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  • 1.
    Appelgren, Henrik
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Kniola, Barbara
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells2003In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 116, no 19, p. 4035-4042Article in journal (Refereed)
    Abstract [en]

    Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one Another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion defect. Thus, S. pombe centromeres have two distinguishable domains even during mitosis, and our functional analyses support the previous observations that the kinetochore/central core and the heterochromatin domains have distinct functions both in interphase and mitosis.

  • 2. Bayne, Elizabeth H.
    et al.
    Portoso, Manuela
    Kagansky, Alexander
    Kos-Braun, Isabelle C.
    Urano, Takeshi
    Ekwall, Karl
    Södertörn University, School of Life Sciences.
    Alves, Flavia
    Rappsilber, Juri
    Allshire, Robin C.
    Splicing factors facilitate RNAi-directed silencing in fission yeast2008In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 322, no 5901, p. 602-606Article in journal (Refereed)
    Abstract [en]

    Heterochromatin formation at fission yeast centromeres is directed by RNA interference (RNAi). Noncoding transcripts derived from centromeric repeats are processed into small interfering RNAs (siRNAs) that direct the RNA-induced transcriptional silencing (RITS) effector complex to engage centromer transcripts, resulting in recruitment of the histone H3 lysine 9 methyltransferase Clr4, and hence silencing. We have found that defects in specific splicing factors, but not splicing itself, affect the generation of centromeric siRNAs and consequently centromeric heterochromatin integrity. Moreover, splicing factors physically associate with Cid12, a component of the RNAi machinery, and with centromeric chromatin, consistent with a direct role in RNAi. We propose that spliceosomal complexes provide a platform for siRNA generation and hence facilitate effective centromere repeat silencing.

  • 3. Bernard, Pascal
    et al.
    Schmidt, Christine Katrin
    Vaur, Sabine
    Dheur, Sonia
    Drogat, Julie
    Genier, Sylvie
    Ekwall, Karl
    Södertörn University, School of Life Sciences.
    Uhlmann, Frank
    Javerzat, Jean-Paul
    Cell-cycle regulation of cohesin stability along fission yeast chromosomes2008In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, no 1, p. 111-121Article in journal (Refereed)
    Abstract [en]

    Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.

  • 4.
    Bjerling, Pernilla
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Centromere domain organization and histone modifications2002In: Brazilian journal of medical and biological research, ISSN 0100-879X, E-ISSN 1414-431X, Vol. 35, no 5, p. 499-507Article in journal (Refereed)
    Abstract [en]

    Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles. Centromere structure appears to be organized in different, separable domains in order to accomplish these functions. Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences. Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms. The recent analysis of the centromere structure in the yeast S. pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere. The structural organization of the centromere is generally multilayered with a heterochromaun domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore. It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histories and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core. Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes.

  • 5.
    Bjerling, Pernilla
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Uppsala University / University of Copenhagen, Denmark .
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Egel, R
    Thon, G
    A novel type of silencing factor, Clr2, is necessary for transcriptional silencing at various chromosomal locations in the fission yeast Schizosaccharomyces pombe2004In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 32, no 15, p. 4421-4428Article in journal (Refereed)
    Abstract [en]

    The mating-type region of the fission yeast Schizosaccharomyces pombe comprises three loci: mat1, mat2-P and mat3-M. mat1 is expressed and determines the mating type of the cell. mat2-P and mat3-M are two storage cassettes located in a 17 kb heterochromatic region with features identical to those of mammalian heterochromatin. Mutations in the swi6(+), clr1(+), clr2(+), clr3(+), clr4(+) and clr6(+) genes were obtained in screens for factors necessary for silencing the mat2-P-mat3-M region. swi6(+) encodes a chromodomain protein, clr3(+) and clr6(+) histone deacetylases, and clr4(+) a histone methyltransferase. Here, we describe the cloning and characterization of clr2(+). The clr2(+) gene encodes a 62 kDa protein with no obvious sequence homologs. Deletion of clr2(+) not only affects transcriptional repression in the mating-type region, but also centromeric silencing and silencing of a PolII-transcribed gene inserted in the rDNA repeats. Using chromatin immunoprecipitation, we show that Clr2 is necessary for histone hypoacetylation in the mating-type region, suggesting that Clr2 acts upstream of histone deacetylases to promote transcriptional silencing.

  • 6.
    Bjerling, Pernilla
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Silverstein, Rebecca A
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Thon, G
    Caudy, A
    Grewal, S
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Functional divergence between histone deacetylases in fission yeast by distinct cellular localization and in vivo specificity2002In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 22, no 7, p. 2170-2181Article in journal (Refereed)
    Abstract [en]

    Histone deacetylases (HDACs) are important for gene regulation and the maintenance of heterochromatin in eukaryotes. Schizosaccharomyces pombe was used as a model system to investigate the functional divergence within this conserved enzyme family. S. pombe has three HDACs encoded by the hda1(+), clr(3+), and clr6(+) genes. Strains mutated in these genes have previously been shown to display strikingly different phenotypes when assayed for viability, chromosome loss, and silencing. Here, conserved differences in the substrate binding pocket identify Clr6 and Hda1 as class I HDACs, while Clr3 belongs in the class II family. Furthermore, these HDACs were shown to have strikingly different subcellular localization patterns. Hda1 was localized to the cytoplasm, while most of Clr3 resided throughout the nucleus. Finally, Clr6 was localized exclusively on the chromosomes in a spotted pattern. Interestingly, Clr3, the only HDAC present in the nucleolus, was required for ribosomal DNA (rDNA) silencing. Clr3 presumably acts directly on heterochromatin, since it colocalized with the centromere, mating-type region, and rDNA as visualized by in situ hybridization. In addition, Clr3 could be cross-linked to mat3 in chromatin immunoprecipitation experiments. Western analysis of bulk histone preparations indicated that Hda1 (class I) had a generally low level of activity in vivo and Clr6 (class 1) had a high level of activity and broad in vivo substrate specificity, whereas Clr3 (class II) displayed its main activity on acetylated lysine 14 of histone H3. Thus, the distinct functions of the S. pombe HDACs are likely explained by their distinct cellular localization and their different in vivo specificities.

