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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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Central core chromatin structure is disrupted in a sim4 mutant. MNase digestion of chromatin from cells (FY3027 and 4536) grown at 25°C or shifted to 36°C for 6 h. Top, ethidium bromide–stained gel with ladder indicative of partial MNase digestion. Bottom, Southern blot hybridized with cnt1 probe, showing that the smear pattern in wild type is replaced by ladder-like pattern in the sim4 mutant.
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fig3: Central core chromatin structure is disrupted in a sim4 mutant. MNase digestion of chromatin from cells (FY3027 and 4536) grown at 25°C or shifted to 36°C for 6 h. Top, ethidium bromide–stained gel with ladder indicative of partial MNase digestion. Bottom, Southern blot hybridized with cnt1 probe, showing that the smear pattern in wild type is replaced by ladder-like pattern in the sim4 mutant.

Mentions: Chromatin was analyzed by limited MNase digestion and hybridization with a cnt1 probe. As shown in Fig. 3 , the unique central core smear pattern seen in wild type (Polizzi and Clarke, 1991; Takahashi et al., 1992) was lost and replaced by a ladder in the sim4 mutant at permissive and restrictive temperatures. Similar observations have been made in other strains that have defects in central core proteins (Saitoh et al., 1997; Goshima et al., 1999; Takahashi et al., 2000; Jin et al., 2002).


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

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

Central core chromatin structure is disrupted in a sim4 mutant. MNase digestion of chromatin from cells (FY3027 and 4536) grown at 25°C or shifted to 36°C for 6 h. Top, ethidium bromide–stained gel with ladder indicative of partial MNase digestion. Bottom, Southern blot hybridized with cnt1 probe, showing that the smear pattern in wild type is replaced by ladder-like pattern in the sim4 mutant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172903&req=5

fig3: Central core chromatin structure is disrupted in a sim4 mutant. MNase digestion of chromatin from cells (FY3027 and 4536) grown at 25°C or shifted to 36°C for 6 h. Top, ethidium bromide–stained gel with ladder indicative of partial MNase digestion. Bottom, Southern blot hybridized with cnt1 probe, showing that the smear pattern in wild type is replaced by ladder-like pattern in the sim4 mutant.
Mentions: Chromatin was analyzed by limited MNase digestion and hybridization with a cnt1 probe. As shown in Fig. 3 , the unique central core smear pattern seen in wild type (Polizzi and Clarke, 1991; Takahashi et al., 1992) was lost and replaced by a ladder in the sim4 mutant at permissive and restrictive temperatures. Similar observations have been made in other strains that have defects in central core proteins (Saitoh et al., 1997; Goshima et al., 1999; Takahashi et al., 2000; Jin et al., 2002).

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