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DNA microarray approaches to understanding the regulation and evolution of gene expression networks
Södertörn University, School of Life Sciences, Molecular biology. Karolinska Intitutet.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA microarray technology allows biological and medical research to shift from investigation of individual functions of a few related genes to the whole genome level. This creates opportunities for discovery of complex and coordinated transcriptional networks in biological systems. The aim of this thesis has been to study gene regulation and evolution using yeast responses to environmental cues as a model system. We first developed and validated a fission yeast cDNA microarray for genome-wide expression analysis (Paper I). It is the first commercially available fission yeast microarray, which presents a useful resouce for yeast researchers and provides information required to contruct the array from scratch. Next, we characterised the gene regulatory networks involved in the pheromone response (Paper II) and investigate the role of Gcn5 transcription co-regulator, a histone acetyltransferase (HAT), in re-programming gene expression during the salt stress response in fission yeast (Paper III). We further investigated evolutionary conservation and divergence of Gcn5 in gene regulation by comparing its role in the evolutionarily distantly related yeast species. The parallel study of the fission yeast and budding yeast showed that Gcn5 has a conserved physiological role in salt stress responses, but it regulates diverged sets of stress response genes potentially via distinct mechanisms (paper IV). Finally, we investigated interactions between different HATs and between HATs and HDACs (histone deacetylases). Phenotypic studies and gene expression profiling revealed that Gcn5 has overlapping functions with another HAT, Mst2, in the stress response and DNA damage repair (Paper V). We found that the HDAC Clr3 acts antagonistically to Gcn5 in transcriptional elongation and stress responses (Paper VI).

Place, publisher, year, edition, pages
Stockholm: Karolinska Institutet , 2009. , 46 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:sh:diva-30873ISBN: 978-91-7409-554-8OAI: oai:DiVA.org:sh-30873DiVA: diva2:971213
Public defence
2009-09-29, 10:00
Available from: 2016-09-15 Created: 2016-09-15 Last updated: 2016-09-15Bibliographically approved
List of papers
1. A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift
Open this publication in new window or tab >>A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift
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2004 (English)In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 21, no 1, 25-39 p.Article in journal (Refereed) Published
Abstract [en]

Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches. We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent >99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25degreesC to 36degreesC, conditions widely used when studying temperature-sensitive mutants. Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here.. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially.

National Category
Biochemistry and Molecular Biology Microbiology
Identifiers
urn:nbn:se:sh:diva-15500 (URN)10.1002/yea.1053 (DOI)000188635600003 ()14745780 (PubMedID)2-s2.0-0742289927 (ScopusID)
Available from: 2012-02-20 Created: 2012-02-20 Last updated: 2016-09-15Bibliographically approved
2. Genomewide identification of pheromone-targeted transcription in fission yeast
Open this publication in new window or tab >>Genomewide identification of pheromone-targeted transcription in fission yeast
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2006 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, 303- p., 303Article in journal (Refereed) Published
Abstract [en]

Background: Fission yeast cells undergo sexual differentiation in response to nitrogen starvation. In this process haploid M and P cells first mate to form diploid zygotes, which then enter meiosis and sporulate. Prior to mating, M and P cells communicate with diffusible mating pheromones that activate a signal transduction pathway in the opposite cell type. The pheromone signalling orchestrates mating and is also required for entry into meiosis. Results: Here we use DNA microarrays to identify genes that are induced by M-factor in P cells and by P-factor in M-cells. The use of a cyr1 genetic background allowed us to study pheromone signalling independently of nitrogen starvation. We identified a total of 163 genes that were consistently induced more than two-fold by pheromone stimulation. Gene disruption experiments demonstrated the involvement of newly discovered pheromone-induced genes in the differentiation process. We have mapped Gene Ontology ( GO) categories specifically associated with pheromone induction. A direct comparison of the M- and P-factor induced expression pattern allowed us to identify cell-type specific transcripts, including three new M- specific genes and one new P-specific gene. Conclusion: We found that the pheromone response was very similar in M and P cells. Surprisingly, pheromone control extended to genes fulfilling their function well beyond the point of entry into meiosis, including numerous genes required for meiotic recombination. Our results suggest that the SteII transcription factor is responsible for the majority of pheromone-induced transcription. Finally, most cell-type specific genes now appear to be identified in fission yeast.

National Category
Microbiology Genetics
Identifiers
urn:nbn:se:sh:diva-14270 (URN)10.1186/1471-2164-7-303 (DOI)000242727700002 ()17137508 (PubMedID)2-s2.0-33845715195 (ScopusID)
Available from: 2011-12-21 Created: 2011-12-20 Last updated: 2016-09-15Bibliographically approved
3. Stress-specific role of fission yeast Gcn5 histone acetyltransferase in programming a subset of stress response genes
Open this publication in new window or tab >>Stress-specific role of fission yeast Gcn5 histone acetyltransferase in programming a subset of stress response genes
2006 (English)In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 5, no 8, 1337-1346 p.Article in journal (Refereed) Published
Abstract [en]

Gcn5 is a coactivator protein that contributes to gene activation by acetylating specific lysine residues within the N termini of histone proteins. Gcn5 has been intensively studied in the budding yeast, Saccharomyces cerevisiae, but the features of genes that determine whether they require Gcn5 during activation have not been conclusively clarified. To allow comparison with S. cerevisiae, we have studied the genome-wide role of Gcn5 in the distantly related fission yeast, Schizosaccharomyces pombe. We show that Gcn5 is specifically required for adaptation to KCl- and CaCl2-mediated stress in S. pombe. We have characterized the genome-wide gene expression responses to KCl stress and show that Gcn5 is involved in the regulation of a subset of stress response genes. Gcn5 is most clearly associated with KCl-induced genes, but there is no correlation between Gcn5 dependence and the extent of their induction. Instead, Gen5-dependent KCl-induced genes are specifically enriched in four different DNA motifs. The Gcn5-dependent KCl-induced genes are also associated with biological process gene ontology terms such as carbohydrate metabolism, glycolysis, and nicotinamide metabolism that together constitute a subset of the ontology parameters associated with KCl-induced genes.