  • 7. Carmichael, J B
    et al.
    Provost, P
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science.
    Hobman, T C
    Ago1 and Dcr1, two core components of the RNA interference pathway, functionally diverge from Rdp1 in regulating cell cycle events in Schizosaccharomyces pombe2004In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 15, no 3, p. 1425-1435Article in journal (Refereed)
    Abstract [en]

    In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1(+), dcr1(+), and rdp1(+), have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1(+) and dcr1(+) but not rdp1(+), exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1(+) and dcr1(+) are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1(+) is dispensable for this process, the functions of ago1(+) and dcr1(+) in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1(+) is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1(+) function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.

  • 8.
    Djupedal, Ingela
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Durand-Dubief, M.
    Babraham Institute, Cambridge, IK.
    Sinha, Indranil
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Differential Genome-wide Occupancies of RNA Polymerase II Subunits Rpb4 and Rpb7 in Fission YeastManuscript (preprint) (Other academic)
  • 9.
    Djupedal, Ingela
    et al.
    Södertörn University, School of Life Sciences.
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology.
    Epigenetics: heterochromatin meets RNAi2009In: Cell Research, ISSN 1001-0602, E-ISSN 1748-7838, Vol. 19, no 3, p. 282-295Article in journal (Refereed)
    Abstract [en]

    The term epigenetics refers to heritable changes not encoded by DNA. The organization of DNA into chromatin fibers affects gene expression in a heritable manner and is therefore one mechanism of epigenetic inheritance. Large parts of eukaryotic genomes consist of constitutively highly condensed heterochromatin, important for maintaining genome integrity but also for silencing of genes within. Small RNA, together with factors typically associated with RNA interference (RNAi) targets homologous DNA sequences and recruits factors that modify the chromatin, commonly resulting in formation of heterochromatin and silencing of target genes. The scope of this review is to provide an overview of the roles of small RNA and the RNAi components, Dicer, Argonaute and RNA dependent polymerases in epigenetic inheritance via heterochromatin formation, exemplified with pathways from unicellular eukaryotes, plants and animals.

  • 10.
    Djupedal, Ingela
    et al.
    Södertörn University, School of Life Sciences.
    Ekwall, Karl
    Södertörn University, School of Life Sciences.
    Molecular biology - The paradox of silent heterochromatin2008In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 320, no 5876, p. 624-625Article in journal (Other academic)
  • 11.
    Djupedal, Ingela
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Kos-Braun, Isabelle C.
    University of Edinburgh, Edinburgh, UK / Universität Heidelberg, Heidelberg, Germany.
    Mosher, Rebecca A.
    University of Cambridge, Cambridge, UK.
    Söderholm, Niklas
    Karolinska Institutet.
    Simmer, Femke
    University of Edinburgh, Edinburgh, UK.
    Hardcastle, Thomas J.
    University of Cambridge, Cambridge, UK.
    Fender, Aurelie
    Uppsala universitet.
    Heidrich, Nadja
    Uppsala universitet.
    Kagansky, Alexander
    University of Edinburgh, Edinburgh, UK.
    Bayne, Elizabeth
    University of Edinburgh, Edinburgh, UK.
    Wagner, E. Gerhart H.
    Uppala universitet.
    Baulcombe, David C.
    University of Cambridge, Cambridge, UK.
    Allshire, Robin C.
    University of Edinburgh, Edinburgh, UK.
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA2009In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 28, no 24, p. 3832-3844Article in journal (Refereed)
    Abstract [en]

    The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 50-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. The EMBO Journal (2009) 28, 3832-3844. doi: 10.1038/emboj.2009.351; Published online 26 November 2009

  • 12.
    Djupedal, Ingela
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Portoso, M
    University of Edinburgh, Edinburgh, UK.
    Spåhr, H
    Karolinska Institutet.
    Bonilla, Carolina
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Gustafsson, C M
    Karolinska Institutet.
    Allshire, R C
    University of Edinburgh, Edinburgh, UK.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing2005In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 19, no 19, p. 2301-2306Article in journal (Refereed)
    Abstract [en]

    Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in presiRNA transcription. We define a centromeric presiRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing.

  • 13.
    Durand-Dubief, Mickael
    et al.
    Södertörn University, School of Life Sciences.
    Ekwall, Karl
    Södertörn University, School of Life Sciences.
    Heterochromatin tells CENP-A where to go2008In: Bioessays, ISSN 0265-9247, E-ISSN 1521-1878, Vol. 30, no 6, p. 526-529Article in journal (Refereed)
    Abstract [en]

    The centromere is the region of the chromosome where the kinetochore forms. Kinetochores are the attachment sites for spindle microtubules that separate duplicated chromosomes in mitosis and meiosis. Kinetochore formation depends on a special chromatin structure containing the histone H3 variant CENP-A. The epigenetic mechanisms that maintain CENP-A chromatin throughout the cell cycle have been studied extensively but little is known about the mechanism that targets CENP-A to naked centromeric DNA templates. In a recent report published in Science,((1)) such de novo centromere assembly of CENP-A is shown to be dependent on heterochromatin and the RNA interference pathway.

  • 14.
    Durand-Dubief, Mickael
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Sinha, Indranil
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Fagerström-Billai, Fredrik
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Bonilla, Carolina
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony
    Södertörn University, School of Life Sciences. Karolinska Instiutet.
    Grunstein, Michael
    University of California, Los Angeles, CA, USA.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation and retrotransposon silencing2007In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 10, p. 2477-2488Article in journal (Refereed)
    Abstract [en]

    Expression profiling, ChiP-CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD(+)-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 colocalizes with the other Sirtuins at silent regions (cen, mat, tel, rDNA), and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function, whereas Hst4 represses amino-acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 50 mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation.

