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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–Mis6 complex and kinetochore dependency relationships. (A) Extracts were prepared from cells expressing Mis6–HA and Sim4–GFP (FY5237). IPs were performed with the indicated antibodies, or beads only as a negative control. IPs were analyzed on Western blots with either α-HA or α-Sim4 antibodies. The positions of Mis6–HA (M), Sim4–GFP (S), IgG (asterisk), and standards are shown. (B–E) Strains (FY3027, 5691, 4536, 5903, and 5900) were grown at 25°C or shifted to 36°C for 6 h before fixation and processing for immunolocalization with the antibodies (green) indicated at right, and DAPI staining (red). Bar, 5 μm. (B) Sim4 localization in wild type and mis6 mutant. (C) Cnp1 localization in wild type and sim4 mutant. (D) Mis6–HA localization in wild type and sim4 mutant. (E) Sim4 localization in wild type and sim4 mutant.
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fig6: Sim4–Mis6 complex and kinetochore dependency relationships. (A) Extracts were prepared from cells expressing Mis6–HA and Sim4–GFP (FY5237). IPs were performed with the indicated antibodies, or beads only as a negative control. IPs were analyzed on Western blots with either α-HA or α-Sim4 antibodies. The positions of Mis6–HA (M), Sim4–GFP (S), IgG (asterisk), and standards are shown. (B–E) Strains (FY3027, 5691, 4536, 5903, and 5900) were grown at 25°C or shifted to 36°C for 6 h before fixation and processing for immunolocalization with the antibodies (green) indicated at right, and DAPI staining (red). Bar, 5 μm. (B) Sim4 localization in wild type and mis6 mutant. (C) Cnp1 localization in wild type and sim4 mutant. (D) Mis6–HA localization in wild type and sim4 mutant. (E) Sim4 localization in wild type and sim4 mutant.

Mentions: The fission yeast kinetochore is likely to be a massive multiprotein complex made up of smaller subcomplexes. To investigate protein–protein interactions at the fission yeast kinetochore, we asked whether Sim4 coimmunoprecipitates with other kinetochore components. Immunoprecipitation with α-HA, α-GFP, or α-Sim4 antibodies was performed on extracts from a strain containing Mis6–HA and Sim4–GFP. Immunoprecipitates (IPs) were analyzed by Western blotting with complementary antibodies. Mis6–HA and Sim4–GFP clearly coimmunoprecipitated (Fig. 6 A). This complex appeared to be very tightly associated, as washing IPs with high concentrations of salt, urea, or nonionic detergents failed to disrupt it (unpublished data). This complex is specific, as Sim4 does not coimmunoprecipitate with other kinetochore proteins, such as Mis12 (unpublished data; Goshima et al., 1999).


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–Mis6 complex and kinetochore dependency relationships. (A) Extracts were prepared from cells expressing Mis6–HA and Sim4–GFP (FY5237). IPs were performed with the indicated antibodies, or beads only as a negative control. IPs were analyzed on Western blots with either α-HA or α-Sim4 antibodies. The positions of Mis6–HA (M), Sim4–GFP (S), IgG (asterisk), and standards are shown. (B–E) Strains (FY3027, 5691, 4536, 5903, and 5900) were grown at 25°C or shifted to 36°C for 6 h before fixation and processing for immunolocalization with the antibodies (green) indicated at right, and DAPI staining (red). Bar, 5 μm. (B) Sim4 localization in wild type and mis6 mutant. (C) Cnp1 localization in wild type and sim4 mutant. (D) Mis6–HA localization in wild type and sim4 mutant. (E) Sim4 localization in wild type and sim4 mutant.
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fig6: Sim4–Mis6 complex and kinetochore dependency relationships. (A) Extracts were prepared from cells expressing Mis6–HA and Sim4–GFP (FY5237). IPs were performed with the indicated antibodies, or beads only as a negative control. IPs were analyzed on Western blots with either α-HA or α-Sim4 antibodies. The positions of Mis6–HA (M), Sim4–GFP (S), IgG (asterisk), and standards are shown. (B–E) Strains (FY3027, 5691, 4536, 5903, and 5900) were grown at 25°C or shifted to 36°C for 6 h before fixation and processing for immunolocalization with the antibodies (green) indicated at right, and DAPI staining (red). Bar, 5 μm. (B) Sim4 localization in wild type and mis6 mutant. (C) Cnp1 localization in wild type and sim4 mutant. (D) Mis6–HA localization in wild type and sim4 mutant. (E) Sim4 localization in wild type and sim4 mutant.
Mentions: The fission yeast kinetochore is likely to be a massive multiprotein complex made up of smaller subcomplexes. To investigate protein–protein interactions at the fission yeast kinetochore, we asked whether Sim4 coimmunoprecipitates with other kinetochore components. Immunoprecipitation with α-HA, α-GFP, or α-Sim4 antibodies was performed on extracts from a strain containing Mis6–HA and Sim4–GFP. Immunoprecipitates (IPs) were analyzed by Western blotting with complementary antibodies. Mis6–HA and Sim4–GFP clearly coimmunoprecipitated (Fig. 6 A). This complex appeared to be very tightly associated, as washing IPs with high concentrations of salt, urea, or nonionic detergents failed to disrupt it (unpublished data). This complex is specific, as Sim4 does not coimmunoprecipitate with other kinetochore proteins, such as Mis12 (unpublished data; Goshima et al., 1999).

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