| 1. |
- Appelgren, Henrik, et al.
(författare)
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Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells
- 2003
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 116:19, s. 4035-4042
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Bayne, Elizabeth H., et al.
(författare)
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Splicing factors facilitate RNAi-directed silencing in fission yeast
- 2008
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 322:5901, s. 602-606
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Bernard, Pascal, et al.
(författare)
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Cell-cycle regulation of cohesin stability along fission yeast chromosomes
- 2008
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 27:1, s. 111-121
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
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| 5. |
- Bjerling, Pernilla, et al.
(författare)
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Centromere domain organization and histone modifications
- 2002
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Ingår i: Brazilian journal of medical and biological research. - 0100-879X .- 1414-431X. ; 35:5, s. 499-507
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Bjerling, Pernilla, et al.
(författare)
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Functional divergence between histone deacetylases in fission yeast by distinct cellular localization and in vivo specificity
- 2002
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 22:7, s. 2170-2181
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Carmichael, J B, et al.
(författare)
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Ago1 and Dcr1, two core components of the RNA interference pathway, functionally diverge from Rdp1 in regulating cell cycle events in Schizosaccharomyces pombe
- 2004
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 15:3, s. 1425-1435
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Djupedal, Ingela, et al.
(författare)
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Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA
- 2009
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 28:24, s. 3832-3844
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Tidskriftsartikel (refereegranskat)abstract
- 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
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| 9. |
- Djupedal, Ingela
(författare)
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Characterization of RNA polymerase II subunit Rpb7 in silencing and transcription
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The DNA in eukaryotes is arranged in fibres of chromatin. The chromatin may be more or less compacted and the degree of condensation of the chromatin affects the accessibility of the DNA. The accessibility of the DNA, in turn, affects transcription and gene regulation. Genes within inaccessible DNA are commonly repressed whereasgenes within accessible DNA are active and expressed. This thesis concerns the interplay between chromatin and transcription with focus on the function of the RNA polymerase II (pol II) subunit Rpb7. We have demonstrated that processing of centromeric transcripts by the ribonuclease III family protein Dcr1 is required for heterochromatin formation at the centromeres of Schizosaccharomyces pombe. A point mutation in the pol II subunit Rpb7 caused a specific defect in centromeric heterochromatin formation. We have shown i) that the centromeric transcripts that accumulate in dcr1delta cells are products of pol II, ii) the rpbG150D mutation is deficient in recognition and/or initiation of transcription from the centromeric promoter. Transcription by pol II within the centromeres was surprising since insertion of marker genes within these loci normally results in repression of pol II transcription. Here, paradoxically, pol II transcription was required for the construction of the inaccessible heterochromatin structure. Our analysis of sRNA in S. pombe revealed that most centromeric siRNA are originating from two clusters, which are repeated several times within the centromeres. This lead us to propose a model in which centromeric transcripts fold into double stranded structures that are processed by Dcr1. The resulting siRNAs may contribute with the starting signal for the RNAi feedback loop required for heterochromatin formation at the centromeres. Finally, we demonstrate that the genome-wide association of Rpb7 is nearly identical to that of the core pol II subunit Rpb2, indicating a general role for Rpb7 in transcription. We further show that the occupancy pattern of Rpb4, a pol II subunit that forms a subcomplex together with Rpb7, differs from those of Rpb2 and Rpb7. Rpb4 may therefore have a less general function in transcription than Rpb7. Hence, transcription by pol II is required not only for gene expression but also for repression via formation of inaccessible heterochromatin.
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| 10. |
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