Condensin association with histone H2A shapes mitotic chromosomes

Journal name:
Nature
Volume:
474,
Pages:
477–483
Date published:
DOI:
doi:10.1038/nature10179
Received
Accepted
Published online
Corrected online

Abstract

Chromosome structure is dynamically regulated during cell division, and this regulation is dependent, in part, on condensin. The localization of condensin at chromosome arms is crucial for chromosome partitioning during anaphase. Condensin is also enriched at kinetochores but its precise role and loading machinery remain unclear. Here we show that fission yeast (Schizosaccharomyces pombe) kinetochore proteins Pcs1 and Mde4—homologues of budding yeast (Saccharomyces cerevisiae) monopolin subunits and known to prevent merotelic kinetochore orientation—act as a condensin ‘recruiter’ at kinetochores, and that condensin itself may act to clamp microtubule binding sites during metaphase. In addition to the regional recruitment factors, overall condensin association with chromatin is governed by the chromosomal passenger kinase Aurora B. Aurora-B-dependent phosphorylation of condensin promotes its association with histone H2A and H2A.Z, which we identify as conserved chromatin ‘receptors’ of condensin. Condensin phosphorylation and its deposition onto chromosome arms reach a peak during anaphase, when Aurora B kinase relocates from centromeres to the spindle midzone, where the separating chromosome arms are positioned. Our results elucidate the molecular basis for the spatiotemporal regulation of mitotic chromosome architecture, which is crucial for chromosome partitioning.

At a glance

Figures

  1. Dissection of condensin function at the kinetochore and chromosome
arm.
    Figure 1: Dissection of condensin function at the kinetochore and chromosome arm.

    a, Serial dilutions growth assay on YE plates. b, A ChIP assay was used to measure Cnd2-HA throughout kinetochore (cnt and imr), pericentric (dg and dh), arm (rpl4301, rps29, pgk1 and msp1) and rDNA (rDNA-N1) regions in the indicated strains arrested at prometaphase by nda3-KM311 inactivation (also see Supplementary Fig. 3). Error bars represent s.d. (n = 3 PCR amplifications). c, Serial dilutions growth assay on YE plates containing 0 or 10μgml−1 TBZ. Photos show the representative localization of Cnd2–tdTomato (left) and Cnd2–tdTomato–Cnp3C (right) at metaphase. Scale bar, 2μm. d, Serial dilutions growth assay on YE plates. e, Schematic depiction illustrating distinct condensin recruitment at kinetochores and chromosome arms (bottom).

  2. Phosphorylation of condensin by Aurora B is required for chromatin
association.
    Figure 2: Phosphorylation of condensin by Aurora B is required for chromatin association.

    a, Frequencies of unresolved DNA at anaphase (n>100 cells) were examined in the indicated cells. b, Wild-type (WT) and ark1-as2 cells expressing Cnd2–GFP were arrested at metaphase by the overexpression of mad2+. For the last 1h, cells were cultured in the presence of 1NM-PP1, then fixed and examined by ChIP assay. Error bars represent s.d. (n = 3 PCR amplifications). c, Schematic diagram of fission yeast Cnd2. Aurora B phosphorylation sites are indicated by arrowheads. GST–Cnd2 and GST–Cnd2-3A (alanine substitution at S5, S41 and S52) were phosphorylated by Ark1 and analysed for phosphate incorporation (32P) and protein levels (Coomassie brilliant blue; CBB). d, Cnd2–GFP proteins were precipitated from asynchronous and mitotic (arrested by the overexpression of mad2+) extracts of wild-type and ark1-as2 cells, and analysed by immunoblot using anti-Cnd2-pS5 or anti-GFP antibodies. For the last 30min of mitotic arrest, cells were cultured in the presence of 5μM 1NM-PP1. The mitotic index was determined by counting nuclear Cnd2–GFP-positive cells. e, Serial dilution growth assay on YE plates. Photo shows a representative cnd2-3A cell at anaphase. f, Serial dilution growth assay on YE plates in the presence or absence of 0.2μM 1NM-PP1. g, The indicated cells ectopically expressing Cnd2–GFP or Cnd2-3A–GFP were arrested at prometaphase by nda3-KM311 inactivation and examined by ChIP assay. Error bars represent s.d. (n = 3 PCR amplifications). Scale bars, 2μm (a, e).

