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Sim4: a novel fission yeast kinetochore protein required for centromeric silencing and chromosome segregation.

Pidoux AL, Richardson W, Allshire RC - J. Cell Biol. (2003)

Bottom Line: Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins.The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region.Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, 6.34 Swann Building, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK. robin.allshire@ed.ac.uk

ABSTRACT
Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.

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Sim4 is associated with the centromere central core. (A) cnt and imr sequences are enriched in α-Sim4 ChIP. Multiplex PCR analysis. The positions of primers in cen1 are indicated; fbp is a control euchromatic locus. Enrichment of cnt and imr sequences in ChIPs is compared with the input PCR and expressed relative to fbp (right). (B) α-Sim4 ChIP performed on strains (FY4835 and 4837) with ura4+ inserted at Rint or cnt1. PCR with ura4 primers assays enrichment of ura4 sequences relative to the ura4-DSE minigene at the endogenous locus. Only ura4 at cnt1 is enriched in Sim4 ChIPs, indicating that Sim4 can coat noncentromeric DNA inserted at this site. Enrichment of ura4 in the α-Sim4 ChIPs relative to the input is indicated.
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fig5: Sim4 is associated with the centromere central core. (A) cnt and imr sequences are enriched in α-Sim4 ChIP. Multiplex PCR analysis. The positions of primers in cen1 are indicated; fbp is a control euchromatic locus. Enrichment of cnt and imr sequences in ChIPs is compared with the input PCR and expressed relative to fbp (right). (B) α-Sim4 ChIP performed on strains (FY4835 and 4837) with ura4+ inserted at Rint or cnt1. PCR with ura4 primers assays enrichment of ura4 sequences relative to the ura4-DSE minigene at the endogenous locus. Only ura4 at cnt1 is enriched in Sim4 ChIPs, indicating that Sim4 can coat noncentromeric DNA inserted at this site. Enrichment of ura4 in the α-Sim4 ChIPs relative to the input is indicated.

Mentions: To determine with which region(s) of the centromere Sim4 is associated, chromatin immunoprecipitation (ChIP) experiments were performed. DNA present in crude extracts and α-Sim4 ChIPs was analyzed using four primer pairs in a multiplex PCR. These primer pairs specifically amplify regions within the central core of cen1 and 3 (cnt), the inner repeats of cen1 (imr), the outer repeat of cen1 (otr), and fbp1+, a control euchromatic gene (fbp). As shown in Fig. 5 A, central core (cnt) sequences and inner repeat (imr) sequences were enriched 6.5- and 6.8-fold, respectively, relative to fbp in Sim4 ChIPs compared with the input control. Outer repeat sequences (otr) were not enriched. These experiments indicate that Sim4 is specifically associated with the central domain of the centromere but is absent from the outer repeat regions.


Sim4: a novel fission yeast kinetochore protein required for centromeric silencing and chromosome segregation.

Pidoux AL, Richardson W, Allshire RC - J. Cell Biol. (2003)

Sim4 is associated with the centromere central core. (A) cnt and imr sequences are enriched in α-Sim4 ChIP. Multiplex PCR analysis. The positions of primers in cen1 are indicated; fbp is a control euchromatic locus. Enrichment of cnt and imr sequences in ChIPs is compared with the input PCR and expressed relative to fbp (right). (B) α-Sim4 ChIP performed on strains (FY4835 and 4837) with ura4+ inserted at Rint or cnt1. PCR with ura4 primers assays enrichment of ura4 sequences relative to the ura4-DSE minigene at the endogenous locus. Only ura4 at cnt1 is enriched in Sim4 ChIPs, indicating that Sim4 can coat noncentromeric DNA inserted at this site. Enrichment of ura4 in the α-Sim4 ChIPs relative to the input is indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172903&req=5

fig5: Sim4 is associated with the centromere central core. (A) cnt and imr sequences are enriched in α-Sim4 ChIP. Multiplex PCR analysis. The positions of primers in cen1 are indicated; fbp is a control euchromatic locus. Enrichment of cnt and imr sequences in ChIPs is compared with the input PCR and expressed relative to fbp (right). (B) α-Sim4 ChIP performed on strains (FY4835 and 4837) with ura4+ inserted at Rint or cnt1. PCR with ura4 primers assays enrichment of ura4 sequences relative to the ura4-DSE minigene at the endogenous locus. Only ura4 at cnt1 is enriched in Sim4 ChIPs, indicating that Sim4 can coat noncentromeric DNA inserted at this site. Enrichment of ura4 in the α-Sim4 ChIPs relative to the input is indicated.
Mentions: To determine with which region(s) of the centromere Sim4 is associated, chromatin immunoprecipitation (ChIP) experiments were performed. DNA present in crude extracts and α-Sim4 ChIPs was analyzed using four primer pairs in a multiplex PCR. These primer pairs specifically amplify regions within the central core of cen1 and 3 (cnt), the inner repeats of cen1 (imr), the outer repeat of cen1 (otr), and fbp1+, a control euchromatic gene (fbp). As shown in Fig. 5 A, central core (cnt) sequences and inner repeat (imr) sequences were enriched 6.5- and 6.8-fold, respectively, relative to fbp in Sim4 ChIPs compared with the input control. Outer repeat sequences (otr) were not enriched. These experiments indicate that Sim4 is specifically associated with the central domain of the centromere but is absent from the outer repeat regions.

Bottom Line: Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins.The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region.Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, 6.34 Swann Building, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK. robin.allshire@ed.ac.uk

ABSTRACT
Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.

Show MeSH