  • 15.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institute.
    'Arc' escorts siRNAs in heterochromatin assembly2007In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 14, no 3, p. 178-179Article in journal (Other academic)
    Abstract [en]

    RNA interference (RNAi) is important in directing heterochromatin assembly at centromeres in fission yeast, which is crucial for maintaining a stable genome through mitotic and meiotic divisions. In this issue, Buker et al. describe a new Argonaute siRNA chaperone (ARC) that converts duplex RNA to single-stranded RNA. This is a previously unknown step in the RNAi-directed heterochromatin-formation pathway.

  • 16.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Epigenetic control of centromere behavior2007In: Annual review of genetics / [ed] Allan Campbell, Wyatt W Anderson, Elizabeth W Jones, Palo Atlo: Annual Reviews , 2007, p. 63-81Chapter in book (Refereed)
    Abstract [en]

    The centromere is the DNA region that ensures genetic stability and is therefore of vital importance. Paradoxically, centromere proteins and centromeric structural domains are conserved despite that fact that centromere DNA sequences are highly variable and are not conserved. Remarkably, heritable states at the centromere can be propagated independent of the underlying centromeric DNA sequences. This review describes the epigenetic mechanisms governing centromere behavior, i.e., the mechanisms that control centromere assembly and propagation. A centromeric histone variant, CenH3, and histone modifications play key roles at centromeric chromatin. Histone modifications and RNA interference are important in assembly of pericentric heterochromatin structures. The molecular machinery that is directly involved in epigenetic control of centromeres is shared with regulation of gene expression. Nucleosome remodeling factors, histone chaperones, histone-modifying enzymes, transcription factors, and even RNA polymerase II itself control epigenetic states at centromeres.

  • 17.
    Ekwall, Karl
    Södertörn University, School of Life Sciences.
    Genome-wide analysis of HDAC function2005In: Trends in Genetics, ISSN 0168-9525, E-ISSN 1362-4555, Vol. 21, no 11, p. 608-615Article in journal (Refereed)
    Abstract [en]

    This article focuses on new developments in the genome-wide analysis of histone deacetylase (HDAC) function in yeast. HDACs are highly conserved in many organisms; therefore, their basic functions can be investigated using experimentally tractable model organisms, such as the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. New microarray techniques have enabled the systematic study of HDACs by identifying their direct and indirect gene targets in addition to their physiological functions and enzymatic specificity. These new approaches have already provided new surprising insights into the basic function of HDACs.

  • 18.
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    The RITS complex - A direct link between small RNA and heterochromatin2004In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 13, no 3, p. 304-305Article in journal (Refereed)
  • 19.
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    The roles of histone modifications and small RNA in centromere function2004In: Chromosome Research, ISSN 0967-3849, E-ISSN 1573-6849, Vol. 12, no 6, p. 535-542Article in journal (Refereed)
    Abstract [en]

    Here, epigenetic regulation of centromeric chromatin in fission yeast (Schizosaccharomyces pombe) is reviewed, focussing on the role of histone modifications and the link to RNA interference (RNAi). Fission yeast centromeres are organized into two structurally and functionally distinct domains, both of which are required for centromere function. The central core domain anchors the kinetochore structure while the flanking heterochromatin domain is important for sister centromere cohesion. The chromatin structure of both domains is regulated epigenetically. In the central core domain, the histone H3 variant Cnp1(CENP-A) plays a key role. In the flanking heterochromatin domain, histones are kept underacetylated by the histone deacetylases (HDACs) Clr3, Clr6 and Sir2, and methylated by Clr4 methyltransferase (HMTase) to create a specific binding site for the Swi6 protein. Swi6 then directly mediates cohesin binding to the centromeric heterochromatin. Recently, a surprising link was made between heterochromatin formation and RNAi. Centromeric flanking repeats are transcribed and the transcripts processed by the RNAse III-like enzyme, Dicer (Dcr1), to produce small interfering RNAs ( siRNA), which direct formation of heterochromatin via the RNA-induced Initiation of Transcriptional Silencing (RITS) protein complex. Consequently Dicer, Argonaute (Ago1), an RNA-dependent RNA polymerase (Rdp1) and several hitherto uncharacterized Csp ( centromere suppressor of position effect) gene products implicated in the RNAi pathway at centromeres are required for sister chromatid cohesion.

  • 20. Facanha, A L O
    et al.
    Appelgren, Henrik
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Tabish, Mohammad
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Okorokov, L
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics2002In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 157, no 6, p. 1029-1039Article in journal (Refereed)
    Abstract [en]

    Here we describe the phenotypic characterization of the cta(4+) gene, encoding a novel member of the P4 family of P-type ATPases of fission yeast. The cta4Delta mutant is temperature sensitive and cold sensitive lethal and displays several morphological defects in cell polarity and cytokinesis. Microtubules are generally destabilized in cells lacking Cta4p. The microtubule length is decreased, and the number of microtubules per cell is increased. This is concomitant with an increase in the number of microtubule catastrophe events in the midzone of the cell. These defects are likely due to a general imbalance in cation homeostasis. Immunofluorescence microscopy and membrane fractionation experiments revealed that green fluorescent protein-tagged Cta4 localizes to the ER. Fluorescence resonance energy transfer experiments in living cells using the yellow cameleon indicator for Ca2+ indicated that Cta4p regulates the cellular Ca2+ concentration. Thus, our results reveal a link between cation homeostasis and the control of cell shape, microtubule dynamics, and cytokinesis, and appoint Ca2+ as a key ion in controlling these processes.

