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Science 327 (5962): 172-177

Copyright © 2010 by the American Association for the Advancement of Science

Phosphorylation of H2A by Bub1 Prevents Chromosomal Instability Through Localizing Shugoshin

Shigehiro A. Kawashima1, Yuya Yamagishi1,2,*, Takashi Honda1,3,*, Kei-ichiro Ishiguro1, and Yoshinori Watanabe1,2,3,{dagger}

1 Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Tokyo 113-0032, Japan.
2 Graduate Program in Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Yayoi, Tokyo 113-0032, Japan.
3 Graduate School of Agricultural and Life Science, University of Tokyo, Yayoi, Tokyo 113-0032, Japan.


Figure 1
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Fig. 1. H2A-S121 is phosphorylated by Bub1. (A) Chromatin fractions were incubated with or without recombinant His-Bub1 in the presence of [{gamma}-32P]ATP. The incorporation of the radioactive phosphate group was visualized by means of autoradiography (32P), and protein loading was analyzed through staining with Coomassie Brilliant Blue (CBB). (B) The phosphorylated chromatin fraction (PCF) was denatured and immunoprecipitated with the indicated antibodies. (C) Chromatin fractions from h2a+ or h2a+-flag cells were phosphorylated as in (A). (D) Recombinant H2A{alpha}, H2Aβ, and H3 were phosphorylated with His-Bub1 or GST-Ark1 and GST-Pic1-C. (E) The indicated mutants of GST-H2A{alpha} were phosphorylated with His-Bub1. (F) Phospho–amino acid analysis of H2A{alpha} and H2A{alpha}-S121T phosphorylated by Bub1. (G) Cell extracts prepared from the indicated cells were immunoblotted for H2A-pS121 and H2A.

 

Figure 2
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Fig. 2. Similar chromosomal instability (CIN) defects of bub1-KD, h2a-SA, and shugoshin mutants. (A) Serial dilution assay (12.5 µg/ml TBZ). (B) The indicated cells carrying a mutation of β-tubulin (nda3-KM311) were cultured at permissive temperature (+) or restrictive temperature (–) and scored for mitotic index (n > 200 cells). (C) The indicated temperature-sensitive cohesin mutants (psc3-1T) were arrested at the G1/S phase by adding hydroxyurea (HU) and released by increasing the temperature. Prometaphase (spindle pole body–duplicated and securin/Cut2 positive) cells were counted at each time point (n > 200 cells). (D) The indicated strains expressing mCherry-Atb2 ({alpha}2-tubulin) were examined for frequencies of lagging chromosomes in anaphase cells (n > 100 cells). Examples are shown at the right. (E) One of the homologs marked with cen2–green fluorescent protein (GFP) was monitored for segregation during meiosis I in the indicated zygotes. The number of cells that had undergone equational segregation in meiosis I was examined by monitoring metaphase II cells (n > 100 zygotes). (F) One of the homologs marked with cen2-GFP was monitored for segregation during meiosis II in the indicated zygotes (n > 200 zygotes).

 

Figure 3
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Fig. 3. Forced enrichment of shugoshins in centromeres suppresses the CIN defects of bub1-KD or h2a-SA cells. (A) The signals of Sgo2-GFP expressed from the endogenous promoter were measured in metaphase in the indicated cells. Error bars represent SEM (n = 16 cells). (B) Sgo1-GFP expressed from the endogenous promoter was detected in metaphase I in the indicated cells. The metaphase I spindle was visualized with mCherry-Atb2. Sgo1 signals were detected only in wild-type cells (n > 50 zygotes). (C) The indicated pcs3-1T strains were arrested at the G1/S phase and released by raising the temperature. Prometaphase cells were counted at each time point (n > 200 cells). (D) The indicated cells were examined for frequencies of lagging chromosomes at anaphase (n > 100 cells). (E) The signal intensity of Ark1-GFP in metaphase cells was measured. Error bars represent SEM (n > 25 cells). (F) One of the homologs marked with cen2-GFP was monitored for segregation during meiosis II in the indicated zygotes (n > 200 zygotes). (G) Par1-mCherry was detected at metaphase I. (H) Schematic depiction of the Bub1 pathway regulating chromosome segregation.

