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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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A genetic screen to identify mutants that alleviate silencing in the centromere central core. (A) S. pombe centromere 1. (Top) Central core (cnt1 and inner part of imr1L and imr1R) surrounded by outer repeat regions (otr). Vertical lines indicate the position of tRNA genes at the transition point between the two domains (Partridge et al., 2000). (Bottom) Structure of the arg3+ insertion at the central core. Restriction sites: N, NcoI; C, ClaI; E, EcoRI. (B) Diagram of strain FY3027 used to isolate mutants defective in central core silencing, showing insertion sites of marker genes used to assay silencing. (C) Serial dilutions of S. pombe strains to assay silencing at various loci. The first spot contains 5 × 103 cells followed by fivefold dilutions. Plates were incubated at 25°C for 3–7 d. Assessment of growth on YES at 36°C and YES containing 0, 10, or 15 mg/ml TBZ at 25°C. (D) RT-PCR of ura4 transcripts from random integrant (Rint), cnt1, and otr1 insertion sites, compared with a ura4 minigene control (ura4-DSE) at the endogenous locus. Strains analyzed were FY4835, 4837, 4841, 5711, 5717, 5674, 5695, 5719, 5683, 5714, 5720, and 5688. (E) Quantification of RT-PCR shown in D. The levels of transcripts normalized to ura4-DSE are expressed relative to the wild type at 25°C for each ura4+ insertion site, Rint, cnt1, and otr1.
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fig1: A genetic screen to identify mutants that alleviate silencing in the centromere central core. (A) S. pombe centromere 1. (Top) Central core (cnt1 and inner part of imr1L and imr1R) surrounded by outer repeat regions (otr). Vertical lines indicate the position of tRNA genes at the transition point between the two domains (Partridge et al., 2000). (Bottom) Structure of the arg3+ insertion at the central core. Restriction sites: N, NcoI; C, ClaI; E, EcoRI. (B) Diagram of strain FY3027 used to isolate mutants defective in central core silencing, showing insertion sites of marker genes used to assay silencing. (C) Serial dilutions of S. pombe strains to assay silencing at various loci. The first spot contains 5 × 103 cells followed by fivefold dilutions. Plates were incubated at 25°C for 3–7 d. Assessment of growth on YES at 36°C and YES containing 0, 10, or 15 mg/ml TBZ at 25°C. (D) RT-PCR of ura4 transcripts from random integrant (Rint), cnt1, and otr1 insertion sites, compared with a ura4 minigene control (ura4-DSE) at the endogenous locus. Strains analyzed were FY4835, 4837, 4841, 5711, 5717, 5674, 5695, 5719, 5683, 5714, 5720, and 5688. (E) Quantification of RT-PCR shown in D. The levels of transcripts normalized to ura4-DSE are expressed relative to the wild type at 25°C for each ura4+ insertion site, Rint, cnt1, and otr1.

Mentions: Understanding these kinetochore properties requires the identification of its protein components. The kinetochore consists of a very large complex of many proteins even in simple organisms (He et al., 2001; Cheeseman et al., 2002). The fission yeast, Schizosaccharomyces pombe, provides an excellent system for the investigation of centromere–kinetochore function because it combines genetic tractability with structurally complex centromeres (Pidoux and Allshire, 2000b). The three fission yeast centromeres share the same structural organization (Fig. 1 A; Hahnenberger et al., 1991; Takahashi et al., 1992; Pidoux and Allshire, 2000b). At each centromere, a central core region (cnt) is surrounded by inverted repeat elements (innermost repeats; imr), which are specific to each centromere. These are flanked by the outer repeat elements (otr), the organization of which differs between the three centromeres. The central cores of cen1 and cen3 share a 4-kb element, cnt1/cnt3, which is partially conserved in cen2 (Wood 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)

A genetic screen to identify mutants that alleviate silencing in the centromere central core. (A) S. pombe centromere 1. (Top) Central core (cnt1 and inner part of imr1L and imr1R) surrounded by outer repeat regions (otr). Vertical lines indicate the position of tRNA genes at the transition point between the two domains (Partridge et al., 2000). (Bottom) Structure of the arg3+ insertion at the central core. Restriction sites: N, NcoI; C, ClaI; E, EcoRI. (B) Diagram of strain FY3027 used to isolate mutants defective in central core silencing, showing insertion sites of marker genes used to assay silencing. (C) Serial dilutions of S. pombe strains to assay silencing at various loci. The first spot contains 5 × 103 cells followed by fivefold dilutions. Plates were incubated at 25°C for 3–7 d. Assessment of growth on YES at 36°C and YES containing 0, 10, or 15 mg/ml TBZ at 25°C. (D) RT-PCR of ura4 transcripts from random integrant (Rint), cnt1, and otr1 insertion sites, compared with a ura4 minigene control (ura4-DSE) at the endogenous locus. Strains analyzed were FY4835, 4837, 4841, 5711, 5717, 5674, 5695, 5719, 5683, 5714, 5720, and 5688. (E) Quantification of RT-PCR shown in D. The levels of transcripts normalized to ura4-DSE are expressed relative to the wild type at 25°C for each ura4+ insertion site, Rint, cnt1, and otr1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172903&req=5

fig1: A genetic screen to identify mutants that alleviate silencing in the centromere central core. (A) S. pombe centromere 1. (Top) Central core (cnt1 and inner part of imr1L and imr1R) surrounded by outer repeat regions (otr). Vertical lines indicate the position of tRNA genes at the transition point between the two domains (Partridge et al., 2000). (Bottom) Structure of the arg3+ insertion at the central core. Restriction sites: N, NcoI; C, ClaI; E, EcoRI. (B) Diagram of strain FY3027 used to isolate mutants defective in central core silencing, showing insertion sites of marker genes used to assay silencing. (C) Serial dilutions of S. pombe strains to assay silencing at various loci. The first spot contains 5 × 103 cells followed by fivefold dilutions. Plates were incubated at 25°C for 3–7 d. Assessment of growth on YES at 36°C and YES containing 0, 10, or 15 mg/ml TBZ at 25°C. (D) RT-PCR of ura4 transcripts from random integrant (Rint), cnt1, and otr1 insertion sites, compared with a ura4 minigene control (ura4-DSE) at the endogenous locus. Strains analyzed were FY4835, 4837, 4841, 5711, 5717, 5674, 5695, 5719, 5683, 5714, 5720, and 5688. (E) Quantification of RT-PCR shown in D. The levels of transcripts normalized to ura4-DSE are expressed relative to the wild type at 25°C for each ura4+ insertion site, Rint, cnt1, and otr1.
Mentions: Understanding these kinetochore properties requires the identification of its protein components. The kinetochore consists of a very large complex of many proteins even in simple organisms (He et al., 2001; Cheeseman et al., 2002). The fission yeast, Schizosaccharomyces pombe, provides an excellent system for the investigation of centromere–kinetochore function because it combines genetic tractability with structurally complex centromeres (Pidoux and Allshire, 2000b). The three fission yeast centromeres share the same structural organization (Fig. 1 A; Hahnenberger et al., 1991; Takahashi et al., 1992; Pidoux and Allshire, 2000b). At each centromere, a central core region (cnt) is surrounded by inverted repeat elements (innermost repeats; imr), which are specific to each centromere. These are flanked by the outer repeat elements (otr), the organization of which differs between the three centromeres. The central cores of cen1 and cen3 share a 4-kb element, cnt1/cnt3, which is partially conserved in cen2 (Wood 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
Related in: MedlinePlus