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Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin.

Pidoux AL, Choi ES, Abbott JK, Liu X, Kagansky A, Castillo AG, Hamilton GL, Richardson W, Rappsilber J, He X, Allshire RC - Mol. Cell (2009)

Bottom Line: Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase.Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin.While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, 6.34 Swann Building, Edinburgh EH93JR, Scotland, UK. alison.pidoux@ed.ac.uk

ABSTRACT
The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

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Localization of Scm3Sp Is Dependent on Mis6, Sim4, Mis16, Mis18, but Not CENP-ACnp1(A) Wild-type, cnp1-1, mis6-302, sim4-193, mis16-53, and mis18-262 strains expressing Scm3-GFP were shifted to 36°C for 6 hr, fixed, and processed for immunofluorescence using anti-GFP (Scm3-GFP; green), anti-Cdc11 antibodies (SPB; red), and DAPI (blue). Identical exposures and processing were performed to ensure that wild-type and mutant images are comparable. Representative images are presented. All cells are in G2. Scale bar 5 μm.(B) ChIP of Scm3-GFP in the indicated strains shifted to 36°C for 6 hr before fixation and analysis by ChIP with anti-Cnp1/CENP-ACnp1 (C) and anti-GFP (G, Scm3-GFP) antibodies. PCR analysis as in Figure 1E. Part of same experiment shown in Figure 3C; wild-type control identical.
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fig5: Localization of Scm3Sp Is Dependent on Mis6, Sim4, Mis16, Mis18, but Not CENP-ACnp1(A) Wild-type, cnp1-1, mis6-302, sim4-193, mis16-53, and mis18-262 strains expressing Scm3-GFP were shifted to 36°C for 6 hr, fixed, and processed for immunofluorescence using anti-GFP (Scm3-GFP; green), anti-Cdc11 antibodies (SPB; red), and DAPI (blue). Identical exposures and processing were performed to ensure that wild-type and mutant images are comparable. Representative images are presented. All cells are in G2. Scale bar 5 μm.(B) ChIP of Scm3-GFP in the indicated strains shifted to 36°C for 6 hr before fixation and analysis by ChIP with anti-Cnp1/CENP-ACnp1 (C) and anti-GFP (G, Scm3-GFP) antibodies. PCR analysis as in Figure 1E. Part of same experiment shown in Figure 3C; wild-type control identical.

Mentions: As Scm3Sp, Mis16, and Mis18 display highly similar temporal localization during the cell cycle, we investigated dependency relationships between these proteins. We examined whether Scm3Sp localization is affected by temperature-sensitive conditional mutations in Mis16 and Mis18 and other kinetochore proteins including CENP-ACnp1, Sim4, and Mis6 (Figure 5A). Reciprocally, we determined if the localization of Mis6-HA, Sim4-GFP, Mis16-GFP, or Mis18-GFP is disrupted in the scm3-139 mutant (Figure 6). Scm3Sp-GFP is lost from centromeres in mis16-53 and mis18-262 mutants and also in sim4-193 and mis6-302 at 36°C. However, in cells with defective CENP-ACnp1 (cnp1-1), which have dramatically reduced levels of CENP-ACnp1 at centromeres, Scm3Sp-GFP remains at centromeres. Cells with defective Mis12, or that lack CENP-CCnp3 (cnp3Δ), or the CENP-ACnp1 escort Sim3 (sim3Δ), also retain Scm3Sp-GFP at centromeres (Figure S9). This indicates that the maintenance of Scm3Sp at centromeres is independent of CENP-ACnp1-containing chromatin but that its localization is dependent on other kinetochore proteins (Sim4 and Mis6) known to affect the maintenance of CENP-ACnp1 at centromeres. This is underscored by the fact that individual cells with strong Scm3Sp-GFP staining but undetectable CENP-ACnp1 are common in cnp1-1 (Figure S6 and data not shown). In agreement with these localization data, ChIP analyses shows that Scm3Sp-GFP levels drop significantly in the central domain in sim4-193, mis6-302, mis16-53, and mis18-262, but not in cnp1-1 (Figure 5B).


Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin.

