Two Histone Marks Establish the Inner Centromere and Chromosome Bi-Orientation

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Science  08 Oct 2010:
Vol. 330, Issue 6001, pp. 239-243
DOI: 10.1126/science.1194498

Location, Location, Location

Cell division is orchestrated by a complex signaling pathway that ensures the correct segregation of newly replicated chromosomes to the two daughter cells. The pathway is controlled in part by restricting the activity of critical regulators to specific subcellular locations. For example, the chromosomal passenger complex (CPC) is recruited to chromosomes during mitosis where it oversees kinetochore activity and cytokinesis (see Perspective by Musacchio). Wang et al. (p. 231, published online 12 August), Kelly et al. (p. 235, published online 12 August), and Yamagishi et al. (p. 239) now show that the phosphorylation of the chromatin protein, histone H3, acts to bring the CPC to chromosomes, thereby activating its aurora B kinase subunit. The Survivin subunit of CPC binds specifically to phosphorylated H3, with the phosphorylation at centromeres being carried out by the mitosis-specific kinase, haspin. Furthermore, Bub1 phosphorylation of histone H2A recruits shugoshin, a centromeric CPC adapter. Thus, these two histone marks in combination define the inner centromere.


For proper partitioning of chromosomes in mitosis, the chromosomal passenger complex (CPC) including Aurora B and survivin must be localized at the center of paired kinetochores, at the site called the inner centromere. It is largely unknown what defines the inner centromere and how the CPC is targeted to this site. Here, we show that the phosphorylation of histone H3–threonine 3 (H3-pT3) mediated by Haspin cooperates with Bub1-mediated histone 2A–serine 121 (H2A-S121) phosphorylation in targeting the CPC to the inner centromere in fission yeast and human cells. H3-pT3 promotes nucleosome binding of survivin, whereas phosphorylated H2A-S121 facilitates the binding of shugoshin, the centromeric CPC adaptor. Haspin colocalizes with cohesin by associating with Pds5, whereas Bub1 localizes at kinetochores. Thus, the inner centromere is defined by intersection of two histone kinases.

The accurate segregation of chromosomes during cell division requires sister chromatids to be captured by microtubules from the opposite spindle poles (bi-orientation), which is accomplished by a trial-and-error process of kinetochore-microtubule attachment that relies on Aurora B kinase locating at centromeres (16). Shugoshin, a conserved inner centromere protein protecting centromeric cohesion (7, 8), also mediates chromosomal passenger complex (CPC) targeting to centromeres (912). In fission yeast, meiosis-specific shugoshin Sgo1 protects cohesin, a sister chromatid cohesion molecule (13), whereas another shugoshin, Sgo2, mediates CPC targeting to centromeres, thus dividing the labors (9). These functions are shared by two shugoshins in human cells (12, 14). Although Bub1-mediated histone 2A–serine 121 (H2A-S121) phosphorylation is important for the centromere targeting of shugoshin and the CPC (15), we are still far from understanding the full picture of CPC targeting to the inner centromere. More fundamentally, it is unknown how the chromatin locus of the inner centromere is established.

Haspin is a highly conserved kinase and might function in the centromeric protection of cohesin in parallel with shugoshin in human cells (16). To examine the functional link of Haspin to shugoshins in fission yeast, we deleted the hrk1+ gene (SPAC23C4.03) encoding Haspin-related kinase (17). Hrk1 is dispensable for centromeric cohesion in both mitosis and meiosis (fig. S1, A and B), meaning that Hrk1 is not relevant to cohesion or Sgo1 function. However, hrk1∆ cells show synthetic lethality (or severe sickness) with the mutation of another shugoshin Sgo2, the centromeric CPC adaptor (Fig. 1A) (9, 12). Like sgo2∆ cells, hrk1∆ cells impair CPC localization to centromeres in metaphase (fig. S1C). The hrk1∆ sgo2∆ double mutant nearly entirely abolishes CPC targeting to centromeres accompanied by a high incidence of chromosome lagging or mis-segregation in anaphase (fig. S1, C and D). Notably, hrk1∆ does not show a synthetic sickness with swi6∆, a mutation in the heterochromatin protein gene that shows a reduction in centromeric CPC concentration and a synthetic sickness with sgo2∆ (Fig. 1A) (9). Thus, Hrk1 acts in CPC targeting to centromeres in a redundant capacity with Sgo2 but possibly in the same pathway with Swi6.