  • 21.
    Fagerström-Billai, Fredrik
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Durand-Dubief, Mikael
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Individual Subunits of the Ssn6-Tup11/12 corepressor are selectively required for repression of different target genes2007In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 27, no 3, p. 1069-1082Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae Ssn6 and Tup1 proteins form a corepressor complex that is recruited to target genes by DNA-bound repressor proteins. Repression occurs via several mechanisms, including interaction with hypoacetylated N termini of histones, recruitment of histone deacetylases (HDACs), and interactions with the RNA polymerase II holoenzyme. The distantly related fission yeast, Schizosaccharomyces pombe, has two partially redundant Tup1-like proteins that are dispensable during normal growth. In contrast, we show that Ssn6 is an essential protein in S. pombe, suggesting a function that is independent of Tup11 and Tup12. Consistently, the group of genes that requires Ssn6 for their regulation overlaps but is distinct from the group of genes that depend on Tup11 or Tup12. Global chip-on-chip analysis shows that Ssn6 is almost invariably found in the same genomic locations as Tup11 and/or Tup12. All three corepressor subunits are generally bound to genes that are selectively regulated by Ssn6 or Tup11/12, and thus, the subunit specificity is probably manifested in the context of a corepressor complex containing all three subunits. The corepressor binds to both the intergenic and coding regions of genes, but differential localization of the corepressor within genes does not appear to account for the selective dependence of target genes on the Ssn6 or Tup11/12 subunits. Ssn6, Tup11, and Tup12 are preferentially found at genomic locations at which histones are deacetylated, primarily by the Clr6 class I HDAC. Clr6 is also important for the repression of corepressor target genes. Interestingly, a subset of corepressor target genes, including direct target genes affected by Ssn6 overexpression, is associated with the function of class II (CIr3) and III (Hst4 and Sir2) HDACs.

  • 22. Hogan, C. J.
    et al.
    Aligianni, S.
    Durand-Dubief, M.
    Persson, J.
    Will, W. R.
    Webster, J.
    Wheeler, L.
    Mathews, C. K.
    Elderkin, S.
    Oxley, D.
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Varga-Weisz, P. D.
    Fission yeast Iec1-Ino80-mediated nucleosome eviction regulates nucleotide and phosphate metabolism2010In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 30, no 3, p. 657-674Article in journal (Refereed)
    Abstract [en]

    Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenineresponsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

  • 23. Isaac, Sara
    et al.
    Walfridsson, Julian
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Zohar, Tal
    Lazar, David
    Kahan, Tamar
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Cohen, Amikam
    Interaction of Epe1 with the heterochromatin assembly pathway in Schizosaccharomyces pombe2007In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 175, no 4, p. 1549-1560Article in journal (Refereed)
    Abstract [en]

    Epe1 is a JmjC domain protein that antagonizes heterochromatization in Schizosaccharomyces pombe. Related JmjC domain proteins catalyze a histone demethylation reaction that depends on Fe(II) and alpha-ketoglutarate. However, no detectable demethylase activity is associated with Epe1, and its JmjC domain lacks conservation of Fe(II)-binding residues. We report that Swi6 recruits Epe1 to heterochromatin and that overexpression of epe1(+), like mutations in silencing genes or overexpression of swi6(+), upregulates expression of certain genes. A significant overlap was observed between the lists of genes that are upregulated by overexpression of epe1(+) and those that are upregulated by mutations in histone deacetylase genes. However, most of the common genes are not regulated by Clr4 histone methyltransferase. This suggests that Epe1 interacts with the heterochromatin assembly pathway at the stage of histone deacetylation. Mutational inactivation of Epe1 downregulates similar to 12% of S. pombe genes, and the list of these genes overlaps significantly with the lists of genes that are upregulated by mutations in silencing genes and genes that are hyperacetylated at their promoter regions in clr6-1 mutants. We propose that an interplay between the repressive HDACs activity and Epe1 helps to regulate gene expression in S. pombe.

  • 24.
    Johnsson, Anna
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Durand-Dubief, Mickael
    Karolinska Intitutet.
    Xue-Franzen, Yongtao
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Rönnerblad, Michelle
    Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Wright, Anthony P. H.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    HAT-HDAC interplay modulates global histone H3K14 acetylation in gene-coding regions during stress2009In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 10, no 9, p. 1009-1014Article in journal (Refereed)
    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.

  • 25. Khorosjutina, Olga
    et al.
    Wanrooij, Paulina H.
    Walfridsson, Julian
    Södertörn University, School of Life Sciences, Molecular biology.
    Szilagyi, Zsolt
    Zhu, Xuefeng
    Baraznenok, Vera
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology.
    Gustafsson, Claes M.
    A Chromatin-remodeling Protein Is a Component of Fission Yeast Mediator2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 39, p. 29729-29737Article in journal (Refereed)
    Abstract [en]

    The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cells. In contrast to the situation in many other eukaryotes, the conserved Med15 protein is not a stable component of Mediator isolated from fission yeast. We here demonstrate that Med15 exists in a protein complex together with Hrp1, a CHD1 ATP-dependent chromatin-remodeling protein. The Med15-Hrp1 subcomplex is not a component of the core Mediator complex but can interact with the L-Mediator conformation. Deletion of med15(+) and hrp1(+) causes very similar effects on global steady-state levels of mRNA, and genome-wide analyses demonstrate that Med15 associates with a distinct subset of Hrp1-bound gene promoters. Our findings therefore indicate that Mediator may directly influence histone density at regulated promoters.

  • 26.
    Kniola, Barbara
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    O'Toole, E
    McIntosh, J R
    Mellone, B
    Allshire, R
    Mengarelli, S
    Hultenby, K
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    The domain structure of centromeres is conserved from fission yeast to humans2001In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 12, no 9, p. 2767-2775Article in journal (Refereed)
    Abstract [en]

    The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes.

  • 27.
    Kärblane, Kairi
    et al.
    Tallinn University of Technology, Tallinn, Estonia / Competence Centre for Cancer Research, Tallinn, Estonia .
    Gerassimenko, Jelena
    Tallinn University of Technology, Tallinn, Estonia / Competence Centre for Cancer Research, Tallinn, Estonia .
    Nigul, Lenne
    Tallinn University of Technology, Tallinn, Estonia.
    Piirsoo, Alla
    Tallinn University of Technology, Tallinn, Estonia.
    Smialowska, Agata
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies. Karolinska Institutet.
    Vinkel, Kadri
    Tallinn University of Technology, Tallinn, Estonia .
    Kylsten, Per
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies.
    Ekwall, Karl
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies. Karolinska Institutet.
    Swoboda, Peter
    Karolinska Institutet.
    Truve, Erkki
    Tallinn University of Technology, Tallinn, Estonia / Competence Centre for Cancer Research, Tallinn, Estonia .
    Sarmiento, Cecilia
    Tallinn University of Technology, Tallinn, Estonia / Competence Centre for Cancer Research, Tallinn, Estonia .
    ABCE1 Is a Highly Conserved RNA Silencing Suppressor2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 2, article id e0116702Article in journal (Refereed)
    Abstract [en]

    ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.

