that hos2 affects primarily the 5' regions, sir2 and clr6 affect middle regions, and clr6 affects 3' regions. Thus, mechanisms involving different HDACs modulate histone acetylation levels to maintain a 5' to 3' polarity within the coding regions.

We have conducted a genomewide investigation into the enzymatic specificity, expression profiles, and binding locations of four histone deacetylases (HDACs), representing the three different phylogenetic classes in fission yeast ( Schizosaccharomyces pombe). By directly comparing nucleosome density, histone acetylation patterns and HDAC binding in both intergenic and coding regions with gene expression profiles, we found that Sir2 ( class III) and Hos2 ( class I) have a role in preventing histone loss; Clr6 ( class I) is the principal enzyme in promoter-localized repression. Hos2 has an unexpected role in promoting high expression of growth-related genes by deacetylating H4K16Ac in their open reading frames. Clr3 ( class II) acts cooperatively with Sir2 throughout the genome, including the silent regions: rDNA, centromeres, mat2/3 and telomeres. The most significant acetylation sites are H3K14Ac for Clr3 and H3K9Ac for Sir2 at their genomic targets. Clr3 also affects subtelomeric regions which contain clustered stress- and meiosis-induced genes. Thus, this combined genomic approach has uncovered different roles for fission yeast HDACs at the silent regions in repression and activation of gene expression.