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Dissection of the essential steps for condensin accumulation at kinetochores and rDNAs during fission yeast mitosis.

Nakazawa N, Nakamura T, Kokubu A, Ebe M, Nagao K, Yanagida M - J. Cell Biol. (2008)

Bottom Line: Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites.Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs.The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells.

View Article: PubMed Central - PubMed

Affiliation: Core Research for Evolutional Science and Technology Research Program, Japan Science and Technology Corporation, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
The condensin complex has a fundamental role in chromosome dynamics. In this study, we report that accumulation of Schizosaccharomyces pombe condensin at mitotic kinetochores and ribosomal DNAs (rDNAs) occurs in multiple steps and is necessary for normal segregation of the sister kinetochores and rDNAs. Nuclear entry of condensin at the onset of mitosis requires Cut15/importin alpha and Cdc2 phosphorylation. Ark1/aurora and Cut17/Bir1/survivin are needed to dock the condensin at both the kinetochores and rDNAs. Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites. Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs. The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells.

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Enrichment of condensin at rDNAs requires a nucleolar protein, Acr1, which interacts with the rDNA upstream activator complex. (A) Cut14-GFP was observed in mitotic cells of the acr1-936 mutant and the control wild type cultured at 36°C for 4 h. (B) A ChIP assay was performed using extracts of asynchronous wild-type and acr1-936 mutant that expressed Cut14-Flag. The wild-type and acr1-936 mutant were cultured at 26°C and shifted to 36°C for 4 h, and immunoprecipitation was performed. Real-time PCR was used to amplify and quantify DNAs with the PCR primers (N1, N2, N3, lys1+, and cnt1). Error bars represent SD. (C) Localization of Acr1 was examined in the strain that expressed Acr1-GFP. (D) A ChIP assay was performed using the strain that expressed Acr1-Flag. Two rDNA probes (N2 and 18S) and four control probes (cnt1, imr1, dg, and lys1+) were used. (E, top) Preparation of proteins coprecipitated with Acr1-Flag and the Flag-only control for liquid chromatography–tandem mass spectrometry. The positions of identified protein names are indicated. The two bands were obtained for Acr1-Flag (Fig. S2 D, available at http://www.jcb.org/cgi/content/full/jcb.200708170/DC1). (bottom) Four proteins (Rrn5, Rrn11, Rrn7, and Spp27) were coprecipitated with Acr1-Flag. emPAI, exponentially modified protein abundance index (Ishihama et al., 2005). Bars: (A) 2 μm; (C) 10 μm.
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fig5: Enrichment of condensin at rDNAs requires a nucleolar protein, Acr1, which interacts with the rDNA upstream activator complex. (A) Cut14-GFP was observed in mitotic cells of the acr1-936 mutant and the control wild type cultured at 36°C for 4 h. (B) A ChIP assay was performed using extracts of asynchronous wild-type and acr1-936 mutant that expressed Cut14-Flag. The wild-type and acr1-936 mutant were cultured at 26°C and shifted to 36°C for 4 h, and immunoprecipitation was performed. Real-time PCR was used to amplify and quantify DNAs with the PCR primers (N1, N2, N3, lys1+, and cnt1). Error bars represent SD. (C) Localization of Acr1 was examined in the strain that expressed Acr1-GFP. (D) A ChIP assay was performed using the strain that expressed Acr1-Flag. Two rDNA probes (N2 and 18S) and four control probes (cnt1, imr1, dg, and lys1+) were used. (E, top) Preparation of proteins coprecipitated with Acr1-Flag and the Flag-only control for liquid chromatography–tandem mass spectrometry. The positions of identified protein names are indicated. The two bands were obtained for Acr1-Flag (Fig. S2 D, available at http://www.jcb.org/cgi/content/full/jcb.200708170/DC1). (bottom) Four proteins (Rrn5, Rrn11, Rrn7, and Spp27) were coprecipitated with Acr1-Flag. emPAI, exponentially modified protein abundance index (Ishihama et al., 2005). Bars: (A) 2 μm; (C) 10 μm.

Mentions: We isolated a ts mutant from a collection of ∼1,000 randomly mutagenized strains (Hayashi et al., 2004) through the search of strains that exhibited the phenotype similar to that of nuc1-632. One strain, 936, was defective in condensin accumulation at rDNAs (Fig. 5 A). Plasmids carrying Spbc17D1.04 (GenBank/EMBL/DDBJ accession no. CAA20428.1) in chromosome II completely rescued the ts phenotype. Tetrad dissection of the cross between 936 and mad2Δ deletion indicated that they were tightly linked (1.2 cM). The mad2+ gene is only 38 kb apart from Spbc17D1.04, so the 936 strain was the most likely mutant in the Spbc17D1.04 gene that encoded a 29-kD protein classified as a sequence orphan in the Sanger Center S. pombe database. The Cys58 residue was changed to Tyr58 in Spbc17D1.04. We hereafter refer to Spbc17D1.04 as Acr1 (accumulation of condensin at rDNA 1). Gene disruption showed that the acr1+ gene was essential for viability (unpublished data). A high-copy suppressor encoding Tbp1/Spac29E6.08 was isolated (unpublished data). Tbp1 is the TATA-binding transcription initiation factor (Mitsuzawa et al., 2001).


Dissection of the essential steps for condensin accumulation at kinetochores and rDNAs during fission yeast mitosis.

