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. Author manuscript; available in PMC: 2017 Mar 7.
Published in final edited form as: Dev Cell. 2016 Mar 7;36(5):487–497. doi: 10.1016/j.devcel.2016.02.008

HP1-assisted Aurora B kinase activity prevents chromosome segregation errors

Yusuke Abe 1, Kosuke Sako 1, Kentaro Takagaki 1, Youko Hirayama 1, Kazuhiko SK Uchida 1, Jacob A Herman 2, Jennifer G DeLuca 2, Toru Hirota 1,*
PMCID: PMC4949072  NIHMSID: NIHMS788615  PMID: 26954544

Summary

Incorrect attachment of kinetochore microtubules is the leading cause of chromosome missegregation in cancers. The highly conserved chromosomal passenger complex (CPC), containing mitotic kinase Aurora B as a catalytic subunit, ensures faithful chromosome segregation through destabilizing incorrect microtubule attachments and promoting bi-orientation of chromosomes on the mitotic spindle. It is unknown whether CPC dysfunction affects chromosome segregation fidelity in cancers and, if so, how. Here we show that heterochromatin protein 1 (HP1) is an essential CPC component required for full Aurora B activity. HP1 binding to the CPC becomes particularly important when Aurora B phosphorylates kinetochore targets to eliminate erroneous microtubule-attachments. Remarkably, a reduced proportion of HP1-bound to CPC is widespread in cancers, which causes an impairment in Aurora B activity. These results indicate that HP1 is an essential modulator for CPC function, and identify a molecular basis for chromosome segregation errors in cancer cells.

eTOC Blurb

Improper kinetochore-microtubule attachments can cause chromosome missegregation, but are normally corrected through an Aurora B-dependent mechanism. Abe et al. find that the heterochromatin protein HP1 is required for full Aurora B activity and that HP1 association with the Aurora B complex is widely impaired in cancer cells with chromosomal instability.

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Introduction

The ultimate aim of mitosis is faithful transmission of the genome. Essential in chromosome segregation is that all chromosomes bi-orient on the mitotic spindle so that two daughter cells receive equal sets of chromosomes as they divide. A chronic failure in chromosome segregation leads to aneuploidy, a widespread pathological feature of malignant tumors (for review see Thompson et al., 2010).

The stochastic nature of kinetochore-microtubule interaction inevitably generates incorrect attachments in prometaphase. Release of such error-prone attachments is crucial to ensure fail-safe segregation of chromosomes (Cimini et al., 2001; Thompson and Compton, 2008; Thompson and Compton, 2011), and the activity of Aurora B is essential for this cellular function (for reviews see Kelly and Funabiki, 2009; Carmena et al., 2012). The core of the kinetochore-microtubule attachment interface is provided by sets of three protein complexes called the KMN network, including the Knl1, Mis12 and Ndc80 complexes (for review see Varma and Salmon, 2012). Aurora B-mediated phosphorylation of the Hec1 subunit (the Ndc80 complex), the Dsn1 subunit (the Mis12 complex), and the Knl1 subunit have been implicated in releasing incorrect attachments (DeLuca et al., 2006; Cheeseman et al., 2006; Ciferri et al., 2008; Welburn et al., 2010; DeLuca et al., 2011). The combinatorial phosphorylation of these proteins creates graded levels of microtubule binding stability (Welburn et al., 2010), suggesting that a proper destabilization of microtubule attachments would require sufficient levels of Aurora B activity. Notably, the ability of Aurora B to release incorrect attachments is indeed impaired by partial inhibition of its kinase activity (Cimini et al., 2006). Any reduction in Aurora B activity may therefore cause chromosome missegregation. The regulatory mechanism responsible for such a pathological condition has not yet been identified, however.