  3. Chromatin association of human condensin I is also regulated by
Aurora-B-dependent phosphorylation.
    Figure 3: Chromatin association of human condensin I is also regulated by Aurora-B-dependent phosphorylation.

    a, GST–CAP-H wild-type and GST–CAP-H-S70A were incubated with GST–Aurora B, and analysed for phosphate incorporation (32P) and protein levels (CBB). b, Whole-cell extracts were obtained from HeLa cells treated with or without the Aurora inhibitor ZM447439 (ZM) at prometaphase or asynchronous cells, and analysed by immunoblot using the indicated antibodies. c, Cell extracts prepared from nocodazole-arrested cells were fractionated into chromatin and cytoplasmic fractions, and analysed by immunoblot. d, Synchronized mitotic HeLa cells expressing GFP–CAP-H were treated with nocodazole in the absence or presence of ZM447439 for 2h. Signals of GFP–CAP-H at prometaphase were examined by immunostaining. Cells expressing GFP–CAP-H-S70A were similarly analysed without ZM447439 treatment. The ratios of nucleus and cytoplasmic signals were quantified in the indicated cells. Error bars represent s.e.m. (n = 15 cells). ***, P<0.001. Scale bar, 5μm. e, Co-immunoprecipitation of GFP–CAP-H and SMC2. Whole-cell extracts (WCE) were prepared from nocodazole-arrested HeLa cells expressing GFP–CAP-H wild type or GFP–CAP-H-S70A. GFP–CAP-H was immunoprecipitated (IP) with anti-GFP antibodies to examine the co-precipitation of SMC2. f, Chromatin fractions were prepared from nocodazole-arrested cells, precipitated with control or anti-SMC2 antibodies, and analysed by silver staining. Asterisks indicate IgG. g, Whole-cell extracts were prepared from mitotic HeLa cells treated with or without ZM447439, pulled down with MBP (maltose-binding protein)–H2A or MBP–H2B, and analysed by immunoblot using antibodies against CAP-H, CAP-H-pS70 or actin.

  4. H2A and H2A.Z act as a chromatin receptor of condensin.
    Figure 4: H2A and H2A.Z act as a chromatin receptor of condensin.

    a, GST–Cnd2 phosphorylated by Ark1 was pulled down with the indicated MBP-fused proteins and analysed by immunoblot using anti-GST antibody. b, GST–Cnd2 wild type or GST–Cnd2-3A reacted with Ark1 in the absence or presence of ATP was pulled down with MBP–H2A. c, GST–Cnd2 phosphorylated by Ark1 was pulled down with the indicated MBP-fused proteins. Mutated residues within the N-terminal tail of H2A are shown in the diagram. d, Serial dilution growth assay on SSA plates containing 0 or 8μM thiamine and 0 or 10μgml−1 TBZ. e, The indicated cells were cultured in SSA medium (−thiamine), transferred to YE medium (+thiamine) and incubated for 36h at 30°C to suppress the expression of H2A.Z. Frequencies of unresolved DNA at anaphase (n>100 cells) were examined. Error bars represent s.d. (n = 3 experiments). f, The indicated cells cultured in YE medium for 24h at 30°C were shifted to 18°C for 6h. Frequencies of lagging chromosomes of cen2-GFP at anaphase (n>100 cells) were examined. Error bars represent s.d. (n = 3 experiments). g, The indicated cells were arrested at G2 phase by cdc25-22 inactivation and released into mitosis. Cells collected at each time point were examined by ChIP assay (average of two PCR amplifications). Anaphase cells were monitored by DAPI staining (n>100 cells). h, Schematic depiction of the pathway that regulates condensin localization on chromatin. Scale bars, 2μm (e, f).

  5. Spatiotemporal regulation of condensin localization by Aurora B.
    Figure 5: Spatiotemporal regulation of condensin localization by Aurora B.

    a, Cells expressing Cnd2–GFP were arrested at G2 phase by cdc25-22 inactivation and released into mitosis. Cell extracts prepared from cells collected at each time point were analysed by immunoblot using the indicated antibodies. Mitotic nuclear division was monitored by nuclear accumulation of Cnd2–GFP and DAPI staining (n>100 cells) (top panels). DIC, differential interference contrast. b, Wild-type and taz1Δ cells expressing Cnd2–GFP were arrested at G2 phase by cdc25-22 inactivation, released into mitosis and examined by ChIP assay. A representative anaphase bridge in a taz1Δ cell is shown compared with normal anaphase in a wild-type cell. c, Schematic depiction of the spatiotemporal regulation of condensin localization by Aurora B. Scale bars, 2μm (a, b).

Change history

Corrected online 12 June 2011
The Supplementary Information PDF was replaced.

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Author information

Affiliations

  1. Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Tokyo 113-0032, Japan

    • Kenji Tada,
    • Hiroaki Susumu,
    • Takeshi Sakuno &
    • Yoshinori Watanabe
  2. Graduate Program in Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Yayoi, Tokyo 113-0032, Japan

    • Kenji Tada,
    • Hiroaki Susumu &
    • Yoshinori Watanabe
  3. Promotion of Independence for Young Investigators, University of Tokyo, Yayoi, Tokyo 113-0032, Japan

    • Takeshi Sakuno

Contributions

K.T., supported by T.S., performed most of the experiments using fission yeast cells and proteins. H.S. performed all experiments using human cells or proteins. Experimental design and interpretation of data were conducted by all authors. Y.W. supervised the project and K.T. and Y.W. wrote the paper.

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The authors declare no competing financial interests.

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