Pidoux AL, Choi ES, Abbott JK, Liu X, Kagansky A, Castillo AG, Hamilton GL, Richardson W, Rappsilber J, He X, Allshire RC - Mol. Cell (2009)

Localization of Scm3Sp Is Dependent on Mis6, Sim4, Mis16, Mis18, but Not CENP-ACnp1(A) Wild-type, cnp1-1, mis6-302, sim4-193, mis16-53, and mis18-262 strains expressing Scm3-GFP were shifted to 36°C for 6 hr, fixed, and processed for immunofluorescence using anti-GFP (Scm3-GFP; green), anti-Cdc11 antibodies (SPB; red), and DAPI (blue). Identical exposures and processing were performed to ensure that wild-type and mutant images are comparable. Representative images are presented. All cells are in G2. Scale bar 5 μm.(B) ChIP of Scm3-GFP in the indicated strains shifted to 36°C for 6 hr before fixation and analysis by ChIP with anti-Cnp1/CENP-ACnp1 (C) and anti-GFP (G, Scm3-GFP) antibodies. PCR analysis as in Figure 1E. Part of same experiment shown in Figure 3C; wild-type control identical.
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Show All Figures
getmorefigures.php?uid=PMC2697330&req=5

fig5: Localization of Scm3Sp Is Dependent on Mis6, Sim4, Mis16, Mis18, but Not CENP-ACnp1(A) Wild-type, cnp1-1, mis6-302, sim4-193, mis16-53, and mis18-262 strains expressing Scm3-GFP were shifted to 36°C for 6 hr, fixed, and processed for immunofluorescence using anti-GFP (Scm3-GFP; green), anti-Cdc11 antibodies (SPB; red), and DAPI (blue). Identical exposures and processing were performed to ensure that wild-type and mutant images are comparable. Representative images are presented. All cells are in G2. Scale bar 5 μm.(B) ChIP of Scm3-GFP in the indicated strains shifted to 36°C for 6 hr before fixation and analysis by ChIP with anti-Cnp1/CENP-ACnp1 (C) and anti-GFP (G, Scm3-GFP) antibodies. PCR analysis as in Figure 1E. Part of same experiment shown in Figure 3C; wild-type control identical.
Mentions: As Scm3Sp, Mis16, and Mis18 display highly similar temporal localization during the cell cycle, we investigated dependency relationships between these proteins. We examined whether Scm3Sp localization is affected by temperature-sensitive conditional mutations in Mis16 and Mis18 and other kinetochore proteins including CENP-ACnp1, Sim4, and Mis6 (Figure 5A). Reciprocally, we determined if the localization of Mis6-HA, Sim4-GFP, Mis16-GFP, or Mis18-GFP is disrupted in the scm3-139 mutant (Figure 6). Scm3Sp-GFP is lost from centromeres in mis16-53 and mis18-262 mutants and also in sim4-193 and mis6-302 at 36°C. However, in cells with defective CENP-ACnp1 (cnp1-1), which have dramatically reduced levels of CENP-ACnp1 at centromeres, Scm3Sp-GFP remains at centromeres. Cells with defective Mis12, or that lack CENP-CCnp3 (cnp3Δ), or the CENP-ACnp1 escort Sim3 (sim3Δ), also retain Scm3Sp-GFP at centromeres (Figure S9). This indicates that the maintenance of Scm3Sp at centromeres is independent of CENP-ACnp1-containing chromatin but that its localization is dependent on other kinetochore proteins (Sim4 and Mis6) known to affect the maintenance of CENP-ACnp1 at centromeres. This is underscored by the fact that individual cells with strong Scm3Sp-GFP staining but undetectable CENP-ACnp1 are common in cnp1-1 (Figure S6 and data not shown). In agreement with these localization data, ChIP analyses shows that Scm3Sp-GFP levels drop significantly in the central domain in sim4-193, mis6-302, mis16-53, and mis18-262, but not in cnp1-1 (Figure 5B).

Bottom Line: Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase.Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin.While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, 6.34 Swann Building, Edinburgh EH93JR, Scotland, UK. alison.pidoux@ed.ac.uk

ABSTRACT
The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

Show MeSH
Related in: MedlinePlus