Fig. 1

Phosphorylation of H3-T3 promotes CPC targeting to centromeres. (A) Serial dilution growth assay. (B) H3–maltose binding protein (MBP) protein was incubated with His-Hrk1 in the absence or presence of adenosine triphosphate (ATP). H3-T3 phosphorylation was detected by immunoblot. (C) Cell extracts prepared from the indicated cells were immunoblotted for H3-pT3 and H3. G2; asynchronous culture, M; prometaphase arrest (by nda3-KM311). (D) Serial dilution growth assay. (E) The signal intensity of Ark1–green fluorescent protein (GFP) in metaphase cells was measured. Error bars represent SD (n = 20 cells). Scale bar, 2 μm. ***P < 0.001 (Student’s t test). AU, arbitrary units. (F) The indicated cells were examined for the frequency of lagging chromosomes at anaphase (n > 100 cells).

Like human Hapsin (16), fission yeast Hrk1 has the ability to phosphorylate histone H3 at Thr3 (T3) in vitro (Fig. 1B). Immunoblot assays revealed that histone H3-T3 is phosphorylated in prometaphase-arrested cells, but little in asynchronous cells. This phosphorylation is abolished in hrk1∆ cells or h3-TA cells in which Thr3 is replaced with alanine (H3-TA) in all histone H3 genes (hht1+, hht2+, and hht3+) (Fig. 1C), suggesting that Hrk1 is the sole kinase phosphorylating H3-T3 in vivo. Although h3-TA cells are viable as hrk1∆ cells, they show a similar level of reduction in centromeric CPC, as well as a defect in chromosome bi-orientation (lagging chromosomes) (Fig. 1, D to F). The hrk1∆ h3-TA double mutant shows no cumulative defects, whereas h3-TA and sgo2∆ show synergetic defects as seen for hrk1∆ and sgo2∆ (Fig. 1, A and D). These results suggest that Hrk1 acts in CPC targeting to centromeres by phosphorylating H3-T3. Consistently, in vitro binding assays revealed that only when H3-T3 is phosphorylated, histone H3 interacts with the baculoviral IAP repeat (BIR) domain of survivin (fig. S2).

In HeLa cells, depletion of Haspin by treatment with small interfering RNA (siRNA) impaired the centromeric CPC localization, as well as H3-T3 phosphorylation, in mitotic chromosomes (Fig. 2A). A pull-down assay using recombinant H3 revealed that wild-type H3, but not the mutant H3-TA, pulled down the CPC from mitotic extracts only when H3 was phosphorylated by Haspin (Fig. 2B). Point mutations at the conserved amino acids (Arg18, Trp25, Cys33, Cys57, and Trp67) in the BIR domain, but not a mutation at a nonconserved residue (Gln56), lost the ability to bind phosphorylated histone H3–threonine 3 (H3-pT3) in vitro (Fig. 2, C and D), whereas they showed intact complex formation with INCENP (inner centromere protein) and borealin (fig. S3). Accordingly, all mutant survivin proteins whose in vitro binding to H3-pT3 was abolished failed to localize at centromeres in vivo (Fig. 2E). These results for human survivin, together with those for fission yeast Bir1 (fig. S2), strongly argue that the conserved BIR domain of survivin plays a crucial role in targeting the CPC to centromeres by binding directly to H3-pT3.

Fig. 2

The interaction between the BIR domain and H3-pT3 is required for CPC targeting to centromeres in HeLa cells. (A) HeLa cells treated with Haspin siRNA were arrested at prometaphase by treatment with nocodazole and MG132 for 3 hours and were immunostained with anti-Aurora B, anti-H3-pT3, and Hoechst 33342. Relative fluorescence intensities of Aurora B and H3-pT3 toward anti-centromere antibodies (ACA) were quantified at 10 centromeres in each cell. Error bars represent SEM (n > 10 cells). RNAi, RNA interference. (B) Chromatin extracts prepared from mitotic HeLa cells were pulled down by H3–glutathione S-transferase (GST) or GST that had been pre-incubated with His-Haspin in the absence or presence of ATP and were analyzed by immunoblot using the indicated antibodies. (C) Alignment of the BIR domain of survivin. Schizosaccharomyces pombe survivin has two BIR domains. Mutation sites used in (D) and (E) are marked at the top, and S. pombe bir1-T1 mutations are denoted by red circles. (D) H3-GST was pre-incubated with His-Haspin and pulled down with MBP-survivin (wild-type and mutant) proteins. (E) Mitotic HeLa cells expressing indicated enhanced GFP–survivin were immunostained with ACA and antibodies against GFP. DNA was costained with Hoechst 33342. Scale bars in (A) and (E), 10 μm.

