Biomedical subjects

Jürg Bähler

Publications and source records attributed to Jürg Bähler.

At least 19 recordsLinked to original sources

PubMed2006

Genome-wide characterization of fission yeast DNA replication origins.

Eukaryotic DNA replication is initiated from multiple origins of replication, but little is known about the global regulation of origins throughout the genome or in different types of cell cycles. Here, we identify 401 strong origins and 503 putative weaker origins spaced in total every 14 kb throughout the genome of the fission yeast Schizosaccharomyces pombe. The same origins are used during premeiotic and mitotic S-phases. We found that few origins fire late in mitotic S-phase and that activating the Rad3 dependent S-phase checkpoint by inhibiting DNA replication had little effect on which origins were fired. A genome-wide analysis of eukaryotic origin efficiencies showed that efficiency was variable, with large chromosomal domains enriched for efficient or inefficient origins. Average efficiency is twice as high during mitosis compared with meiosis, which can account for their different S-phase lengths. We conclude that there is a continuum of origin efficiency and that there is differential origin activity in the mitotic and meiotic cell cycles.

Chromosomes, Fungal↗

PubMed2006

Simplified primer design for PCR-based gene targeting and microarray primer database: two web tools for fission yeast.

PCR-based gene targeting is a popular method for manipulating yeast genes in their normal chromosomal locations. The manual design of primers, however, can be cumbersome and error-prone. We have developed a straightforward web-based tool that applies user-specified inputs to automate and simplify the task of primer selection for deletion, tagging and/or regulated expression of genes in Schizosaccharomyces pombe. This tool, named PPPP (for Pombe PCR Primer Programs), is available at http://www.sanger.ac.uk/PostGenomics/S_pombe/software/. We also present a searchable Microarray Primer Database to retrieve the sequences and accompanying information for primers and PCR products used to build our in-house Sz. pombe microarrays. This database contains information on both coding and intergenic regions to provide context for the microarray data, and it should be useful also for other applications, such as quantitative PCR. The database can be accessed at http://www.sanger.ac.uk/PostGenomics/S_pombe/microarray/.

DNA Primers↗

PubMed2006

Global roles of Ste11p, cell type, and pheromone in the control of gene expression during early sexual differentiation in fission yeast.

Fission yeast cells belong to one of two specialized cell types, M or P. Specific environmental conditions trigger sexual differentiation, which leads to an internal program starting with pheromone signaling between M and P cells, followed by mating, meiosis, and sporulation. The initial steps of this process are controlled by Ste11p, a master transcriptional regulator that activates the expression of cell type-specific genes (only expressed in either M or P cells) as well as genes expressed in both M and P cells. Pheromone signaling is activated by Ste11p-dependent transcription and, in turn, enhances some of this transcription in a positive feedback. To obtain a genomewide view of Ste11p target genes, their cell-type specificity, and their dependence on pheromone, we used DNA microarrays along with different genetic and environmental manipulations of fission yeast cells. We identified 78 Ste11p-dependent genes, 12 and 4 of which are only expressed in M and P cells, respectively. These genes show differing grades of pheromone dependencies for Ste11p-activated transcription, ranging from complete independence to complete dependence on pheromone. We systematically deleted all novel cell type-specific genes and characterized their phenotype during sexual differentiation. A comparison with a similar data set from the distantly related budding yeast reveals striking conservation in both number and types of the proteins that define cell types. Given the divergent mechanisms regulating cell type-specific gene expression, our results highlight the plasticity of regulatory circuits, which evolve to allow adaptation to changing environments and lifestyles.

Cell Differentiation↗

PubMed2006

The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation.

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division.

Cell Division↗

PubMed2006

YOGY: a web-based, integrated database to retrieve protein orthologs and associated Gene Ontology terms.