  • 28. Lejeune, Erwan
    et al.
    Bortfeld, Miriam
    White, Sharon A.
    Pidoux, Alison L.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Allshire, Robin C.
    Ladurner, Andreas G.
    The chromatin-remodeling factor FACT contributes to centromeric heterochromatin independently of RNAi2007In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 17, no 14, p. 1219-1224Article in journal (Refereed)
    Abstract [en]

    Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms, including the fission yeast, Schizosaccharomyces pombe, the RNAi pathway contributes to the formation of silent chromatin in pericentromeric regions. Little is known about how chromatin-remodeling factors contribute to heterochromatic integrity and centromere function. Here we show that the histone chaperone and remodeling complex FACT is required for centromeric-heterochromatin integrity and accurate chromosome segregation. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for pob3+ are viable and alleviate gene silencing at centromeric repeats and at the silent mating-type locus. Importantly, like heterochromatin and RNAi pathway mutants, Pob3 null strains exhibit lagging chromosomes on anaphase spindles. Whereas the processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, Swi6-association with the centromere is reduced. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in heterochromatin integrity and in centromere function.

  • 29. Okorokova-Facanha, A L
    et al.
    Okorokov, L A
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    An inventory of the P-type ATPases in the fission yeast Schizosaccharomyces pombe2003In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 43, no 4, p. 273-280Article in journal (Refereed)
    Abstract [en]

    The analysis of the Schizosaccharomyces pombe genome revealed the presence of 14 putative P-type ATPases. The clustering of ATPases resembles that of Saccharomyces cerevisiae, indicating that the main classes of pumps were already present before the split of the Archiascomycetes from the other Ascomycota. The overall amino acid identity between fission and budding yeast P-type ATPases is generally low (30-50%). This is similar to the fungus-plant and fungus-animal comparisons.. suggesting that fungal ATPases underwent an extensive process of diversification. Unlike Sac. cerevisiae. fission yeast lacks Na+-ATPases, has a single heavy-metal ATPase and three ATPases of unknown specificity. The observed divergence within these fungi might reflect physiological differences, including adaptation to environmental stresses.

  • 30.
    Olsson, T G S
    et al.
    Göteborg University.
    Silverstein, Rebecka A.
    Karolinska Institutet.
    Ekwall, Karl
    Karolinska Institutet.
    Sunnerhagen, P
    Göteborg University.
    Transient inhibition of histone deacetylase activity overcomes silencing in the mating-type region in fission yeast1999In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 35, no 2, p. 82-87Article in journal (Refereed)
    Abstract [en]

    We have investigated the effects of inhibition of histone de-acetylase activity on silencing at the silent mating-type loci in fission yeast. Treatment of exponentially growing cells with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in derepression of a marker gene inserted 150 bp distal from the silent mat3-M locus. The natural targets for the silencing mechanism in this region were only partially derepressed and the activation appeared to be asymmetric. i.e. the mat2-P cassette remained silent at concentrations that clearly partially derepressed the mat3-M cassette. We further noted that treatment of wild-type h(90) cells resulted in the generation of altered sporulation phenotypes, indicating that the treatment affected the expression of mating-type genes and/or mating-type switching. The results are discussed in the light of recent accumulated data regarding the role of deacetylation for silencing in other species.

  • 31.
    Opel, Michael
    et al.
    University of Cambridge, Cambridge, United Kingdom.
    Lando, David
    Gurdon Institute and Department of Pathology, Cambridge, United Kingdom.
    Bonilla, Carolina
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Trewick, Sarah C.
    University of Edinburgh, Edinburgh, United Kingdom.
    Boukaba, Abdelhalim
    University of Edinburgh, Edinburgh, United Kingdom.
    Walfridsson, Julian
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Cauwood, James
    University of Cambridge, Cambridge, United Kingdom.
    Werler, Petra J. H.
    University of Sussex, Falmer, Sussex, United Kingdom.
    Carr, Antony M.
    University of Sussex, Falmer, Sussex, United Kingdom.
    Kouzarides, Tony
    Gurdon Institute and Department of Pathology, Cambridge, United Kingdom .
    Murzina, Natalia V.
    University of Cambridge, Cambridge, United Kingdom.
    Allshire, Robin C.
    University of Edinburgh, Edinburgh, United Kingdom.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Laue, Ernest D.
    University of Cambridge, Cambridge, United Kingdom.
    Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD12007In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 2, no 4, article id e386Article in journal (Refereed)
    Abstract [en]

    In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up-and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+) gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1 Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression.

  • 32.
    Persson, Jenna
    et al.
    Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation2010In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 316, no 8, p. 1316-1323Article in journal (Refereed)
    Abstract [en]

    Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.

  • 33.
    Provost, P
    et al.
    Karolinska Institute.
    Silverstein, Rebecca A
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Dishart, D
    Karolinska Institute.
    Walfridsson, Julian
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Djupedal, Ingela
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Kniola, Barbara
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Samuelsson, B
    Karolinska Institute.
    Radmark, O
    Karolinska Institute.
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Dicer is required for chromosome segregation and gene silencing in fission yeast cells2002In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 99, no 26, p. 16648-16653Article in journal (Refereed)
    Abstract [en]

    RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1(+)) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into approximate to23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1(+) was required for silencing of two genes containing a conserved motif.