Given that the interaction between Bir1/survivin and H3-pT3 is required for centromere targeting of the CPC, the phosphorylation of H3-T3 might be observed at the inner centromere. Chromatin immunoprecipitation (ChIP) assays in fission yeast indicates that H3-T3 phosphorylation localizes, not only at the pericentromeric region (inner centromere in metazoan cells), but also at the mating-type locus and teleomere-adjacent regions in mitosis (Fig. 3A and fig. S4A), whereas Bir1 is enriched mostly at the pericentromeric region (Fig. 3A). Although Hrk1 localizes to all heterochromatic regions like H3-pT3, additional localization appears in the euchromatic regions (Fig. 3A; also see below). As Hrk1 might function in the Swi6 pathway (Fig. 1A), Hrk1 localization was abolished specifically in the heterochromatic regions by swi6∆, whereas it was preserved at the euchromatic regions (Fig. 3A and fig. S4B). Thus, heterochromatin assembly is an important, but not the sole, requirement for chromatin association of Hrk1.

Fig. 3

Hrk1 is recruited to cohesin sites through the interaction with Pds5. (A) ChIP analysis was used to measure Bir1, Hrk1, Swi6, H3-pT3, and H3 throughout the kinetochore (imr1); heterochromatic centromere (cen), mating type locus (mat), and telomere (tel); and euchromatic arm region (mps1, 56F2, mes1, and zfs1) in the indicated strains arrested at prometaphase (by nda3-KM311). Average of two polymerase chain reaction (PCR) amplifications. WT, wild type. (B) Yeast two-hybrid assay with Hrk1 fragments. The Ras and Raf pair acts as a positive control. (C) Hrk1-GFP was detected in the indicated cells arrested at prometaphase. Scale bar, 2 μm. (D) ChIP analysis was used to measure Bir1, H3-pT3, H3, H2A-pS121, H2A Hrk1, Swi6, and Psc3 in the indicated strains arrested at prometaphase (by nda3-KM311). Error bars represent SD (n = 3 PCR amplifications). Note that msp1 and 56F2 correspond to the cohesin peaks, whereas mes1 and zfs1 do not.

Cohesin is the crucial factor involved in chromosome segregation downstream of heterochromatin assembly (18) and might be required for CPC localization (19, 20). We therefore screened cohesin-related proteins for interaction with Hrk1 by the yeast two-hybrid system, revealing that the N terminus of Hrk1 interacts with the cohesin-associating protein Pds5, as well as with Swi6 (Fig. 3B). pds5∆ cells lost Hrk1 localization, as well as H3-T3 phosphorylation, along the entire chromosome region (Fig. 3, C and D), whereas Pds5 localization did not depend on Hrk1 (fig. S5A). Consistently, the localization of Hrk1 and Pds5 (and cohesin) coincide at all tested chromosomal sites (Fig. 3D and fig. S5C). By tethering Pds5 at a specific chromosomal site, we observed accumulation of Hrk1 at this site (fig. S6), validating that Pds5 acts as a primary basis for Hrk1 localization. The interaction of Hrk1 with Swi6 might also be important, because Hrk1 localization at the heterochromatic region partly depends on this interaction (fig. S7).

The aforementioned results suggest that the Swi6-Pds5-Hrk1-H3-survivin pathway acts in CPC targeting to centromeres in addition to the previously identified Bub1-H2A-Sgo2 pathway (15). Genetic analyses of mutations involved in these pathways show that intrapathway double mutations cause no cumulative defects, whereas interpathway double mutations cause synthetic defects in chromosome bi-orientation and cell growth (Fig. 4A). Therefore, the two histone phosphorylations of H3-T3 and H2A-S121 may act in parallel to accomplish targeting the CPC to centromeres. Although either histone mutation h3-TA or h2a-SA causes moderate defects in CPC targeting to centromeres, the double mutant abolishes CPC localization at centromeres, causing a severe bi-orientation defect (Fig. 4, B and C). If centromere targeting of the CPC is the major function of the phosphorylation of H3-T3 and H2A-S121, then the forced localization of the CPC should restore the defects in the h3-TA h2a-SA double mutant or any interpathway double mutant. To test this, we used the Bir1–chromodomain (CD) protein, a fusion of the Bir1 C-terminal end to the CD, which binds to Lys-9-methylated histone H3 principally located at the pericentromeric heterochromatin (fig. S8) (9). Remarkably, Bir1-CD restored cell viability as well as chromosome bi-orientation of h3-TA h2a-SA cells, as it restored viability of all interpathway double mutants tested (Fig. 4, A and C). These results strongly argue that the primary readout of the histone phosphorylation of H3-T3 and H2A-S121 is the localization of the CPC to the inner centromere, a process essential for setting up chromosome bi-orientation. Although H3-pT3 associates with the CPC, H2A-pS121 may associate with shugoshin (15). A recent study has revealed that the Cdk1-dependent phosphorylation of the CPC promotes the binding to shugoshin, and neither protein can fully localize to chromatin without this phosphorylation (12), suggesting that a physical interaction between the CPC and shugoshin creates a synergetic association of these proteins with nucleosomes (Fig. 4D).

