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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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ChIP analysis of dependency relationships for kinetochore localization. Strains (FY3027, 5691, 4536, 5903, and 5900) were grown at 25°C or shifted to 36°C for 6 h for ChIP with the indicated antibodies. Left, multiplex PCR analysis of ChIPs. Right, quantification of ChIP PCR data. cnt and imr enrichment is measured relative to the fbp euchromatic control and normalized to the input PCR. For each site/temperature, the mutant value has been normalized to the wild-type value. Data in A and B are from four to six separate ChIP measurements, and data in C are from three separate ChIPs. (A) Sim4 and Cnp1 ChIP in wild type and mis6 mutant at 25°C and 36°C. (B) Mis6–HA and Cnp1 in wild type and sim4 mutant at 25°C and 36°C. (C) Sim4 ChIP in wild type and sim4 mutant at 25°C and 36°C. The PCR of wild-type input at 36°C and, to a lesser extent, at 25°C shows bias of extracted chromatin (little cnt and imr chromatin). The ChIP PCR is compared with this input, and therefore, there is a relative enrichment of cnt and imr sequences.
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fig7: ChIP analysis of dependency relationships for kinetochore localization. Strains (FY3027, 5691, 4536, 5903, and 5900) were grown at 25°C or shifted to 36°C for 6 h for ChIP with the indicated antibodies. Left, multiplex PCR analysis of ChIPs. Right, quantification of ChIP PCR data. cnt and imr enrichment is measured relative to the fbp euchromatic control and normalized to the input PCR. For each site/temperature, the mutant value has been normalized to the wild-type value. Data in A and B are from four to six separate ChIP measurements, and data in C are from three separate ChIPs. (A) Sim4 and Cnp1 ChIP in wild type and mis6 mutant at 25°C and 36°C. (B) Mis6–HA and Cnp1 in wild type and sim4 mutant at 25°C and 36°C. (C) Sim4 ChIP in wild type and sim4 mutant at 25°C and 36°C. The PCR of wild-type input at 36°C and, to a lesser extent, at 25°C shows bias of extracted chromatin (little cnt and imr chromatin). The ChIP PCR is compared with this input, and therefore, there is a relative enrichment of cnt and imr sequences.

Mentions: Because Mis6 and Sim4 exist in a kinetochore subcomplex, we investigated whether they are required for each other's localization at the centromere. Centromere localization of Sim4 was greatly reduced at the permissive temperature (25°C) and abolished at the restrictive temperature (36°C) in a mis6 mutant (Fig. 6 B). To quantify this effect, ChIP experiments were performed (Fig. 7 A). At 36°C, there was a large reduction in Sim4 associated with cnt and imr sequences in the mis6 mutant compared with wild type (approximately fivefold reduction, within the limits of the multiplex PCR quantification method). Western blotting confirmed that there were similar amounts of Sim4 protein in wild-type and mis6 cells (unpublished data). As Mis6 has been proposed to function as a loading factor for Cnp1 in fission yeast, we also performed Cnp1 ChIP, using antiserum raised to a Cnp1 NH2-terminal peptide (amino acids 1–19). A decrease in the amount of endogenous Cnp1 associated with cnt and imr was observed in the mis6 mutant incubated at 36°C (Fig. 7 A), consistent with results obtained with HA-tagged Cnp1 (Takahashi et al., 2000).


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

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

ChIP analysis of dependency relationships for kinetochore localization. Strains (FY3027, 5691, 4536, 5903, and 5900) were grown at 25°C or shifted to 36°C for 6 h for ChIP with the indicated antibodies. Left, multiplex PCR analysis of ChIPs. Right, quantification of ChIP PCR data. cnt and imr enrichment is measured relative to the fbp euchromatic control and normalized to the input PCR. For each site/temperature, the mutant value has been normalized to the wild-type value. Data in A and B are from four to six separate ChIP measurements, and data in C are from three separate ChIPs. (A) Sim4 and Cnp1 ChIP in wild type and mis6 mutant at 25°C and 36°C. (B) Mis6–HA and Cnp1 in wild type and sim4 mutant at 25°C and 36°C. (C) Sim4 ChIP in wild type and sim4 mutant at 25°C and 36°C. The PCR of wild-type input at 36°C and, to a lesser extent, at 25°C shows bias of extracted chromatin (little cnt and imr chromatin). The ChIP PCR is compared with this input, and therefore, there is a relative enrichment of cnt and imr sequences.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172903&req=5

fig7: ChIP analysis of dependency relationships for kinetochore localization. Strains (FY3027, 5691, 4536, 5903, and 5900) were grown at 25°C or shifted to 36°C for 6 h for ChIP with the indicated antibodies. Left, multiplex PCR analysis of ChIPs. Right, quantification of ChIP PCR data. cnt and imr enrichment is measured relative to the fbp euchromatic control and normalized to the input PCR. For each site/temperature, the mutant value has been normalized to the wild-type value. Data in A and B are from four to six separate ChIP measurements, and data in C are from three separate ChIPs. (A) Sim4 and Cnp1 ChIP in wild type and mis6 mutant at 25°C and 36°C. (B) Mis6–HA and Cnp1 in wild type and sim4 mutant at 25°C and 36°C. (C) Sim4 ChIP in wild type and sim4 mutant at 25°C and 36°C. The PCR of wild-type input at 36°C and, to a lesser extent, at 25°C shows bias of extracted chromatin (little cnt and imr chromatin). The ChIP PCR is compared with this input, and therefore, there is a relative enrichment of cnt and imr sequences.
Mentions: Because Mis6 and Sim4 exist in a kinetochore subcomplex, we investigated whether they are required for each other's localization at the centromere. Centromere localization of Sim4 was greatly reduced at the permissive temperature (25°C) and abolished at the restrictive temperature (36°C) in a mis6 mutant (Fig. 6 B). To quantify this effect, ChIP experiments were performed (Fig. 7 A). At 36°C, there was a large reduction in Sim4 associated with cnt and imr sequences in the mis6 mutant compared with wild type (approximately fivefold reduction, within the limits of the multiplex PCR quantification method). Western blotting confirmed that there were similar amounts of Sim4 protein in wild-type and mis6 cells (unpublished data). As Mis6 has been proposed to function as a loading factor for Cnp1 in fission yeast, we also performed Cnp1 ChIP, using antiserum raised to a Cnp1 NH2-terminal peptide (amino acids 1–19). A decrease in the amount of endogenous Cnp1 associated with cnt and imr was observed in the mis6 mutant incubated at 36°C (Fig. 7 A), consistent with results obtained with HA-tagged Cnp1 (Takahashi et al., 2000).

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