Histone deacetylases and their co-regulators in Schizosaccharomyces pombe

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

The role of fission yeast nuclear actin-related protein in mitosis.

Nuclear actin-related proteins (Arps) have 20-30% identity to conventional actin and many are found to be in chromatin remodelling complexes. There are two families of chromatin remodelling complexes, one of which carries out covalent modification on histones such as acetylation. The other is an ATPase complex, which alters the nucleosomal spacing, and nuclear Arps are found in both complexes. These complexes are believed to be required for transcriptional activation by increasing the accessibility of the transcription machinery to the target DNA. The alp5-1134 mutant was isolated from a screen for temperature-sensitive (ts) mutants with altered polarity and shows severe mitotic defects. Cloning of alp5+ revealed that Alp5 is an essential actin-related protein, most similar to budding yeast Arp4 and human BAF53. Alp5 localises to the nucleus and immunoprecipitates with Mst1, a histone acetyltransferase. These results strongly indicate the role of Alp5 in chromatin remodelling process, as its homologues. Given the interaction between Alp5 and Mst1, its function in histone acetylation was investigated both genetically and biochemically. It was found that Alp5 is required for acetylating the N-terminus tail of histone H4 lysine residues, and functionally counteracts with the histone deacetylases Clr6, Hst4 and Sir2. At the restrictive temperature, the alp5-1134 mutant shows a mitotic delay due to the activation of a spindle assembly checkpoint, which suggests a defect in the kinetochore-spindle interaction. This study also reveals that the function of Alp5 is required for the transcriptional repression at the core centromere region. Possible roles of Alp5 in mitosis are discussed

L'acide valproïque inhibe la progression dans le cycle cellulaire chez Saccharomyces cerevisiae

L’acétylation est une modification post-traductionnelle des protéines essentielles. Elle est impliquée dans bon nombre de processus cellulaires importants comme la régulation de la structure de la chromatine et le recrutement de protéines. Deux groupes d’enzymes, soient les lysines acétyltransférases et les lysines désacétylases, régulent cette modification, autant sur les histones que sur les autres protéines. Au cours des dernières années, de petites molécules inhibitrices des désacétylases ont été découvertes. Certaines d’entre elles semblent prometteuses contre diverses maladies telles le cancer. L’acide valproïque, un inhibiteur de deux des trois classes des désacétylases, a un effet antiprolifératif chez plusieurs organismes modèles. Toutefois, les mécanismes cellulaires sous-jacents à cet effet restent encore méconnus. Ce mémoire met en lumière l’effet pH dépendant de l’acide valproïque sur différentes voies cellulaires importantes chez la levure Saccharomyces cerevisiae. Il démontre que ce composé a la capacité d’inhiber la transition entre les phases G1 et S par son action sur l’expression des cyclines de la phase G1. De plus, il inhibe l’activation de la kinase principale de la voie activée suite à un stress à la paroi cellulaire. L’acide valproïque occasionne également un arrêt dans la réplication de l’ADN sans y causer de dommage. Il s’agit là d’un effet unique qui, à notre connaissance, n’est pas observable avec d’autres agents qui inhibent la progression en phase S.Acetylation is an essential post-translational modification involved in many important cellular processes such as regulation of chromatin structure and proteins interactions. Two enzyme families, lysine acetyltransferases and lysine deacetylases, allow proper regulation of this modification both on histones and non-histones proteins. In recent years, the discovery of small deacetylase inhibitors has led to promising novel therapy in the treatment against various diseases such as cancer. Valproic acid, a class I and II deacetylase inhibitor, has been shown to have antiproliferative effects in various models. However, the cellular mechanisms underlying this effect remain unknown. This thesis highlights the pH-dependent effects of VPA on numerous important cellular pathways in the yeast Saccharomyces cerevisiae. Our results demonstrate that VPA inhibits the transition from G1 to S phase of the cell cycle by its action on the expression of G1 cyclins. Moreover, VPA inhibits the activation of the main kinase involved in the cell wall integrity pathway. Furthermore, VPA exposure also leads to DNA replication arrest in a DNA damage-independent manner. This is a unique effect that, to our knowledge, is not observable with other agents that inhibit S phase progression

Investigating the role of histones in fission yeast centromere function

The centromere is the chromosomal region which is responsible for the accurate segregation of chromosomes during mitosis and meiosis. Failure to properly segregate replicated chromosomes causes aneuploidy and contributes to cancer progression. Fission Yeast centromeres display several features in common with the centromeres of higher eukaryotes. They are assembled in a specialised silent heterochromatin composed of underacetylated histones and methylated histone H3. To investigate the role of histone H3 and H4 N-tails in centromere structure and function, conserved lysines of histone N-termini were mutated to mimic hyperacetylated or non-methylated states. Since the fission yeast haploid genome contains three copies of both histones H3 and H4, initially a strain harbouring a single H3 and H4 gene was generated and analysed. This phenotypically wild type strain provided a genetic background in which to perform site-directed mutagenesis of the histone tails. These mutants showed that the H4 tail is not critical for silencing, while the H3 tail plays an essential role and is required for centromere function. Centromeric nucleosomes contain an essential histone H3-like protein, CENP-A. Antibodies were raised against the fission yeast CENP-A homologue Cnpl and were used to map Cnp1 association with the central domain of the centromere. Cnp1 appears to replace histone H3 in this domain and can coat a large fragment of non-centromeric DMA artificially inserted within this centromeric domain. Futhermore, strains expressing more histone H3 than H4 showed delocalisation of Cnp1, alleviation of centromeric silencing and missegregation of chromosomes in mitosis indicating that a fine balance between histone H3 and H4 is important for centromere function and that histone H3 can compete with Cnp1 in nucleosome assembly