The highly conserved chromosomal passenger complex (CPC) represents a functional entity for Aurora B, composed of INCENP, Survivin and Borealin/Dasra. These proteins have specific functions in controlling the localization and activity of Aurora B: Survivin and Borealin/Dasra associate with N-terminus of INCENP and mediate chromosomal association by directly binding to phosphorylated histone tails (Kelly et al., 2010; Wang et al., 2010; Yamagishi et al., 2010). Aurora B interacts with the C-terminus of INCENP through a domain termed the IN-box. This interaction leads to an allosteric activation of Aurora B, involving (auto)phosphorylation of the catalytic activation loop of Aurora B and the TSS-motif in the IN-box (Bishop and Schumacher, 2002; Honda et al., 2003; Kelly et al., 2007). Of the proteins known to associate with the CPC, heterochromatin protein 1 (HP1) is known to directly bind INCENP (Ainsztein et al., 1998; Kang et al., 2011), although the significance of this interaction for CPC function remains unclear.

Here we show that HP1 enhances the enzymatic activity of Aurora B. This HP1-mediated allosteric effect is required to phosphorylate kinetochore substrates to eliminate kinetochore-microtubule mal-attachments and thus to prevent chromosome segregation errors. Our data suggest that HP1 confers increased Aurora B kinase activity by accelerating the rate of the kinase reaction. Remarkably, the significance of this HP1 function is crucially underscored by the finding that a wide-range of cancer cells reveal consistent reduction of HP1-bound CPC, which causally relates to a reduced output of Aurora B activity.

Results

The amount of HP1-bound CPC is affected in cancer cells

Immunofluorescence microscopy demonstrated that HP1 localizes to mitotic centromeres similarly to the CPC (Figure 1A). We set out to examine the interaction of the CPC with HP1 proteins by immunoprecipitating INCENP-containing complexes, and found not only HP1α but also HP1β and HP1γ to co-immunoprecipitate with the CPC from mitotic cell extracts (Figures S1A, B). Specific depletion of any HP1 subtype caused a compensatory increase in the binding of the other HP1 subtypes to INCENP, suggesting that the same binding mode is shared by all three subunits (Figure 1B). We estimated that approximately 10% of the CPC pool is stably bound to HP1 in mitotic HeLa cells (Figure 1C and Figures S1C–H). These results suggested that all three HP1 subtypes may influence CPC function at centromeres in mitosis.

Figure 1. Characterization of HP1 association with the CPC in mitosis.

Figure 1

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(A) HP1 proteins localize to centromeres in mitosis. Immunofluorescence microscopy of mitotic chromosomes from HeLa cells stained for HP1α, HP1β, HP1γ or Aurora B. Scale bar indicates 10 μm.

(B) All three types of HP1 coimmunoprecipitate with the CPC. The CPC from mitotic HeLa cell extracts, with or without depletion of HP1, was immunoprecipitated using INCENP antibodies. Note that HP1 proteins bind to the CPC in a compensatory manner.

(C) The amount of HP1 proteins stably bound to CPC. Quantities of the indicated proteins in immunoprecipitates of CPC from mitotic HeLa cell extracts were estimated by comparing to their recombinant proteins of known concentrations (Figures S1C–H). The proportion of HP1-bound CPC is calculated assuming that “dimerized” HP1 proteins associate with the CPC.

(D) The CPC was immunoprecipitated from mitotic RPE1 and HeLa cell extracts using INCENP antibodies. Comparable amounts of INCENP complexes from both cell lines were assessed for co-immunoprecipitated HP1 proteins. The HP1:INCENP ratio in protein complexes was compared in HeLa and RPE1 cells (The ratio in RPE1 cells is set to 1.0).

(E) Mitotic chromosome spreads from RPE1 and HeLa cells were costained with antibodies to HP1α, HP1β, or HP1γ and INCENP. Relative fluorescence intensities of HP1α, HP1β, or HP1γ, normalized to INCENP, are shown in the histogram. n > 140 centromeres from 4 cells were analyzed. Error bars indicate SEM. Scale bar indicates 10 μm.