We present YOGY a web-based resource for orthologous proteins from nine eukaryotic organisms: Homo sapiens, Mus musculus, Rattus norvegicus, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, Plasmodium falciparum, Schizosaccharomyces pombe and Saccharomyces cerevisiae. Using a gene name from any of these organisms as a query, this database provides comprehensive, combined information on orthologs in other species using data from five independent resources: KOGs, Inparanoid, HomoloGene, OrthoMCL and a table of curated fission and budding yeast orthologs. Associated Gene Ontology (GO) terms of orthologs can also be retrieved for functional inference. Integrating these different and complementary datasets provides a straightforward tool to identify known and predicted orthologs of proteins from a variety of species. This resource should be useful for bench scientists looking for functional clues for their genes of interest as well as for curators looking for information that can be transferred based on orthology and for rapidly identifying the relevant GO terms as an aid to literature curation. YOGY is accessible online at http://www.sanger.ac.uk/PostGenomics/S_pombe/YOGY/.

Animals↗

PubMed2006

Cip1 and Cip2 are novel RNA-recognition-motif proteins that counteract Csx1 function during oxidative stress.

Eukaryotic cells reprogram their global patterns of gene expression in response to stress. Recent studies in Schizosaccharomyces pombe showed that the RNA-binding protein Csx1 plays a central role in controlling gene expression during oxidative stress. It does so by stabilizing atf1(+) mRNA, which encodes a subunit of a bZIP transcription factor required for gene expression during oxidative stress. Here, we describe two related proteins, Cip1 and Cip2, that were identified by multidimensional protein identification technology (MudPIT) as proteins that coprecipitate with Csx1. Cip1 and Cip2 are cytoplasmic proteins that have RNA recognition motifs (RRMs). Neither protein is essential for viability, but a cip1Delta cip2Delta strain grows poorly and has altered cellular morphology. Genetic epistasis studies and whole genome expression profiling show that Cip1 and Cip2 exert posttranscriptional control of gene expression in a manner that is counteracted by Csx1. Notably, the sensitivity of csx1Delta cells to oxidative stress and their inability to induce expression of Atf1-dependent genes are partially rescued by cip1Delta and cip2Delta mutations. This study emphasizes the importance of a modulated mRNA stability in the eukaryotic stress response pathways and adds new information to the role of RNA-binding proteins in the oxidative stress response.

Amino Acid Motifs↗

PubMed2006

The more the merrier: comparative analysis of microarray studies on cell cycle-regulated genes in fission yeast.

The last two years have seen the publication of three genome-wide gene expression studies of the fission yeast cell cycle. While these microarray papers largely agree on the main patterns of cell cycle-regulated transcription and its control, there are discrepancies with regard to the identity and numbers of periodically expressed genes. We present benchmark and reproducibility analyses showing that the main discrepancies do not reflect differences in the data themselves (microarray or synchronization methods seem to lead only to minor biases) but rather in the interpretation of the data. Our reanalysis of the three datasets reveals that combining all independent information leads to an improved identification of periodically expressed genes. These evaluations suggest that the available microarray data do not allow reliable identification of more than about 500 cell cycle-regulated genes. The temporal expression pattern of the top 500 periodically expressed genes is generally consistent across experiments and the three studies, together with our integrated analysis, provide a coherent and rich source of information on cell cycle-regulated gene expression in Schizosaccharomyces pombe. The reanalysed datasets and other supplementary information are available from an accompanying website: http://www.cbs.dtu.dk/cellcycle/. We hope that this paper will resolve the apparent discrepancies between the previous studies and be useful both for wet-lab biologists and for theoretical scientists who wish to take advantage of the data for follow-up work.

Cell Cycle↗

PubMed2006

Upf1, an RNA helicase required for nonsense-mediated mRNA decay, modulates the transcriptional response to oxidative stress in fission yeast.

