Project description:SWI/SNF chromatin remodeling complexes play critical roles in transcription and other chromatin-related processes. The analysis of the two members of this class in Saccharomyces cerevisiae, SWI/SNF and RSC, has heavily contributed to our understanding of these complexes. To understand the in vivo functions of SWI/SNF and RSC in an evolutionarily distant organism, we have characterized these complexes in Schizosaccharomyces pombe. While core components are conserved between the two yeasts, the compositions of S. pombe SWI/SNF and RSC differ from their S. cerevisiae counterparts and in some ways are more similar to metazoan complexes. Furthermore, several of the conserved proteins, including actin-like proteins, are strikingly different between the two yeasts with respect to their requirement for viability. Finally, phenotypic and microarray analyses identified widespread requirements for SWI/SNF and RSC on transcription including strong evidence that SWI/SNF directly represses iron transport genes.
Project description:The assembly of nucleosomes by histone chaperones is an important component of transcriptional regulation. Here we have assessed the global roles of the S. pombe HIRA histone chaperone complex. Microarray analysis indicates that inactivation of the HIRA complex results in increased expression of at least 4% of fission yeast genes. HIRA-regulated genes overlap with those which are normally repressed in vegetatively growing cells, such as targets of the Clr6 histone deacetylase and silenced genes located in subtelomeric regions. HIRA is also required for silencing of all 13 intact copies of the Tf2 long terminal repeat (LTR) retrotransposon. However, the role of HIRA is not restricted to bona fide promoters, because it also suppresses non-coding transcripts from solo LTR elements and spurious antisense transcripts from cryptic promoters associated with transcribed regions. Furthermore, the HIRA complex is essential in the absence of the quality control provided by nuclear exosome-mediated degradation of illegitimate transcripts. This suggests that HIRA restricts genomic accessibility, and, consistent with this, the chromosomes of cells lacking HIRA are more susceptible to genotoxic agents that cause double strand breaks. Thus the HIRA histone chaperone is required to maintain the protective functions of chromatin.
Project description:The cooperation of transcriptional and post-transcriptional controls to shape gene regulation is poorly understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modelling, afford insight into the multi-layered regulation of gene expression dynamics in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional control in response to stress, and it points to the first minutes after stress induction as a critical time when control of mRNA turnover can support transcriptional control for rapid gene regulation. In addition, we uncover specialized gene expression strategies such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for the ribosome biogenesis regulon, dominant roles of mRNA stabilisation for genes participating in protein degradation, and adjustment of mRNA turnover during stress adaptation. We also show that genes regulated independently of the Atf1p transcription factor are mainly controlled by mRNA turnover during oxidative stress.
Project description:The SAGA complex is a conserved multifunctional coactivator known to play broad roles in eukaryotic transcription. To gain new insights into its functions, we have performed biochemical and genetic analyses of SAGA in the fission yeast, Schizosaccharomyces pombe. Purification of the S. pombe SAGA complex showed that its subunit composition is identical to that of Saccharomyces cerevisiae. Analysis of S. pombe SAGA mutants revealed that SAGA has two opposing roles regulating sexual differentiation. First, in nutrient rich conditions, the SAGA histone acetyltransferase, Gcn5, represses ste11+, which encodes the master regulator of the mating pathway. In contrast, the SAGA subunit Spt8 is required for the induction of ste11+ upon nutrient starvation. Chromatin immunoprecipitation experiments suggest that these regulatory effects are direct, as SAGA is physically associated with the ste11+ promoter independent of nutrient levels. Genetic tests suggest that nutrient levels do cause a switch in SAGA function, as spt8? suppresses gcn5? with respect to ste11+ derepression in rich medium, whereas the opposite relationship, gcn5? suppression of spt8?, occurs during starvation. Thus, SAGA plays distinct roles in the control of the switch from proliferation to differentiation in S. pombe through the dynamic and opposing activities of Gcn5 and Spt8.
Project description:Spt6 is a conserved factor, critically required for several transcription and chromatin related processes. We now show that Spt6 and its binding partner, Iws1, are required for heterochromatic silencing in Schizosaccharomyces pombe. Our studies demonstrate that Spt6 is required for silencing of all heterochromatic loci and that an spt6 mutant has an unusual combination of heterochromatic phenotypes compared to previously studied silencing mutants. Unexpectedly, we find normal nucleosome positioning over heterochromatin and normal levels of histone H3K9 dimethylation. However, we also find greatly reduced levels of H3K9 trimethylation, elevated levels of H3K14 acetylation, and reduced recruitment of several silencing factors. Our evidence suggests that Spt6 plays a role at both the transcriptional and post-transcriptional levels; in an spt6 mutant, RNA polymerase II (RNAPII) occupancy at the pericentric regions is only modestly increased, while production of small interfering RNAs (siRNAs) is lost. Taken together, our results suggest that Spt6 is required for multiple steps in heterochromatic silencing by controlling chromatin, transcriptional, and post-transcriptional processes.
Project description:The SAGA complex is a conserved, multifunctional coactivator that plays broad roles in eukaryotic transcription. Previous studies suggested that Tra1, the largest SAGA component, is required either for SAGA assembly or for recruitment by DNA-bound transcriptional activators. In contrast to S. cerevisiae and mouse, a tra1? mutant is viable in S. pombe, allowing us to test these issues in vivo. We find that, in a tra1? mutant, SAGA assembles and is recruited to some, but not all promoters. Consistent with these findings, Tra1 regulates the expression of only a subset of SAGA-dependent genes. We previously reported that the SAGA subunits Gcn5 and Spt8 have opposing regulatory roles during S. pombe sexual differentiation. We show here that, like Gcn5, Tra1 represses this pathway, although by a distinct mechanism. Thus, our study reveals that Tra1 has specific regulatory roles, rather than global functions, within SAGA.
Project description:Gene expression is controlled globally and at multiple levels in response to environmental stress, but the relationships among these dynamic regulatory changes are not clear. Here we analyzed genome regulation in response to stress in the fission yeast, Schizosaccharomyces pombe, combining dynamic genome-wide data on mRNA levels and translational profiles with data on the levels of 2148 proteins (43% of proteome).
Project description:Although much is known about the regulation of eukaryotic transcription, the global controls which determine rates of total transcription within a cell are not well understood. We have investigated the effects that the DNA to protein ratio has on both total cellular transcription and the transcription of individual mRNA genes in the unicellular eukaryote fission yeast. Mutants altered in cell size and those blocked in cell cycle progression were used to vary the DNA to protein ratio over a fivefold range around the wild type value. We find that cells of sizes within twofold of wild type value regulate global transcription to maintain similar transcription rates per protein regardless of the cellular DNA content. These changes in total transcription were correlated with coordinated changes in gene occupancy by RNA polymerase II. In large cell cycle arrested mutants, when the DNA to protein ratio falls to a low level, total transcription rates plateau as DNA becomes limiting for transcription1. Unexpectedly, expression levels of individual genes remained tightly coordinated with each other over the entire range of cell sizes. We propose that there is a coordinated, global cellular control which determines the rate of transcription of most genes in the genome and that this control plays a role in regulating the overall growth rate of the cell. Normalized data generated using protocol P-TABM-1335 is available in file ZHURINSKY_NORMALISED_DATA.txt in the additional data archive.
Project description:The relative amount of RNA polymerase II (Pol II) associated with a given ORF provides an estimate for transcriptional efficiency. We therefore established a systematic approach to measure Pol II occupancy using chromatin immunoprecipitation followed by analysis on microarrays