  • 34. Rhind, Nicholas
    et al.
    Chen, Zehua
    Yassour, Moran
    Thompson, Dawn A.
    Haas, Brian J.
    Habib, Naomi
    Wapinski, Ilan
    Roy, Sushmita
    Lin, Michael F.
    Heiman, David I.
    Young, Sarah K.
    Furuya, Kanji
    Guo, Yabin
    Pidoux, Alison
    Chen, Huei Mei
    Robbertse, Barbara
    Goldberg, Jonathan M.
    Aoki, Keita
    Bayne, Elizabeth H.
    Berlin, Aaron M.
    Desjardins, Christopher A.
    Dobbs, Edward
    Dukaj, Livio
    Fan, Lin
    FitzGerald, Michael G.
    French, Courtney
    Gujja, Sharvari
    Hansen, Klavs
    Keifenheim, Dan
    Levin, Joshua Z.
    Mosher, Rebecca A.
    Mueller, Carolin A.
    Pfiffner, Jenna
    Priest, Margaret
    Russ, Carsten
    Smialowska, Agata
    Södertörn University, School of Life Sciences, Molecular biology.
    Swoboda, Peter
    Sykes, Sean M.
    Vaughn, Matthew
    Vengrova, Sonya
    Yoder, Ryan
    Zeng, Qiandong
    Allshire, Robin
    Baulcombe, David
    Birren, Bruce W.
    Brown, William
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology.
    Kellis, Manolis
    Leatherwood, Janet
    Levin, Henry
    Margalit, Hanah
    Martienssen, Rob
    Nieduszynski, Conrad A.
    Spatafora, Joseph W.
    Friedman, Nir
    Dalgaard, Jacob Z.
    Baumann, Peter
    Niki, Hironori
    Regev, Aviv
    Nusbaum, Chad
    Comparative Functional Genomics of the Fission Yeasts2011In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 332, no 6032, p. 930-936Article in journal (Refereed)
    Abstract [en]

    The fission yeast clade-comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus-occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

  • 35. Schramke, V
    et al.
    Sheedy, D M
    Denli, A M
    Bonilla, Carolina
    Södertörn University, School of Life Sciences.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Hannon, G J
    Allshire, R C
    RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription2005In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 435, no 7046, p. 1275-1279Article in journal (Refereed)
    Abstract [en]

    RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes(1-3). In plants(3-5), fission yeast(6-8), ciliates(9,10), flies(11) and mammalian cells(12,13), short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination(14). siRNAs may direct DNA or chromatin modification by siRNA - DNA interactions at the homologous locus(4,5). Alternatively, they may act by interactions between siRNA and nascent transcript(15,16). Here we show that in fission yeast ( Schizosaccharomyces pombe), chromatin modifications are only directed by RNAi if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNApol II disrupts transcriptional silencing, indicating that, like other RNA processing events(17-19), RNAi-directed chromatin modification is coupled to transcription.

  • 36.
    Silverstein, Rebecca A
    et al.
    Södertörn University, Avdelning Naturvetenskap.
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap.
    Similar mechanisms drive centromeric silencing and gene repression2003In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 2, no 2, p. 73-75Article in journal (Refereed)
  • 37.
    Silverstein, Rebecca A
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Sin3: a flexible regulator of global gene expression and genome stability2005In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 47, no 1, p. 1-17Article in journal (Refereed)
    Abstract [en]

    SIN3 was first identified genetically as a global regulator of transcription. Sin3 is a large protein composed mainly of protein-interaction domains, whose function is to provide structural support for a heterogeneous Sin3/histone deacetylase (HDAC) complex. The core Sin3/HDAC complex is conserved from yeast to man and consists of eight proteins. In addition to HDACs, Sin3 can sequester other enzymatic functions, including nucleosome remodeling, DNA methylation, N-acetylglucoseamine transferase activity, and histone methylation. Since the Sin3/HDAC complex lacks any DNA-binding activity, it must be targeted to gene promoters by interacting with DNA-binding proteins. Although most research on Sin3 has focused on its role as a corepressor, mounting evidence suggests that Sin3 can also positively regulate transcription. Furthermore, Sin3 is key to the propagation of epigenetically silenced domains and is required for centromere function. Thus, Sin3 provides a platform to deliver multiple combinations modifications to the chromatin, using both sequence-specific and sequence-independent mechanisms.

  • 38.
    Silverstein, Rebecka A.
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Richardson, W
    Levin, H
    Allshire, R
    Ekwall, Karl
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    A new role for the transcriptional corepressor SIN3; Regulation of centromeres2003In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 13, no 1, p. 68-72Article in journal (Refereed)
    Abstract [en]

    Centromeres play a vital role in maintaining the genomic stability of eukaryotes by coordinating the equal distribution of chromosomes to daughter cells during mitosis and meiosis. Fission yeast (S. pombe) centromeres consist of a 4-9 kb central core region and 30-100 kb of flanking inner (imr/B) and outer (otr/K) repeats [1-3]. These sequences direct a laminar kinetochore structure similar to that of human centromeres [4, 5]. Centromeric heterochromatin is generally underacetylated [6, 7]. We have previously shown that inhibition of histone deacetylases (HDACs) caused hyperacetylation of centromeres and defective chromosome segregation [8]. SIN3 is a HDAC corepressor that has the ability to mediate HDAC targeting in the repression of promoters. In this study, we have characterized S. pombe sin three corepressors (Pst1p and Pst2p) to investigate whether SIN3-HDAC is required in the regulation of centromeres. We show that only pst1-1 and not pst2Delta cells displayed anaphase defects and thiabendazole sensitivity. pst1-1 cells showed reduced centromeric silencing, increased histone acetylation in centromeric chromatin, and defective centromeric sister chromatid cohesion. The HDAC Clr6p and Pst1p coimmunoprecipitated, and Pst1p colocalized with centromeres, particularly in binucleate cells. These data are consistent with a model in which Pst1 pClr6p temporally associate with centromeres to carry out the initial deacetylation necessary for subsequent steps in heterochromatin formation.