The Aurora B-mediated error correction function is found to be more robust in non-transformed diploid retinal pigment epithelial RPE1 cells than in HeLa cells (Salimian et al., 2011). We therefore asked whether the proportion of HP1 bound to CPC in mitotic cells might differ between these two cell lines. Immunoprecipitation assays revealed that the amount of HP1-bound CPC is two- to fourfold higher in RPE1 cells than that in HeLa cells, but is not necessarily proportional to expression levels of HP1 proteins (Figure 1D). To examine if the difference in HP1-bound CPC levels reflects a difference in its enrichment at centromeres, we performed immunofluorescence microscopy of mitotic chromosomes. We found the fluorescence intensities of HP1 at centromeres were significantly lower in HeLa cells than in RPE1 cells, for all three subtypes (Figure 1E). These results imply that cancer cells contain less HP1-bound CPC at centromeres than non-cancerous cells do.

To address if the amount of HP1-bound CPC is consistently reduced in a wide-range of cancers, we extended the investigation into other types of cell lines. Remarkably, we found that all cancer lines tested contained a lower proportion of HP1-bound CPC than non-transformed diploid cell lines (Figure 2A). These observations raise the possibility that reduced HP1 binding to the CPC may be a general feature of cancer cells that is causally related to chromosome segregation errors. The finding that HP1 binding is also reduced in cancer cell lines with modest missegregation rates (Bakhoum et al., 2014; Figure S2) implies that sufficient levels of HP1-bound CPC are required to prevent segregation errors (Figure 2B).

Figure 2. Proportion of HP1-bound CPC is reduced in cancer cells.

Figure 2

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(A) The CPC was immunoprecipitated with INCENP antibodies from mitotic cell extracts of various types of cell lines, and immunoblotted with the indicated antibodies. The amount of co-immunoprecipitated HP1 proteins in RPE1 cells is set to 1.0 in the histogram.

(B) Proportion of HP1-bound CPC is analyzed in cancer cell lines characterized with modest chromosome missegregation rates (HCT 116 and DLD-1) as in (A).

HP1 is required to attain mitotic activity of Aurora B in full

To investigate the functional significance of HP1 binding to the CPC, we first depleted all three HP1 subtypes (HP1αβγ) by RNAi in which the complex formation of CPC was not detectably affected (Figure 3A). When we assessed Aurora B activity by monitoring phosphorylation of histone H3 (Ser10) and CENP-A (Ser7), well-known Aurora B substrates (Hsu et al., 2000; Zeitlin et al., 2001), the extent of these phosphorylations was not grossly affected in HP1αβγ-depleted mitotic cells (Figures 3B and S3A). By contrast, there was a significant reduction in the phosphorylation of Hec1 (Ndc80 complex) and Dsn1 (Mis12 complex), established Aurora B substrates at kinetochores that have been implicated in controlling interaction to microtubules (DeLuca et al., 2006; Cheeseman et al., 2006; Welburn et al., 2010; DeLuca et al., 2011) (Figures 3C and S3B, C). The requirement of HP1 in phosphorylating these kinetochore substrates was mostly pronounced in prometaphase, presumably indicating the period when destabilization of incorrect microtubule attachments typically occurs. Of note, the effect of HP1 was smaller or negligible under conditions in which kinetochores were positioned too close to (i.e., early prometaphase or nocodazole-treated cells; Figures S3D, E) or too far from (i.e., metaphase; data not shown) the CPC at inner centromeres. Because treatment with low concentrations of the Aurora B inhibitor ZM447439 also caused a reduction in Hec1 and Dsn1 phosphorylation, we predicted that the kinase activity of Aurora B is impaired in the absence of HP1. This prediction was also supported by the localization of CPC, which was less clearly defined at the centromere and became partially redistributed along the chromosome arms in HP1αβγ-depleted cells, likewise in cells treated with a low dose of the Aurora B kinase inhibitor (Figures S3F, G).

Figure 3. HP1 is required to attain mitotic activity of Aurora B in full.

Figure 3

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(A) Depletion of HP1 from the CPC. Three HP1 subtypes (HP1αβγ) were simultaneously depleted from HeLa cells using RNA interference (RNAi)-mediated knockdown, and the CPC was immunoprecipitated with antibodies to INCENP.