 

Figure 4
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Fig. 4. Association between the SGO motif and nucleosomes containing H2A-pS121 is required for the chromosomal localization of shugoshin. (A) ChIP analysis was used to measure Sgo1, Swi6, H2A-pS121, and H2A throughout the heterochromatic centromere (dg and dh), mating type locus (mat), and telomere (tel); euchromatic arm region (msp1 and zfs1); and outer subtelomere (ostel1 and ostel2) in the indicated strains at metaphase I. Values of H2A-pS121/H2A multiplied by Swi6 ChIP (in percent) are compared with those of Sgo1 ChIP (in percent), revealing a good correlation along the chromosome. (B) Bub1-GFP and Bub1{triangleup}N-GFP were detected in cells at metaphase I. (C) ChIP analysis was used to measure Sgo2, Swi6, H2A-pS121, and H2A in asynchronous (G2) or M phase–arrested (M) nda3-KM311 cells. (D) Immunostaining for Sgo2, H2A-pS121, tubulin, and DNA in wild-type cells (top) and for H2A-pS121 and DNA in the indicated cells (bottom). Arrowheads indicate anaphase cells. (E) A schematic of the Sgo1 protein showing the PP2A-interacting coiled-coil region (CC), Swi6/HP1–interacting motif (black box), and SGO motif (SGO). Arrowheads indicate the mutations isolated in a genetic screening (fig. S13), which abolish Sgo1 localization. (F) Sgo1 localization was examined at metaphase I in the indicated strains. (G) Cell extracts prepared from the indicated strains were pulled down with GST-Sgo1 or GST and analyzed by means of immunoblotting with antibodies to H3, H2A, and tubulin. Input extracts are also shown (0.5%). (H) Cell extracts prepared from wild-type cells were pulled down with the indicated Sgo1 mutant proteins fused with GST and analyzed as in (G). (I) One of the homologs marked with cen2-GFP was monitored for segregation during meiosis II in the indicated zygotes at 26.5°C (n > 200 zygotes).

 

Figure 5
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Fig. 5. Bub1-H2A-shugoshin pathway is conserved in budding yeast. (A) Alignment of the C-tail of histone H2A (amino acids are numbered excluding the first methionine). Ser 121 (arrowhead) and the preceding sequence are widely conserved among eukaryotes. (B) GST-ScH2A (wild-type or S121A) proteins were incubated with His-ScBub1 in the presence of [{gamma}-32P]ATP for 30 min at 30°C. (C) Serial dilution assay (10 µg/ml Benomyl). (D) ScSgo1-GFP was detected at metaphase (mitotic spindle indicated by separated Spc42 signals) in wild-type, h2a-SA, and bub1 cells. Quantification of Sgo1-GFP–positive cells at metaphase is shown (n > 100 cells).

 

Figure 6
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Fig. 6. Bub1-H2A-shugoshin pathway is conserved in humans. (A) Cycling HeLa cells were spun onto glass slides after fixation and stained with antibody to H2A-pT120 and anti-centromere antibodies (ACA) as well as antibodies to hBub1 (left) or hSgo1 (right). DNA was counterstained with Hoechst 33342. (B) HeLa cells treated with Bub1 siRNA were arrested at prometaphase by nocodazole and MG132 for 3 hours. Immunostaining was performed as in (A). Arrowheads indicate a Bub1-negative cell (left) and an H2A-pT120 negative cell (right), respectively. (C) Cells injected with antibody to H2A-pT120 or control immunoglobulin G (IgG) were arrested at prometaphase by nocodazole and stained with antibody to hSgo1 and ACA. The injected antibodies and DNA were also stained. The ratio of centromere and arm Sgo1 signals was quantified. (D) HeLa cells expressing H2B-Bub1{triangleup}N-GFP were depleted for endogenous Bub1 by means of RNA interference, subjected to chromosome spreads, and stained with antibody to hSgo1 and ACA. Magnified images of paired sister chromatids are shown on the left. Scale bars, 10 µm.

 


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