Nakazawa N, Nakamura T, Kokubu A, Ebe M, Nagao K, Yanagida M - J. Cell Biol. (2008)

Enrichment of condensin at rDNAs requires a nucleolar protein, Acr1, which interacts with the rDNA upstream activator complex. (A) Cut14-GFP was observed in mitotic cells of the acr1-936 mutant and the control wild type cultured at 36°C for 4 h. (B) A ChIP assay was performed using extracts of asynchronous wild-type and acr1-936 mutant that expressed Cut14-Flag. The wild-type and acr1-936 mutant were cultured at 26°C and shifted to 36°C for 4 h, and immunoprecipitation was performed. Real-time PCR was used to amplify and quantify DNAs with the PCR primers (N1, N2, N3, lys1+, and cnt1). Error bars represent SD. (C) Localization of Acr1 was examined in the strain that expressed Acr1-GFP. (D) A ChIP assay was performed using the strain that expressed Acr1-Flag. Two rDNA probes (N2 and 18S) and four control probes (cnt1, imr1, dg, and lys1+) were used. (E, top) Preparation of proteins coprecipitated with Acr1-Flag and the Flag-only control for liquid chromatography–tandem mass spectrometry. The positions of identified protein names are indicated. The two bands were obtained for Acr1-Flag (Fig. S2 D, available at http://www.jcb.org/cgi/content/full/jcb.200708170/DC1). (bottom) Four proteins (Rrn5, Rrn11, Rrn7, and Spp27) were coprecipitated with Acr1-Flag. emPAI, exponentially modified protein abundance index (Ishihama et al., 2005). Bars: (A) 2 μm; (C) 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2290841&req=5

fig5: Enrichment of condensin at rDNAs requires a nucleolar protein, Acr1, which interacts with the rDNA upstream activator complex. (A) Cut14-GFP was observed in mitotic cells of the acr1-936 mutant and the control wild type cultured at 36°C for 4 h. (B) A ChIP assay was performed using extracts of asynchronous wild-type and acr1-936 mutant that expressed Cut14-Flag. The wild-type and acr1-936 mutant were cultured at 26°C and shifted to 36°C for 4 h, and immunoprecipitation was performed. Real-time PCR was used to amplify and quantify DNAs with the PCR primers (N1, N2, N3, lys1+, and cnt1). Error bars represent SD. (C) Localization of Acr1 was examined in the strain that expressed Acr1-GFP. (D) A ChIP assay was performed using the strain that expressed Acr1-Flag. Two rDNA probes (N2 and 18S) and four control probes (cnt1, imr1, dg, and lys1+) were used. (E, top) Preparation of proteins coprecipitated with Acr1-Flag and the Flag-only control for liquid chromatography–tandem mass spectrometry. The positions of identified protein names are indicated. The two bands were obtained for Acr1-Flag (Fig. S2 D, available at http://www.jcb.org/cgi/content/full/jcb.200708170/DC1). (bottom) Four proteins (Rrn5, Rrn11, Rrn7, and Spp27) were coprecipitated with Acr1-Flag. emPAI, exponentially modified protein abundance index (Ishihama et al., 2005). Bars: (A) 2 μm; (C) 10 μm.
Mentions: We isolated a ts mutant from a collection of ∼1,000 randomly mutagenized strains (Hayashi et al., 2004) through the search of strains that exhibited the phenotype similar to that of nuc1-632. One strain, 936, was defective in condensin accumulation at rDNAs (Fig. 5 A). Plasmids carrying Spbc17D1.04 (GenBank/EMBL/DDBJ accession no. CAA20428.1) in chromosome II completely rescued the ts phenotype. Tetrad dissection of the cross between 936 and mad2Δ deletion indicated that they were tightly linked (1.2 cM). The mad2+ gene is only 38 kb apart from Spbc17D1.04, so the 936 strain was the most likely mutant in the Spbc17D1.04 gene that encoded a 29-kD protein classified as a sequence orphan in the Sanger Center S. pombe database. The Cys58 residue was changed to Tyr58 in Spbc17D1.04. We hereafter refer to Spbc17D1.04 as Acr1 (accumulation of condensin at rDNA 1). Gene disruption showed that the acr1+ gene was essential for viability (unpublished data). A high-copy suppressor encoding Tbp1/Spac29E6.08 was isolated (unpublished data). Tbp1 is the TATA-binding transcription initiation factor (Mitsuzawa et al., 2001).

Bottom Line: Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites.Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs.The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells.

View Article: PubMed Central - PubMed

Affiliation: Core Research for Evolutional Science and Technology Research Program, Japan Science and Technology Corporation, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
The condensin complex has a fundamental role in chromosome dynamics. In this study, we report that accumulation of Schizosaccharomyces pombe condensin at mitotic kinetochores and ribosomal DNAs (rDNAs) occurs in multiple steps and is necessary for normal segregation of the sister kinetochores and rDNAs. Nuclear entry of condensin at the onset of mitosis requires Cut15/importin alpha and Cdc2 phosphorylation. Ark1/aurora and Cut17/Bir1/survivin are needed to dock the condensin at both the kinetochores and rDNAs. Furthermore, proteins that are necessary to form the chromatin architecture of the kinetochores (Mis6, Cnp1, and Mis13) and rDNAs (Nuc1 and Acr1) are required for condensin to accumulate specifically at these sites. Acr1 (accumulation of condensin at rDNA 1) is an rDNA upstream sequence binding protein that physically interacts with Rrn5, Rrn11, Rrn7, and Spp27 and is required for the proper accumulation of Nuc1 at rDNAs. The mechanism of condensin accumulation at the kinetochores may be conserved, as human condensin II fails to accumulate at kinetochores in hMis6 RNA interference-treated cells.

Show MeSH
Related in: MedlinePlus