National Category
Microbiology
Identifiers
urn:nbn:se:sh:diva-14285 (URN)10.1128/EC.00101-06 (DOI)000239778700015 ()16896217 (PubMedID)2-s2.0-33747361359 (ScopusID)
Available from: 2011-12-20 Created: 2011-12-20 Last updated: 2016-09-15Bibliographically approved
4. Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles
Open this publication in new window or tab >>Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles
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2010 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, 200Article in journal (Refereed) Published
Abstract [en]

Background: Gcn5 is a transcriptional coactivator with histone cetyltransferase activity that is conserved with regard to structure as ell as its histone substrates throughout the eukaryotes. Gene egulatory networks within cells are thought to be evolutionarily iverged. The use of evolutionarily divergent yeast species, such as S. erevisiae and S. pombe, which can be studied under similar nvironmental conditions, provides an opportunity to examine the nterface between conserved regulatory components and their cellular pplications in different organisms. esults: We show that Gcn5 is important for a common set of stress esponses in evolutionarily diverged yeast species and that the activity f the conserved histone acetyltransferase domain is required. We define group of KCl stress response genes in S. cerevisiae that are pecifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent enes including Gcn5 repressed targets such as FLO8. Gcn5 regulates ivergent sets of KCl responsive genes in S. cerevisiae and S. pombe. enome-wide localization studies showed a tendency for redistribution of cn5 during KCl stress adaptation in S. cerevisiae from short genes to he transcribed regions of long genes. An analogous redistribution was ot observed in S. pombe. onclusions: Gcn5 is required for the regulation of divergent sets of Cl stress-response genes in S. cerevisiae and S. pombe even though it s required a common group of stress responses, including the response o KCl. Genes that are physically associated with Gcn5 require its ctivity for their repression or activation during stress adaptation, roviding support for a role of Gcn5 as a corepressor as well as a oactivator. The tendency of Gcn5 to re-localise to the transcribed egions of long genes during KCl stress adaptation suggests that Gcn5 lays a specific role in the expression of long genes under adaptive onditions, perhaps by regulating transcriptional elongation as has been een for Gcn5 in S. pombe. Interestingly an analogous redistribution of cn5 is not seen in S. pombe. The study thus provides important new nsights in relation to why coregulators like Gcn5 are required for the orrect expression of some genes but not others.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:sh:diva-13712 (URN)10.1186/1471-2164-11-200 (DOI)000277270600002 ()2861062 (PubMedID)2-s2.0-77952260641 (ScopusID)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Functional aspects of the Gcn5 histone acetyltransferase in stress responses of evolutionarily diverged yest species

Available from: 2011-12-06 Created: 2011-12-06 Last updated: 2016-09-15Bibliographically approved
5. Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation
Open this publication in new window or tab >>Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation
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2010 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, 59Article in journal (Refereed) Published
Abstract [en]

Background: Histone acetyltransferase enzymes (HATs) are implicated in egulation of transcription. HATs from different families may overlap in arget and substrate specificity. esults: We isolated the elp3(+) gene encoding the histone cetyltransferase subunit of the Elongator complex in fission yeast and haracterized the phenotype of an Delta elp3 mutant. We examined genetic nteractions between Delta elp3 and two other HAT mutants, Delta mst2 nd Delta gcn5 and used whole genome microarray analysis to analyze heir effects on gene expression. onclusions: Comparison of phenotypes and expression profiles in single, ouble and triple mutants indicate that these HAT enzymes have verlapping functions. Consistent with this, overlapping specificity in istone H3 acetylation is observed. However, there is no evidence for verlap with another HAT enzyme, encoded by the essential mst1(+) gene.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:sh:diva-13716 (URN)10.1186/1471-2164-11-59 (DOI)000274660300002 ()2823694 (PubMedID)2-s2.0-76949101471 (ScopusID)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Expression profiling of S. pombe acetyltransferase identifies redundant pathways of gene regulation

Available from: 2011-12-06 Created: 2011-12-06 Last updated: 2016-09-15Bibliographically approved
6. HAT-HDAC interplay modulates global histone H3K14 acetylation in gene-coding regions during stress
Open this publication in new window or tab >>HAT-HDAC interplay modulates global histone H3K14 acetylation in gene-coding regions during stress
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2009 (English)In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 10, no 9, 1009-1014 p.Article in journal (Refereed) Published
Abstract [en]

Histone acetylation and deacetylation are important for gene regulation. The histone acetyltransferase, Gcn5, is an activator of transcriptional initiation that is recruited to gene promoters. Here, we map genome-wide Gcn5 occupancy and histone H3K14ac at high resolution. Gcn5 is predominantly localized to coding regions of highly transcribed genes, where it collaborates antagonistically with the class-II histone deacetylase, Clr3, to modulate H3K14ac levels and transcriptional elongation. An interplay between Gcn5 and Clr3 is crucial for the regulation of many stress-response genes. Our findings suggest a new role for Gcn5 during transcriptional elongation, in addition to its known role in transcriptional initiation.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-13886 (URN)10.1038/embor.2009.127 (DOI)000269449500020 ()19633696 (PubMedID)2-s2.0-69949172827 (ScopusID)
Available from: 2011-12-14 Created: 2011-12-14 Last updated: 2016-09-15Bibliographically approved

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