Fig. 4

H3-pT3 and H2A-pS121 act synergistically in CPC targeting to the inner centromere. (A) Summary of genetic analyses. (B) The signals of Ark1-GFP expressed from the endogenous promoter were measured in metaphase in the indicated cells. Error bars represent SD (n = 20 cells). ***P < 0.001 (Student’s t test). Scale bar, 2 μm. (C) Serial dilution assay. Frequencies of lagging chromosomes were measured in the indicated cells at anaphase (n > 100 cells). (D) Schematic depiction of the pathways that regulate CPC targeting to centromeres.

Although Bub1 and Hrk1 kinases cooperate in targeting the CPC to centromeres, Bub1 locates at kinetochores (15), whereas Hrk1 locates at cohesin sites along the chromosome (Fig. 3D and fig. S5C). H2A-S121 is phosphorylated, not only in the kinetochore region, but also in the adjacent pericentromeric region, whereas the phosphorylation of H3-T3 is distributed to all heterochromatic regions (Fig. 3D). Crucially, Bir1 is enriched mostly at the pericentromeric region, the intersection of H3-pT3 and H2A-pS121 distributions, and this enrichment is abolished by either hrk1∆ or bub1-KD (a kinase-dead mutant) (Fig. 3, A and D). Residual localization of Bir1 is detected at the noncentromeric heterochromatin regions, which is preserved in bub1-KD cells (Fig. 3D). These results imply that the single histone mark H3-pT3 has a moderate ability to recruit the CPC, which otherwise is enriched in the pericentromeric region by the synergetic action of H2A-pS121.

Immunostaining of HeLa cells with antibodies against H2A-pT120 (equivalent to yeast H2A-pS121), H3-pT3, and Aurora B indicates that the H2A-pT120 signals increase as Bub1 accumulates at kinetochores during prophase, and, concomitantly, the distributions of H3-pT3 and Aurora B narrow into regions adjacent to H2A-pT120 (fig. S9A). A zoomed-in view of prophase and prometaphase chromosomes reveals that H3-pT3 signals localize to the intersister region, whereas H2A-pT120 signals locate along the interkinetochore axis, with signals culminating near the kinetochores (Fig. 5A and figs. S9A and S10). Importantly, Aurora B signals are enriched in the merged region of these signals (Fig. 5A), and this Aurora B localization is disrupted by depleting either phosphorylation of histone by RNA interference (Fig. 5B and fig. S11). These results in HeLa cells, together with those in fission yeast, advocate the notion that the inner centromere in eukaryotes is principally defined by the intersection of two histone marks, H2A-pS121 and H3-pT3, that are mediated by cohesin-associated kinase Haspin/Hrk1 and kinetochore-associated kinase Bub1 (Fig. 5C and supporting online material text).

Fig. 5

Aurora B locates at the intersection of H2A-pT120 and H3-pT3. (A) Chromosome spreads were prepared from HeLa cells and stained with antibodies against H3-pT3, H2A-pT120, and Aurora B. DNA was costained with Hoechst 33342. Prophase and prometaphase chromosomes are shown (see fig. S9B). Magnified images of paired sister chromatids are shown (insets). (B) HeLa cells treated with Haspin or Bub1 siRNAs were arrested at prometaphase by treatment with nocodazole and MG-132 and were examined in chromosome spreads by staining with antibodies against H3-pT3 and H2A-pT120 and Aurora B. More than 90% of spreads in each sample showed the same staining pattern. Magnified images of paired sister chromatids are shown (insets). Scale bars in (A) and (B), 10 μm. (C) Schematic depiction of the definition of the inner centromere.

In HeLa cells, the prophase pathway causes a dynamic narrowing of cohesin localization to the centromere (13, 21). For this redistribution, Bub1 kinase plays an important role by localizing H2A-pT120 and shugoshin to the centromere (15, 22), because human shugoshins play a crucial role in cohesin protection (14, 2224) in addition to binding to the CPC (12). Because Haspin or H3-pT3 localization may also depend on cohesin in HeLa cells (fig. S12), this redistribution of cohesin to centromeres will further attract the CPC-shugoshin, thus constructing a positive feedback loop. Localization interdependencies are observed, not only between the CPC and shugoshin, but also between cohesin and shugoshin in several organisms (9, 16, 21, 2528). Thus, our study uncovers a fundamental molecular network centered on shugoshin-cohesin-CPC, which couples sister chromatid cohesion to chromosome bi-orientation in eukaryotes.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S12

Table S1


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank S. Hauf for critical reading of the manuscript, the Yeast Genetic Resource Centre for yeast strains, and all the members of our laboratory for their valuable support and discussion. This work was supported in part by Japan Society for the Promotion of Science research fellowships (to Y.Y., T.H., and Y.T.); the Global Center of Excellence Program; and a Grant-in-Aid for Specially Promoted Research (to Y.W.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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