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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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Scm3Sp Is a Kinetochore Protein that Is Recruited in Late Anaphase(A) Immunofluorescence of cells expressing Scm3-GFP stained with antibodies to GFP (green) and Cdc11 (SPB; red) and DAPI (blue). Based on morphology and SPB separation, cells were assigned to cell cycle stages: G2; PM, premetaphase (prophase and prometaphase); M, metaphase; A, anaphase; T, telophase; G1; S phase. Scale bar, 5 μm.(B) Immunofluorescence of cells expressing Scm3-GFP and Mis16-myc stained with antibodies to GFP (green) and myc (red) and DAPI. Cells shown are in mid to late anaphase.(C) ChIP of Scm3-GFP using anti-GFP antibodies. Multiplex PCR indicates that Scm3-GFP is associated with central core domain (cnt) but not a euchromatic control locus (fbp1) or centromeric outer repeats (otr).
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fig2: Scm3Sp Is a Kinetochore Protein that Is Recruited in Late Anaphase(A) Immunofluorescence of cells expressing Scm3-GFP stained with antibodies to GFP (green) and Cdc11 (SPB; red) and DAPI (blue). Based on morphology and SPB separation, cells were assigned to cell cycle stages: G2; PM, premetaphase (prophase and prometaphase); M, metaphase; A, anaphase; T, telophase; G1; S phase. Scale bar, 5 μm.(B) Immunofluorescence of cells expressing Scm3-GFP and Mis16-myc stained with antibodies to GFP (green) and myc (red) and DAPI. Cells shown are in mid to late anaphase.(C) ChIP of Scm3-GFP using anti-GFP antibodies. Multiplex PCR indicates that Scm3-GFP is associated with central core domain (cnt) but not a euchromatic control locus (fbp1) or centromeric outer repeats (otr).

Mentions: To examine the localization of Scm3Sp, the endogenous gene was fused with GFP. Our initial analyses indicated that Scm3Sp-GFP colocalizes with CENP-ACnp1 during interphase, but in contrast to CENP-ACnp1 it appears to dissociate from centromeres at the onset of mitosis and to reassociate in telophase (Figure S4). Single kinetochore signals are clearly seen for CENP-ACnp1 during mitosis, whereas no signal is observed for Scm3Sp. Similar localization patterns were seen upon costaining with anti-Scm3Sp antibodies (Figure S4). To pinpoint more precisely the timing of Scm3Sp departure/arrival at centromeres, wild-type cells expressing Scm3Sp-GFP were stained with anti-Cdc11 antibodies to decorate SPBs (Krapp et al., 2001) and anti-GFP to detect Scm3Sp. Pole-to-pole separation allows the stages of mitosis to be determined; Scm3-GFP is at centromeres in G2 but dissociates just after spindle formation (SPB separation) and reassociates following sister-chromatid segregation to the poles, in mid-late anaphase B (Figure 2A). This temporal pattern of localization is reminiscent of that described previously for Mis16 and Mis18 (Fujita et al., 2007; Hayashi et al., 2004). Examination of cells expressing Scm3Sp-GFP and Mis16-myc revealed that they colocalize at centromeres and that their relative timing of reassociation in mitosis is similar (Figure 2B). This and previous analyses (Fujita et al., 2007) suggest that dissociation of Scm3Sp, Mis16, and Mis18 from centromeres is coincident with spindle formation in early mitosis and that they reassemble at centromeres in mid-anaphase B after chromosome disjunction. Thus, Scm3Sp, Mis16, and Mis18 may function together. This dynamic behavior contrasts with that of CENP-ACnp1, which remains at centromeres throughout mitosis (Figure S4) (Takahashi et al., 2000; Takayama et al., 2008).


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)

Scm3Sp Is a Kinetochore Protein that Is Recruited in Late Anaphase(A) Immunofluorescence of cells expressing Scm3-GFP stained with antibodies to GFP (green) and Cdc11 (SPB; red) and DAPI (blue). Based on morphology and SPB separation, cells were assigned to cell cycle stages: G2; PM, premetaphase (prophase and prometaphase); M, metaphase; A, anaphase; T, telophase; G1; S phase. Scale bar, 5 μm.(B) Immunofluorescence of cells expressing Scm3-GFP and Mis16-myc stained with antibodies to GFP (green) and myc (red) and DAPI. Cells shown are in mid to late anaphase.(C) ChIP of Scm3-GFP using anti-GFP antibodies. Multiplex PCR indicates that Scm3-GFP is associated with central core domain (cnt) but not a euchromatic control locus (fbp1) or centromeric outer repeats (otr).
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fig2: Scm3Sp Is a Kinetochore Protein that Is Recruited in Late Anaphase(A) Immunofluorescence of cells expressing Scm3-GFP stained with antibodies to GFP (green) and Cdc11 (SPB; red) and DAPI (blue). Based on morphology and SPB separation, cells were assigned to cell cycle stages: G2; PM, premetaphase (prophase and prometaphase); M, metaphase; A, anaphase; T, telophase; G1; S phase. Scale bar, 5 μm.(B) Immunofluorescence of cells expressing Scm3-GFP and Mis16-myc stained with antibodies to GFP (green) and myc (red) and DAPI. Cells shown are in mid to late anaphase.(C) ChIP of Scm3-GFP using anti-GFP antibodies. Multiplex PCR indicates that Scm3-GFP is associated with central core domain (cnt) but not a euchromatic control locus (fbp1) or centromeric outer repeats (otr).
Mentions: To examine the localization of Scm3Sp, the endogenous gene was fused with GFP. Our initial analyses indicated that Scm3Sp-GFP colocalizes with CENP-ACnp1 during interphase, but in contrast to CENP-ACnp1 it appears to dissociate from centromeres at the onset of mitosis and to reassociate in telophase (Figure S4). Single kinetochore signals are clearly seen for CENP-ACnp1 during mitosis, whereas no signal is observed for Scm3Sp. Similar localization patterns were seen upon costaining with anti-Scm3Sp antibodies (Figure S4). To pinpoint more precisely the timing of Scm3Sp departure/arrival at centromeres, wild-type cells expressing Scm3Sp-GFP were stained with anti-Cdc11 antibodies to decorate SPBs (Krapp et al., 2001) and anti-GFP to detect Scm3Sp. Pole-to-pole separation allows the stages of mitosis to be determined; Scm3-GFP is at centromeres in G2 but dissociates just after spindle formation (SPB separation) and reassociates following sister-chromatid segregation to the poles, in mid-late anaphase B (Figure 2A). This temporal pattern of localization is reminiscent of that described previously for Mis16 and Mis18 (Fujita et al., 2007; Hayashi et al., 2004). Examination of cells expressing Scm3Sp-GFP and Mis16-myc revealed that they colocalize at centromeres and that their relative timing of reassociation in mitosis is similar (Figure 2B). This and previous analyses (Fujita et al., 2007) suggest that dissociation of Scm3Sp, Mis16, and Mis18 from centromeres is coincident with spindle formation in early mitosis and that they reassemble at centromeres in mid-anaphase B after chromosome disjunction. Thus, Scm3Sp, Mis16, and Mis18 may function together. This dynamic behavior contrasts with that of CENP-ACnp1, which remains at centromeres throughout mitosis (Figure S4) (Takahashi et al., 2000; Takayama et al., 2008).

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