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 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
Project description:Genome-wide translational profiling of rng3-65 compared to wild type cells. We used sucrose gradients to separate RNAs according to the number of associated ribosomes (a surrogate for translational efficiency). Preparation of the extracts and fractionation was carried out as described in Lackner et al, 2007 (Mol Cell 26(1):145-55). The fractions were pooled into four groups (1 closest to the top, i.e. not associated with ribosomes and 4 closest to the bottom, i.e., associated with polysomes). RNA was extracted from the pools and the corresponding pools from wild type and mutant cells were directly compared using DNA microarrays. Changes in translation are expected to alter the number of ribosomes associated with specific transcripts, and therefore result in a redistribution of the RNAs across the different fractions.
Project description:The fission yeast Mcs6-Mcs2-Pmh1 complex (homologous to metazoan Cdk7-cyclin HMat1) performs dual functions: in cell cycle control, as a CDK-activating kinase; and in transcription by RNA polymerase (Pol) II, as part of TFIIH. It is unknown whether that linkage serves to coordinate gene expression with cell division. Mutants in mcs6 and pmh1 arrest with incomplete cell separation and decreased phosphorylation of the Pol II large subunit. Gene expression profiling by microarray hybridization revealed that a defined subset (~5%) of genes was repressed by Mcs6 complex impairment, whereas the majority was refractory. The repression signature included a cell-cycle cluster implicated in cytokinesis and cell separation, and overlapped with those of the cell-separation mutants sep1, sep10 and sep15 (relative to wild-type cells). The gene sep10 encodes the homolog of a protein found in metazoan Mediator-like complexes, and sep15 encodes an established component of the Mediator. In mcs6 or pmh1 mutants, sep10+, which also encodes a component of the transcriptional machinery, becomes essential for viability. Finally, mcs6+ also interacts genetically with sep1+, which encodes a forkhead transcription factor required for periodic transcription during mitosis and cell division. Thus, the Mcs6 complex (a direct activator of the cell-cycle engine) also helps govern the cell-cycle transcriptional program.
Project description:The fission yeast Mcs6-Mcs2-Pmh1 complex (homologous to metazoan Cdk7-cyclin HMat1) performs dual functions: in cell cycle control, as a CDK-activating kinase; and in transcription by RNA polymerase (Pol) II, as part of TFIIH. It is unknown whether that linkage serves to coordinate gene expression with cell division. Mutants in mcs6 and pmh1 arrest with incomplete cell separation and decreased phosphorylation of the Pol II large subunit. Gene expression profiling by microarray hybridization revealed that a defined subset (~5%) of genes was repressed by Mcs6 complex impairment, whereas the majority was refractory. The repression signature included a cell-cycle cluster implicated in cytokinesis and cell separation, and overlapped with those of the cell-separation mutants sep1, sep10 and sep15 (relative to wild-type cells). The gene sep10 encodes the homolog of a protein found in metazoan Mediator-like complexes, and sep15 encodes an established component of the Mediator. In mcs6 or pmh1 mutants, sep10+, which also encodes a component of the transcriptional machinery, becomes essential for viability. Finally, mcs6+ also interacts genetically with sep1+, which encodes a forkhead transcription factor required for periodic transcription during mitosis and cell division. Thus, the Mcs6 complex (a direct activator of the cell-cycle engine) also helps govern the cell-cycle transcriptional program.
Project description:The fission yeast Mcs6-Mcs2-Pmh1 complex (homologous to metazoan Cdk7-cyclin HMat1) performs dual functions: in cell cycle control, as a CDK-activating kinase; and in transcription by RNA polymerase (Pol) II, as part of TFIIH. It is unknown whether that linkage serves to coordinate gene expression with cell division. Mutants in mcs6 and pmh1 arrest with incomplete cell separation and decreased phosphorylation of the Pol II large subunit. Gene expression profiling by microarray hybridization revealed that a defined subset (~5%) of genes was repressed by Mcs6 complex impairment, whereas the majority was refractory. The repression signature included a cell-cycle cluster implicated in cytokinesis and cell separation, and overlapped with those of the cell-separation mutants sep1, sep10 and sep15 (relative to wild-type cells). The gene sep10 encodes the homolog of a protein found in metazoan Mediator-like complexes, and sep15 encodes an established component of the Mediator. In mcs6 or pmh1 mutants, sep10+, which also encodes a component of the transcriptional machinery, becomes essential for viability. Finally, mcs6+ also interacts genetically with sep1+, which encodes a forkhead transcription factor required for periodic transcription during mitosis and cell division. Thus, the Mcs6 complex (a direct activator of the cell-cycle engine) also helps govern the cell-cycle transcriptional program.
Project description:Time course experiment to watch RNA expression profiles as telomeres shorten in trt1- strains, and to compare survivors with linear and circular chromosomes.
Project description:To obtain global data on polyadenylation of mRNAs, we fractionated the mRNAs according to their poly(A) tail length using a poly-U sepharose column followed by differential elution at five temperatures. Five mRNA fractions with distinct ranges of poly(A) tail length were then hybridized to microarrays using total eluate as a reference.
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:E. coli steady-state cultures were grown at different aerobiosis levels; transcriptomic profiling was carried out of these cultures to measure oxygen-dependent gene expression using a reference-design microarray format.