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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 Does Not Extract with CENP-ACnp1 from ChromatinExtraction of CENP-ACnp1 from chromatin by MNase digestion. Nuclei were prepared from cells in which endogenous scm3+ and cnp1+ genes were TAP-tagged. After progressive MNase digestion as labeled, total nuclei digestion mixtures (T) were separated into soluble supernatant (S) and nuclei pellets (P) by centrifugation. Samples were subject to DNA extraction and electrophoresis (upper panel) or protein extraction and western blotting (lower panel). Bands labeled with an asterisk are nonspecific proteins detected by the antibody. Figure S8 shows that bulk histones (H4) are also released into supernatant.
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fig4: Scm3Sp Does Not Extract with CENP-ACnp1 from ChromatinExtraction of CENP-ACnp1 from chromatin by MNase digestion. Nuclei were prepared from cells in which endogenous scm3+ and cnp1+ genes were TAP-tagged. After progressive MNase digestion as labeled, total nuclei digestion mixtures (T) were separated into soluble supernatant (S) and nuclei pellets (P) by centrifugation. Samples were subject to DNA extraction and electrophoresis (upper panel) or protein extraction and western blotting (lower panel). Bands labeled with an asterisk are nonspecific proteins detected by the antibody. Figure S8 shows that bulk histones (H4) are also released into supernatant.

Mentions: To investigate further whether unusual nucleosomes such as those proposed for S. cerevisiae might exist at fission yeast centromeres, we performed a MNase digestion time course on nuclei and, following centrifugation, assessed the relative proportions of Scm3Sp-TAP and CENP-ACnp1-TAP released from the insoluble chromatin pellet into the soluble fraction. Most CENP-ACnp1-TAP and bulk histones are released into the pellet after 2–10 min of MNase treatment, whereas Scm3Sp-TAP remains tightly associated with the pellet even after 20 min digestion (Figures 4 and S8). This demonstrates that Scm3Sp and CENP-ACnp1 do not extract from chromatin under the same conditions; Scm3Sp-TAP remains insoluble, whereas CENP-ACnp1 is released into the supernatant along with oligomeric nucleosome particles and other histones. Taken together, our analyses suggest that Scm3Sp might act to assemble CENP-ACnp1 into subkinetochore chromatin at these regional centromeres, but it is unlikely to be an integral component of CENP-ACnp1 nucleosomes themselves in fission yeast.


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 Does Not Extract with CENP-ACnp1 from ChromatinExtraction of CENP-ACnp1 from chromatin by MNase digestion. Nuclei were prepared from cells in which endogenous scm3+ and cnp1+ genes were TAP-tagged. After progressive MNase digestion as labeled, total nuclei digestion mixtures (T) were separated into soluble supernatant (S) and nuclei pellets (P) by centrifugation. Samples were subject to DNA extraction and electrophoresis (upper panel) or protein extraction and western blotting (lower panel). Bands labeled with an asterisk are nonspecific proteins detected by the antibody. Figure S8 shows that bulk histones (H4) are also released into supernatant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697330&req=5

fig4: Scm3Sp Does Not Extract with CENP-ACnp1 from ChromatinExtraction of CENP-ACnp1 from chromatin by MNase digestion. Nuclei were prepared from cells in which endogenous scm3+ and cnp1+ genes were TAP-tagged. After progressive MNase digestion as labeled, total nuclei digestion mixtures (T) were separated into soluble supernatant (S) and nuclei pellets (P) by centrifugation. Samples were subject to DNA extraction and electrophoresis (upper panel) or protein extraction and western blotting (lower panel). Bands labeled with an asterisk are nonspecific proteins detected by the antibody. Figure S8 shows that bulk histones (H4) are also released into supernatant.
Mentions: To investigate further whether unusual nucleosomes such as those proposed for S. cerevisiae might exist at fission yeast centromeres, we performed a MNase digestion time course on nuclei and, following centrifugation, assessed the relative proportions of Scm3Sp-TAP and CENP-ACnp1-TAP released from the insoluble chromatin pellet into the soluble fraction. Most CENP-ACnp1-TAP and bulk histones are released into the pellet after 2–10 min of MNase treatment, whereas Scm3Sp-TAP remains tightly associated with the pellet even after 20 min digestion (Figures 4 and S8). This demonstrates that Scm3Sp and CENP-ACnp1 do not extract from chromatin under the same conditions; Scm3Sp-TAP remains insoluble, whereas CENP-ACnp1 is released into the supernatant along with oligomeric nucleosome particles and other histones. Taken together, our analyses suggest that Scm3Sp might act to assemble CENP-ACnp1 into subkinetochore chromatin at these regional centromeres, but it is unlikely to be an integral component of CENP-ACnp1 nucleosomes themselves in fission yeast.

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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