In the fission yeast Schizosaccharomyces pombe, oxidative stress triggers the activation of the Spc1/Sty1 mitogen-activated protein kinase, which in turn phosphorylates the Atf1/Pcr1 heterodimeric transcription factor to effect global changes in the patterns of gene expression. This transcriptional response is also controlled by Csx1, an RNA-binding protein that directly associates with and stabilizes atf1(+) mRNA. Here we report the surprising observation that this response also requires Upf1, a component of the nonsense-mediated mRNA decay (NMD) system. Accordingly, upf1Delta and csx1Delta strains are similarly sensitive to oxidative stress, and the effects of the mutations are not additive, suggesting that Upf1 and Csx1 work in the same pathway to stabilize atf1(+) mRNA during oxidative stress. Consistent with these observations, whole-genome expression profiling studies have shown that Upf1 controls the expression of more than 100 genes that are transcriptionally induced in response to oxidative stress, the large majority of which are also controlled by Atf1 and Csx1. The unexpected connection between an NMD factor and the oxidative stress response in fission yeast may provide important new clues about the physiological function of NMD in other species.

Activating Transcription Factor 1↗

PubMed2006

Autoregulation of ribosome biosynthesis by a translational response in fission yeast.

Maintaining the appropriate balance between the small and large ribosomal subunits is critical for translation and cell growth. We previously identified the 40S ribosomal protein S2 (rpS2) as a substrate of the protein arginine methyltransferase 3 (RMT3) and reported a misregulation of the 40S/60S ratio in rmt3 deletion mutants of Schizosaccharomyces pombe. For this study, using DNA microarrays, we have investigated the genome-wide biological response of rmt3-null cells to this ribosomal subunit imbalance. Whereas little change was observed at the transcriptional level, a number of genes showed significant alterations in their polysomal-to-monosomal ratios in rmt3Delta mutants. Importantly, nearly all of the 40S ribosomal protein-encoding mRNAs showed increased ribosome density in rmt3 disruptants. Sucrose gradient analysis also revealed that the ribosomal subunit imbalance detected in rmt3-null cells is due to a deficit in small-subunit levels and can be rescued by rpS2 overexpression. Our results indicate that rmt3-null fission yeast compensate for the reduced levels of small ribosomal subunits by increasing the ribosome density, and likely the translation efficiency, of 40S ribosomal protein-encoding mRNAs. Our findings support the existence of autoregulatory mechanisms that control ribosome biosynthesis and translation as an important layer of gene regulation.

Base Sequence↗

PubMed2005

A logical circuit for the regulation of fission yeast growth modes.

Growth of fission yeast at the ends of its cylindrical cells switches from a monopolar to a bipolar mode, before it ceases during mitosis and cell division. Here we assume that these growth modes correspond to three stable states of an underlying regulatory circuit, which is a relatively simple and to a large degree autonomous subsystem of an otherwise complex cellular control system. We develop a switch-like logical circuit based on three elements defined as binary variables. Effects of circuit variables on each other are expressed in terms of logical operations. We analyse this circuit for its behavior ("phenotypes") after removing single or multiple operations ("mutants"). Known fission yeast polarity mutants such as those defective in the switch to bipolar growth can be classified based on these predicted 'phenotypes'. Differences in growth patterns between daughter cells in different bipolar growth mutants are also predicted by the circuit model. The model presented here should provide a useful framework to guide future experiments into mechanisms of cellular polarity. This paper illustrates the usefulness of simple logical circuits to describe and dissect features of complex regulatory processes such as the fission yeast growth patterns in both wild type and mutant cells.

Cell Cycle↗

PubMed2005

Impairment of the TFIIH-associated CDK-activating kinase selectively affects cell cycle-regulated gene expression in fission yeast.

