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  • 1.
    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)
  • 2.
    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.

  • 3.
    Sinha, Indranil
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Intstitutet.
    Genome-wide patterns of histone modifications in fission yeast2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA is wrapped almost two times around a group of proteins called histones to form a chromosomal structure known as the nucleosome. Both DNA and histones can be modified with different chemical tags by several enzymes to activate or suppress a particular gene or group of genes. Histones can be covalently modified at several places. Among many different types of post-translational histone modifications, histone acetylation and methylation are two important modification types that are associated with transcriptional activation and repression. Histone acetylation and methylation can be added by histone acetyltransferases (HATs) and histone methyletransferases (HMTs), whereas these modifications can be removed by histone deacetylases (HDACs) and histone demethylases (HDMs). Histone modifications are not only involved in the regulation of gene expression, but also in DNA-based processes, such as replication, repair, and the formation and maintenance of heterochromatin. Combinations of modified and unmodified states of histones can form distinct histone modification patterns. In many different genome-wide studies, it was observed that a distinctive pattern of histone modification in various organisms is important for gene regulation, DNA replication, chromosome segregation and heterochromatin-mediated silencing. In this thesis, we have conducted several genome-wide investigations to uncover different histone modification patterns and their roles in transcriptional control in fission yeast. Our analysis of six different HDACs in fission yeast showed that Clr6 and Clr3 are mainly involved in keeping repressed genes silent; Sir2 and Hst2 repress non-expressed genes, and Hst4 acts globally to reduce gene expression, whereas Hos2 is required for the activation of gene expression. By investigating the influence of each HDAC on nucleosome density, we found that all sirtuins and Hos2 enzymes are required to maintain normal nucleosome density and distribution in the S. pombe genome. We have reported that histone acetylation patterns show a 5` to 3` polarity, i.e., the modification levels peak near the ATG and gradually decrease in the coding regions. We also found that histone acetylation patterns depend on gene expression but are independent of gene length. Comparing our data with other published datasets, we observed that different HDAC mutants affect acetylation in different parts of open reading frames (ORFs). We have demonstrated that histone H4 acetylation proceeds in the direction from K16 to K5, consistent with a `zip` model that may be involved in transcriptional control. Our analysis revealed antagonistic crosstalk between H3K36me2/me3 and H3K27ac at promoter regions. We observed that histone H3 K18, K27 and K9 acetylation positively correlate with gene expression, and a conserved pattern was also reported in other organisms. Finally, we report that histone H4K20me1 is strongly linked to active genes, whereas H4K20me3 is associated with weakly expressed genes. Our analysis further shows that H4K20me1 modification levels peak at 3‟UTR regions in active genes. Thus, our analysis revealed many different aspects of histone modification patterns and their roles in transcriptional control in fission yeast.

  • 4.
    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.

  • 5.
    Sinha, Indranil
    et al.
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Institutet.
    Jemt, Elisabeth
    Göteborgs universitet.
    Durand-Dubief, Mickaël
    Baraham Institute, Cambridge, UK.
    Stålfors, Annelie
    Karolinska Institutet.
    Sanders, Steven
    Case Western Reserve university, Cleveland, USA.
    Ekwall, Karl
    Karolinska Institutet.
    Gnome wide mapping suggests different roles for H4K20me1 and H4K20me3 in gene expressionManuscript (preprint) (Other academic)
  • 6.
    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.

  • 7.
    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.

  • 8.
    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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