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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 colocalizes with centromeres. (A) Cells expressing Sim4–GFP (FY5077), showing from left to right: interphase (the three centromeres are clustered); early mitosis (prometaphase or metaphase or anaphase A), four centromeric spots are visible; early, mid, and late anaphase B (centromeres are at the spindle poles). (B) Colocalization of Sim4–GFP (green) with Mis6–HA (red) and merged image (right) in interphase (top row) and early mitosis (bottom row). Strain FY5237. (C) Colocalization of Sim4 (α-Sim4, green) with Mis6–HA (red), the merged image is shown on the right. Strain FY2929. Bar, 5 μm.
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fig4: Sim4 colocalizes with centromeres. (A) Cells expressing Sim4–GFP (FY5077), showing from left to right: interphase (the three centromeres are clustered); early mitosis (prometaphase or metaphase or anaphase A), four centromeric spots are visible; early, mid, and late anaphase B (centromeres are at the spindle poles). (B) Colocalization of Sim4–GFP (green) with Mis6–HA (red) and merged image (right) in interphase (top row) and early mitosis (bottom row). Strain FY5237. (C) Colocalization of Sim4 (α-Sim4, green) with Mis6–HA (red), the merged image is shown on the right. Strain FY2929. Bar, 5 μm.

Mentions: The sim4+ ORF was COOH-terminally tagged at its genomic locus with GFP (Bahler et al., 1998). A single GFP spot was seen in the nucleus of living cells in interphase, reminiscent of clustered centromeres (Fig. 4 A). Several spots were seen in early mitotic cells, likely to be individual (or replicated) centromeres (Fig. 4, A and B); in anaphase B cells, spots were observed at the leading edges of the daughter nuclei (Fig. 4 A). Cells expressing both Sim4–GFP and Mis6–HA (Saitoh et al., 1997) were fixed and stained with α-GFP and α-HA antibodies, revealing colocalization of the two epitopes (Fig. 4 B). Colocalization of endogenous Sim4 and Mis6–HA was also observed (Fig. 4 C). Thus, the Sim4 localization pattern and colocalization with the bona fide kinetochore protein Mis6 indicate that Sim4 is a novel centromere-associated protein.


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 colocalizes with centromeres. (A) Cells expressing Sim4–GFP (FY5077), showing from left to right: interphase (the three centromeres are clustered); early mitosis (prometaphase or metaphase or anaphase A), four centromeric spots are visible; early, mid, and late anaphase B (centromeres are at the spindle poles). (B) Colocalization of Sim4–GFP (green) with Mis6–HA (red) and merged image (right) in interphase (top row) and early mitosis (bottom row). Strain FY5237. (C) Colocalization of Sim4 (α-Sim4, green) with Mis6–HA (red), the merged image is shown on the right. Strain FY2929. Bar, 5 μm.
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Related In: Results  -  Collection

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fig4: Sim4 colocalizes with centromeres. (A) Cells expressing Sim4–GFP (FY5077), showing from left to right: interphase (the three centromeres are clustered); early mitosis (prometaphase or metaphase or anaphase A), four centromeric spots are visible; early, mid, and late anaphase B (centromeres are at the spindle poles). (B) Colocalization of Sim4–GFP (green) with Mis6–HA (red) and merged image (right) in interphase (top row) and early mitosis (bottom row). Strain FY5237. (C) Colocalization of Sim4 (α-Sim4, green) with Mis6–HA (red), the merged image is shown on the right. Strain FY2929. Bar, 5 μm.
Mentions: The sim4+ ORF was COOH-terminally tagged at its genomic locus with GFP (Bahler et al., 1998). A single GFP spot was seen in the nucleus of living cells in interphase, reminiscent of clustered centromeres (Fig. 4 A). Several spots were seen in early mitotic cells, likely to be individual (or replicated) centromeres (Fig. 4, A and B); in anaphase B cells, spots were observed at the leading edges of the daughter nuclei (Fig. 4 A). Cells expressing both Sim4–GFP and Mis6–HA (Saitoh et al., 1997) were fixed and stained with α-GFP and α-HA antibodies, revealing colocalization of the two epitopes (Fig. 4 B). Colocalization of endogenous Sim4 and Mis6–HA was also observed (Fig. 4 C). Thus, the Sim4 localization pattern and colocalization with the bona fide kinetochore protein Mis6 indicate that Sim4 is a novel centromere-associated protein.

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
Related in: MedlinePlus