The fission yeast Mcs6-Mcs2-Pmh1 complex, homologous to metazoan Cdk7-cyclin H-Mat1, has dual functions in cell division and transcription: as a partially redundant cyclin-dependent kinase (CDK)-activating kinase (CAK) that phosphorylates the major cell cycle CDK, Cdc2, on Thr-167; and as the RNA polymerase (Pol) II carboxyl-terminal domain (CTD) kinase associated with transcription factor (TF) IIH. We analyzed conditional mutants of mcs6 and pmh1, which activate Cdc2 normally but cannot complete cell division at restrictive temperature and arrest with decreased CTD phosphorylation. Transcriptional profiling by microarray hybridization revealed only modest effects on global gene expression: a one-third reduction in a severe mcs6 mutant after prolonged incubation at 36 degrees C. In contrast, a small subset of transcripts ( approximately 5%) decreased by more than twofold after Mcs6 complex function was compromised. The signature of repressed genes overlapped significantly with those of cell separation mutants sep10 and sep15. Sep10, a component of the Pol II Mediator complex, becomes essential in mcs6 or pmh1 mutant backgrounds. Moreover, transcripts dependent on the forkhead transcription factor Sep1, which are expressed coordinately during mitosis, were repressed in Mcs6 complex mutants, and Mcs6 also interacts genetically with Sep1. Thus, the Mcs6 complex, a direct activator of Cdc2, also influences the cell cycle transcriptional program, possibly through its TFIIH-associated kinase function.

Cell Cycle↗

PubMed2005

A transcriptional pathway for cell separation in fission yeast.

Numerous genes are transcriptionally activated and repressed in a cell cycle-dependent manner. We have recently reported the global gene expression program during the cell cycle in fission yeast (S. pombe). Among the periodically expressed fission yeast genes, a large proportion shows peak transcript levels during mitosis. Many of these genes are regulated by a transcriptional cascade involving two transcription factors: the forkhead protein Sep1p which activates the zinc finger protein Ace2p. A main function of the Sep1p-Ace2p transcriptional pathway is to trigger the separation of daughter cells after cytokinesis. Absence of Sep1p, Ace2p, or some of their target genes leads to a hyphal-like growth pattern with chains of connected cells. Yeast cells probably evolved from filamentous fungi. It is possible that the Sep1p-Ace2p pathway contributed to the emergence of proliferation through single cells, and that this regulatory pathway can still be modulated to adjust growth modes depending on environmental conditions. Here, various properties of the Sep1p-Ace2p transcriptional pathway and mechanisms for cell separation are discussed in the context of recent findings.

Cell Cycle↗

PubMed2005

SCF(Pof1)-ubiquitin and its target Zip1 transcription factor mediate cadmium response in fission yeast.

Ubiquitin-dependent proteolysis regulates gene expression in many eukaryotic systems. Pof1 is an essential fission yeast F-box protein that is homologous to budding yeast Met30. Temperature-sensitive pof1 mutants display acute growth arrest with small cell size. Extragenic suppressor analysis identified Zip1, a bZIP (basic leucine zipper) transcription factor, as a target for Pof1. We show Zip1 is stabilized in pof1 mutants, Pof1 binds only phosphorylated forms of Zip1, and Zip1 is ubiquitylated in vivo, indicating that Zip1 is a substrate of SCF(Pof1). Genome-wide DNA microarray assay shows that many cadmium-induced genes are under the control of Zip1, suggesting Zip1 plays a role in cadmium response. Consistently, zip1 mutants are hypersensitive to cadmium and unlike wild type, lose cell viability under this stress. Intriguingly, cadmium exposure results in upregulation of Zip1 levels and leads wild-type cells to growth arrest with reduced cell size, reminiscent of pof1 phenotypes. Our results indicate that Zip1 mediates growth arrest in cadmium response, which is essential to maintain viability. Normally growing cells prevent this response through constitutive ubiquitylation and degradation of Zip1 via SCF(Pof1).

Basic-Leucine Zipper Transcription Factors↗

PubMed2005

Post-transcriptional control of gene expression: a genome-wide perspective.

Gene expression is regulated at multiple levels, and cells need to integrate and coordinate different layers of control to implement the information in the genome. Post-transcriptional levels of regulation such as transcript turnover and translational control are an integral part of gene expression and might rival the sophistication and importance of transcriptional control. Microarray-based methods are increasingly used to study not only transcription but also global patterns of transcript decay and translation rates in addition to comprehensively identify targets of RNA-binding proteins. Such large-scale analyses have recently provided supplementary and unique insights into gene expression programs. Integration of several different datasets will ultimately lead to a system-wide understanding of the varied and complex mechanisms for gene expression control.