  • 39.
    Sinha, Indranil
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Buchanan, Luke
    Technische Universität Dresden, Dresden, Germany / Max Planck Institute, Dresten, Germany.
    Rönnerblad, Michelle
    Karolinska Institutet.
    Bonilla, Carolina
    Karolinska Institutet.
    Durand-Dubief, Mickael
    Karolinska Institutet.
    Shevchenko, Andrej
    Max Planck Institute, Dresten, Germany.
    Grunstein, Michael
    Geffen School of Medicine at UCLA, & the Molecular Biology Institute, Los Angeles, USA.
    Stewart, A. Francis
    Technische Universität Dresden, Dresden, Germany.
    Ekwall, Karl
    Karolinska Institutet.
    Genome-wide mapping of histone modifications and mass spectrometry reveal H4 acetylation bias and H3K36 methylation at gene promoters in fission yeast2010In: Epigenomics, ISSN 1750-1911, Vol. 2, no 3, p. 377-393Article in journal (Refereed)
    Abstract [en]

    To map histone modifications with unprecedented resolution both globally and locus-specifically, and to link modification patterns to gene expression. Materials & methods: Using correlations between quantitative mass spectrometry and chromatin immunoprecipitation/microarray analyses, we have mapped histone post-translational modifications in fission yeast (Schizosaccharomyces pombe). Results: Acetylations at lysine 9, 18 and 27 of histone H3 give the best positive correlations with gene expression in this organism. Using clustering analysis and gene ontology search tools, we identified promoter histone modification patterns that characterize several classes of gene function. For example, gene promoters of genes involved in cytokinesis have high H3K36me2 and low H3K4me2, whereas the converse pattern is found ar promoters of gene involved in positive regulation of the cell cycle. We detected acetylation of H4 preferentially at lysine 16 followed by lysine 12, 8 and 5. Our analysis shows that this H4 acetylation bias in the coding regions is dependent upon gene length and linked to gene expression. Our analysis also reveals a role for H3K36 methylation at gene promoters where it functions in a crosstalk between the histone methyltransferase Set2(KMT3) and the histone deacetylase Clr6, which removes H3K27ac leading to repression of transcription. Conclusion: Histone modification patterns could be linked to gene expression in fission yeast.

  • 40.
    Sinha, Indranil
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Wirén, Marianna
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Genome-wide patterns of histone modifications in fission yeast2006In: Chromosome Research, ISSN 0967-3849, E-ISSN 1573-6849, Vol. 14, no 1, p. 95-105Article in journal (Refereed)
    Abstract [en]

    We have used oligonucleotide tiling arrays to construct genome-wide high-resolution histone acetylation maps for fission yeast. The maps are corrected for nucleosome density and reveal surprisingly uniform patterns of modifications for five different histone acetylation sites. We found that histone acetylation and methylation patterns are generally polar, i.e. they change as a function of distance from the ATG codon. A typical fission yeast gene shows a distinct peak of histone acetylation around the ATG and gradually decreased acetylation levels in the coding region. The patterns are independent of gene length but dependent on the gene expression levels. H3K9Ac shows a stronger peak near the ATG and is more reduced in the coding regions of genes with high expression compared with genes with low expression levels. H4K16Ac is strongly reduced in coding regions of highly expressed genes. A second microarray platform was used to confirm the 5' to 3' polarity effects observed with tiling microarrays. By comparing coding region histone acetylation data in HDAC mutants and wild type, we found that hos2 affects primarily the 5' regions, sir2 and clr6 affect middle regions, and clr6 affects 3' regions. Thus, mechanisms involving different HDACs modulate histone acetylation levels to maintain a 5' to 3' polarity within the coding regions.

  • 41.
    Smialowska, Agata
    et al.
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology. Karolinska instiutet.
    Djupedal, Ingela
    Karolinska instiutet.
    Wang, Jingwen
    Karolinska instiutet.
    Kylsten, Per
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology.
    Swoboda, Peter
    Karolinska instiutet.
    Ekwall, Karl
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Biology. Karolinska instiutet.
    RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe2014In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 444, no 2, p. 254-259Article in journal (Other academic)
    Abstract [en]

    RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

  • 42. Strålfors, Annelie
    et al.
    Walfridsson, Julian
    Södertörn University, School of Life Sciences, Molecular biology.
    Bhuiyan, Hasanuzzaman
    Södertörn University, School of Life Sciences, Molecular biology.
    Ekwall, Karl
    Södertörn University, School of Life Sciences, Molecular biology.
    The FUN30 Chromatin Remodeler, Fft3, Protects Centromeric and Subtelomeric Domains from Euchromatin Formation2011In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 7, no 3, article id e1001334Article in journal (Refereed)
    Abstract [en]

    The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. This implies the presence of insulator elements that separate adjacent domains, allowing them to maintain different chromatin structures. We show that the Fun30 chromatin remodeler, Fft3, is essential for maintaining a proper chromatin structure at centromeres and subtelomeres. Fft3 is localized to insulator elements and inhibits euchromatin assembly in silent chromatin domains. In its absence, euchromatic histone modifications and histone variants invade centromeres and subtelomeres, causing a mis-regulation of gene expression and severe chromosome segregation defects. Our data strongly suggest that Fft3 controls the identity of chromatin domains by protecting these regions from euchromatin assembly.

  • 43.
    Walfridsson, Julian
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Bjerling, Pernilla
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Thalen, Maria
    Södertörn University, School of Life Sciences. Karolinska Institute.
    Yoo, Eung-Jae
    Seoul National University, Seoul, Korea.
    Park, Sang Dai
    Seoul national University, Seoul, Korea.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institute.
    The CHD remodeling factor Hrp1 stimulates CENP-A loading to centromeres2005In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 33, no 9, p. 2868-2879Article in journal (Refereed)
    Abstract [en]

    Centromeres of fission yeast are arranged with a central core DNA sequence flanked by repeated sequences. The centromere-associated histone H3 variant Cnp1 ( SpCENP-A) binds exclusively to central core DNA, while the heterochromatin proteins and cohesins bind the surrounding outer repeats. CHD (chromo-helicase/ ATPase DNA binding) chromatin remodeling factors were recently shown to affect chromatin assembly in vitro. Here, we report that the CHD protein Hrp1 plays a key role at fission yeast centromeres. The hrp1&UDelta; mutant disrupts silencing of the outer repeats and central core regions of the centromere and displays chromosome segregation defects characteristic for dysfunction of both regions. Importantly, Hrp1 is required to maintain high levels of Cnp1 and low levels of histone H3 and H4 acetylation at the central core region. Hrp1 interacts directly with the centromere in early S-phase when centromeres are replicated, suggesting that Hrp1 plays a direct role in chromatin assembly during DNA replication.

