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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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Chromosome segregation defects in sim mutants. (A–D) Strains (FY3027, 4484, 4502, and 4536) were shifted to 36°C for 6 h before fixation and immunofluorescence with TAT1 α-tubulin antibody (green) and DAPI staining of DNA (red). Bar, 5 μm. (A) Equal chromosome segregation in wild-type cells. (B) sim1-106, uneven segregation and lagging chromosomes. (C) sim3-143, similar phenotypes and star-shaped spindles (first two cells). (D) sim4-193, uneven segregation and lagging chromosomes. (E) Table of chromosome segregation phenotypes in wild type and the sim4 mutant at restrictive temperature. The top set of numbers is the percentage of cells in that category (early mitosis [prometaphase, metaphase, anaphase A], early anaphase B, late anaphase B) that displayed the phenotype diagrammed. The bottom figures indicate the percentage each category makes to the total of mitotic cells. (F) FISH with a probe to the ribosomal DNA clusters on the ends of chromosome 3 (rDNA [red], DAPI [green]). (G and H) Spindle length in cut9 (G) and cut9sim4 (H) cells incubated at 36°C for 4 h (α-tubulin [green], DAPI [red]).
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fig2: Chromosome segregation defects in sim mutants. (A–D) Strains (FY3027, 4484, 4502, and 4536) were shifted to 36°C for 6 h before fixation and immunofluorescence with TAT1 α-tubulin antibody (green) and DAPI staining of DNA (red). Bar, 5 μm. (A) Equal chromosome segregation in wild-type cells. (B) sim1-106, uneven segregation and lagging chromosomes. (C) sim3-143, similar phenotypes and star-shaped spindles (first two cells). (D) sim4-193, uneven segregation and lagging chromosomes. (E) Table of chromosome segregation phenotypes in wild type and the sim4 mutant at restrictive temperature. The top set of numbers is the percentage of cells in that category (early mitosis [prometaphase, metaphase, anaphase A], early anaphase B, late anaphase B) that displayed the phenotype diagrammed. The bottom figures indicate the percentage each category makes to the total of mitotic cells. (F) FISH with a probe to the ribosomal DNA clusters on the ends of chromosome 3 (rDNA [red], DAPI [green]). (G and H) Spindle length in cut9 (G) and cut9sim4 (H) cells incubated at 36°C for 4 h (α-tubulin [green], DAPI [red]).

Mentions: sim mutants showed enhanced loss rates of a minichromosome (Fig. S1). sim1, sim3, and sim4 showed greater sensitivity to TBZ than wild type, but lower sensitivity than otr mutants such as rik1, clr4, and swi6 (Fig. 1 C; Ekwall et al., 1996). Neither sim2 nor mis6 showed supersensitivity to TBZ. To investigate the chromosome segregation defects, sim mutants were grown at permissive (25°C) or restrictive temperature (36°C for 6 h), fixed, and processed for immunofluorescence with α-tubulin antibody. sim1, 3, and 4 displayed lagging chromosomes on late anaphase spindles (Fig. 2 , B–D) and uneven segregation of chromosomes. Short spindles with hypercondensed chromatin were common. Star or V-shaped spindles were seen frequently in sim3 mutants, which may indicate defects in the organization of a bipolar spindle.


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

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

Chromosome segregation defects in sim mutants. (A–D) Strains (FY3027, 4484, 4502, and 4536) were shifted to 36°C for 6 h before fixation and immunofluorescence with TAT1 α-tubulin antibody (green) and DAPI staining of DNA (red). Bar, 5 μm. (A) Equal chromosome segregation in wild-type cells. (B) sim1-106, uneven segregation and lagging chromosomes. (C) sim3-143, similar phenotypes and star-shaped spindles (first two cells). (D) sim4-193, uneven segregation and lagging chromosomes. (E) Table of chromosome segregation phenotypes in wild type and the sim4 mutant at restrictive temperature. The top set of numbers is the percentage of cells in that category (early mitosis [prometaphase, metaphase, anaphase A], early anaphase B, late anaphase B) that displayed the phenotype diagrammed. The bottom figures indicate the percentage each category makes to the total of mitotic cells. (F) FISH with a probe to the ribosomal DNA clusters on the ends of chromosome 3 (rDNA [red], DAPI [green]). (G and H) Spindle length in cut9 (G) and cut9sim4 (H) cells incubated at 36°C for 4 h (α-tubulin [green], DAPI [red]).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172903&req=5

fig2: Chromosome segregation defects in sim mutants. (A–D) Strains (FY3027, 4484, 4502, and 4536) were shifted to 36°C for 6 h before fixation and immunofluorescence with TAT1 α-tubulin antibody (green) and DAPI staining of DNA (red). Bar, 5 μm. (A) Equal chromosome segregation in wild-type cells. (B) sim1-106, uneven segregation and lagging chromosomes. (C) sim3-143, similar phenotypes and star-shaped spindles (first two cells). (D) sim4-193, uneven segregation and lagging chromosomes. (E) Table of chromosome segregation phenotypes in wild type and the sim4 mutant at restrictive temperature. The top set of numbers is the percentage of cells in that category (early mitosis [prometaphase, metaphase, anaphase A], early anaphase B, late anaphase B) that displayed the phenotype diagrammed. The bottom figures indicate the percentage each category makes to the total of mitotic cells. (F) FISH with a probe to the ribosomal DNA clusters on the ends of chromosome 3 (rDNA [red], DAPI [green]). (G and H) Spindle length in cut9 (G) and cut9sim4 (H) cells incubated at 36°C for 4 h (α-tubulin [green], DAPI [red]).
Mentions: sim mutants showed enhanced loss rates of a minichromosome (Fig. S1). sim1, sim3, and sim4 showed greater sensitivity to TBZ than wild type, but lower sensitivity than otr mutants such as rik1, clr4, and swi6 (Fig. 1 C; Ekwall et al., 1996). Neither sim2 nor mis6 showed supersensitivity to TBZ. To investigate the chromosome segregation defects, sim mutants were grown at permissive (25°C) or restrictive temperature (36°C for 6 h), fixed, and processed for immunofluorescence with α-tubulin antibody. sim1, 3, and 4 displayed lagging chromosomes on late anaphase spindles (Fig. 2 , B–D) and uneven segregation of chromosomes. Short spindles with hypercondensed chromatin were common. Star or V-shaped spindles were seen frequently in sim3 mutants, which may indicate defects in the organization of a bipolar spindle.

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