Gene Expression Profiling↗

PubMed2005

Activation of AP-1-dependent transcription by a truncated translation initiation factor.

Int6/eIF3e is a highly conserved subunit of eukaryotic translation initiation factor 3 (eIF3) that has also been reported to interact with subunits of the proteasome and the COP9 signalosome. Overexpression of full-length Int6 or a 13-kDa C-terminal fragment, Int6CT, in the fission yeast Schizosaccharomyces pombe causes multidrug resistance that requires the otherwise inessential AP-1 transcription factor Pap1. Here we show for the first time that Int6CT acts to increase the transcriptional activity of Pap1. Microarray hybridization data indicate that Int6CT overexpression resulted in the up-regulation of 67 genes; this expression profile closely matched that of cells overexpressing Pap1. Analysis of the upstream regulatory sequences of these genes showed that the majority contained AP-1 consensus binding sites. Partial defects in ubiquitin-dependent proteolysis have been suggested to confer Pap1-dependent multidrug resistance, but no such defect was seen on Int6CT overexpression. Indeed, none of the previously identified interactions of endogenous Int6 was required for the activation of Pap1 transcription described here. Moreover, Int6CT-induced activation of Pap1-responsive gene expression was independent of the ability of Pap1 to undergo a redox-regulated conformational change which mediates its relocalization to the nucleus and expression of oxidative stress response genes. Int6CT therefore activates Pap1-dependent transcription by a novel mechanism.

Basic-Leucine Zipper Transcription Factors↗

PubMed2005

Global effects on gene expression in fission yeast by silencing and RNA interference machineries.

Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing.

Cell Cycle Proteins↗

PubMed2005

Cell-cycle control of gene expression in budding and fission yeast.

Cell-cycle control of transcription seems to be a universal feature of proliferating cells, although relatively little is known about its biological significance and conservation between organisms. The two distantly related yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have provided valuable complementary insight into the regulation of periodic transcription as a function of the cell cycle. More recently, genome-wide studies of proliferating cells have identified hundreds of periodically expressed genes and underlying mechanisms of transcriptional control. This review discusses the regulation of three major transcriptional waves, which roughly coincide with three main cell-cycle transitions (initiation of DNA replication, entry into mitosis, and exit from mitosis). I also compare and contrast the transcriptional regulatory networks between the two yeasts and discuss the evolutionary conservation and possible roles for cell cycle-regulated transcription.

Cell Cycle↗

PubMed2004

Global expression changes resulting from loss of telomeric DNA in fission yeast.

BACKGROUND: Schizosaccharomyces pombe cells lacking the catalytic subunit of telomerase (encoded by trt1+) lose telomeric DNA and enter crisis, but rare survivors arise with either circular or linear chromosomes. Survivors with linear chromosomes have normal growth rates and morphology, but those with circular chromosomes have growth defects and are enlarged. We report the global gene-expression response of S. pombe to loss of trt1+. RESULTS: Survivors with linear chromosomes had expression profiles similar to cells with native telomeres, whereas survivors with circular chromosomes showed continued upregulation of core environmental stress response (CESR) genes. In addition, survivors with circular chromosomes had altered expression of 51 genes compared to survivors with linear chromosomes, providing an expression signature. S. pombe progressing through crisis displayed two waves of altered gene expression. One coincided with crisis and consisted of around 110 genes, 44% of which overlapped with the CESR. The second was synchronized with the emergence of survivors and consisted of a single class of open reading frames (ORFs) with homology both to RecQ helicases and to dh repeats at centromeres targeted for heterochromatin formation via an RNA interference (RNAi) mechanism. Accumulation of transcript from the ORF was found not only in trt1- cells, but also in dcr1- and ago1- RNAi mutants, suggesting that RNAi may control its expression. CONCLUSIONS: These results demonstrate a correlation between a state of cellular stress, short telomeres and growth defects in cells with circular chromosomes. A putative new RecQ helicase was expressed as survivors emerged and appears to be transcriptionally regulated by RNAi, suggesting that this mechanism operates at telomeres.

Adenosine Triphosphatases↗