  • 44.
    Walfridsson, Julian
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Khorosjutina, Olga
    Karolinska Institutet.
    Gustafsson, Claes M.
    Karolinska Institutet.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    A genome wide role for CHD remodelling factors and Nap1 in nucleosome disassemblyManuscript (preprint) (Other academic)
  • 45.
    Walfridsson, Julian
    et al.
    Södertörn University, School of Life Sciences.
    Khorosjutina, Olga
    Matikainen, Paulina
    Gustafsson, Claes M.
    Ekwall, Karl
    Södertörn University, School of Life Sciences.
    A genome-wide role for CHD remodelling factors and Nap1 in nucleosome disassembly2007In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 12, p. 2868-2879Article in journal (Refereed)
    Abstract [en]

    Chromatin remodelling factors and histone chaperones were previously shown to cooperatively affect nucleosome assembly and disassembly processes in vitro. Here, we show that Schizosaccharomyces pombe CHD remodellers, the Hrp1 and Hrp3 paralogs physically interact with the histone chaperone Nap1. Genome- wide analysis of Hrp1, Hrp3 and Nap1 occupancy, combined with nucleosome density measurements revealed that the CHD factors and Nap1 colocalized in particular to promoter regions where they remove nucleosomes near the transcriptional start site. Hrp1 and Hrp3 also regulate nucleosome density in coding regions, where they have redundant roles to stimulate transcription. Previously, DNA replication-dependent and -independent nucleosome disassembly processes have been described. We found that nucleosome density increased in the hrp1 mutant in the absence of DNA replication. Finally, regions where nucleosome density increased in hrp1, hrp3 and nap1 mutants also showed nucleosome density and histone modification changes in HDAC and HAT mutants. Thus, this study revealed an important in vivo role for CHD remodellers and Nap1 in nucleosome disassembly at promoters and coding regions, which are linked to changes in histone acetylation.

  • 46.
    Wiren, Marianna
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Silverstein, Rebecca A
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Sinha, Indranil
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Walfridsson, Julian
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Lee, Hang-mao
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Laurenson, P
    University of California, San Diego, USA.
    Pillus, L
    University of California, San Diego, USA.
    Robyr, D
    University of California, Los Angeles, USA.
    Grunstein, M
    University of California, Los Angeles, USA.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Genomewide analysis of nucleosome density histone acetylation and HDAC function in fission yeast2005In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 24, no 16, p. 2906-2918Article in journal (Refereed)
    Abstract [en]

    We have conducted a genomewide investigation into the enzymatic specificity, expression profiles, and binding locations of four histone deacetylases (HDACs), representing the three different phylogenetic classes in fission yeast ( Schizosaccharomyces pombe). By directly comparing nucleosome density, histone acetylation patterns and HDAC binding in both intergenic and coding regions with gene expression profiles, we found that Sir2 ( class III) and Hos2 ( class I) have a role in preventing histone loss; Clr6 ( class I) is the principal enzyme in promoter-localized repression. Hos2 has an unexpected role in promoting high expression of growth-related genes by deacetylating H4K16Ac in their open reading frames. Clr3 ( class II) acts cooperatively with Sir2 throughout the genome, including the silent regions: rDNA, centromeres, mat2/3 and telomeres. The most significant acetylation sites are H3K14Ac for Clr3 and H3K9Ac for Sir2 at their genomic targets. Clr3 also affects subtelomeric regions which contain clustered stress- and meiosis-induced genes. Thus, this combined genomic approach has uncovered different roles for fission yeast HDACs at the silent regions in repression and activation of gene expression.

  • 47.
    Xue, Yongtao
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Haas, S A
    Max-Plank Institute for Molecular Genetics, Berlin, Germany.
    Brino, L
    Eurogentec SA, Seraing, Belgium.
    Gusnanto, A
    Karolinska Institute.
    Reimers, M
    Karolinska Institute.
    Talibi, D
    Eurogentec SA, Seraing, Belgium.
    Vingron, M
    Max-Plank Institute for Molecular Genetics, Berlin, Germany.
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Wright, Anthony P H
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift2004In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 21, no 1, p. 25-39Article in journal (Refereed)
    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.

  • 48.
    Zhu, Xuefeng
    et al.
    Karolinska Institutet.
    Wirén, Marianna
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Sinha, Indranil
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Rasmussen, Nina N
    Institute of Molecular Biology, Copenhagen, Denmark.
    Linder, Tomas
    Karolinska Institutet.
    Holmberg, Steen
    Institute of Molecular Biology, Copenhagen, Denmark.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Gustafsson, Claes M
    Karolinska Institutet.
    Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions2006In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 22, no 2, p. 169-178Article in journal (Refereed)
    Abstract [en]

    Mediator exists in a free form containing the Med12, Med13, CDK8, and CycC subunits (the Srb8-11 module) and a smaller form, which lacks these four subunits and associates with RNA polymerase II (Pol II), forming a holoenzyme. We use chromatin immunoprecipitation (ChIP) and DNA microarrays to investigate genome-wide localization of Mediator and the Srb8-11 module in fission yeast. Mediator and the Srb8-11 module display similar binding patterns, and interactions with promoters and upstream activating sequences correlate with increased transcription activity. Unexpectedly, Mediator also interacts with the downstream coding region of many genes. These interactions display a negative bias for positions closer to the 5' ends of open reading frames (ORFs) and appear functionally important, because downregulation of transcription in a temperature-sensitive med17 mutant strain correlates with increased Mediator occupancy in the coding region. We propose that Mediator coordinates transcription initiation with transcriptional events in the coding region of eukaryotic genes.

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