(B and C) Immunofluorescence microscopy of Aurora B-mediated phospho-proteins. Following HP1 triple knockdown (HP1αβγ) or treatment with either a low dose (0.4 μM) or high-dose (2.0 μM) of Aurora B inhibitor ZM447439 (ZM), HeLa cells were fixed and stained with antibodies to H3 phospho-Ser10, pS10 (B), to Hec1 phospho-Ser44, pS44 and to Hec1 that do not distinguish phosphorylation status (Hec1) Relative fluorescence intensities of Hec1-pS44 are normalized to Hec1, and are summarized in the histogram. n >150 kinetochores from 10 cells were analyzed for each condition; error bars indicate SEM (C). Representative cells in prometaphase are shown. Scale bar indicates 5 μm.

(D) HP1α domain structure, highlighting the conserved mitotic phosphorylation site Ser92 in the hinge domain (left panel). A single alanine mutation at this site, S92A, significantly inhibits its Aurora B-mediated phosphorylation in vitro (right panel).

(E) Antibodies to HP1α phosphor-Ser92 (HP1α-pS92) preferentially immunoprecipitate HP1-bound CPC from mitotic HeLa cell extracts. A serial dilution of the sample indicates that far more HP1 proteins were immunoprecipitated with the CPC with HP1α-pS92 antibodies (left panel). Both CPC populations were assayed for kinase activity (right panel). Note that most part of the kinase activity was sensitive to Aurora B inhibitor (+ ZM).

HP1 binding to INCENP supports the kinase activity of Aurora B

We therefore wished to assess the kinase activity of Aurora B in vitro, and to ask how kinase activity might be affected by HP1. To do this, we immunopurified the HP1α-bound CPC from mitotic extract using phospho-specific HP1 antibodies, pS92 (Figures 3D and S3H, I; Hengeveld et al., 2012), based on the finding that phosphorylated HP1 at Ser92 is enriched in the CPC-bound fraction and predominantly localized at centromeres (Figures S3J–M). As expected, we could differentially enrich CPC pools with or without HP1 by immunoprecipitation with anti-pS92 or anti-INCENP antibodies, respectively (Figures S3N, O). The kinase activity of Aurora B was found to be significantly higher in the former CPC pool than in the latter, consistent with the idea that HP1 positively supports the activity of Aurora B (Figure 3E).

To specifically ask if the binding of HP1 to INCENP affects Aurora B kinase activity, we generated cell lines stably expressing either wild-type (WT) INCENP or a mutant lacking the HP1-binding region (ΔHP) and the 3A mutant, in which three residues required for HP1 binding, PxVxI (x indicates any amino acid), were changed to alanine (Kang et al., 2011; Figure 4A). In cells expressing these mutants in place of endogenous protein (Figures 4A and S4A–D), INCENP-associated Aurora B activities were measured consistently lower than that in WT cells (Figures 4B and S4E). To address the impact of HP1 on Aurora B activity more directly, we set up in vitro kinase assays using purified recombinant proteins (Figures S4G, H). Kinase reactions with INCENP-Aurora B in the presence of HP1 revealed higher efficiency of substrate phosphorylation than in its absence (Figure 4C). Similar results were obtained by the addition of HP1β and HPγ (Figures 4D and S4I). We could reason that this higher phosphorylation level was achieved by direct binding of HP1 to INCENP because the HP1 dimerization-deficient mutant, I165E (Brasher et al., 2000), which does not bind INCENP, had no such effect (Figures 4E and S4J). That addition of HP1α did not enhance the kinase activity if the enzyme lacks the HP1 binding domain (ΔHP1 INCENP-Aurora B) further verifies the significance of the binding (Figure S4K).

Figure 4. HP1 assists the activity of Aurora B by increasing its affinity to substrates.

Figure 4

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(A) Schematic diagram of INCENP mutants (left). The sequence targeted by RNAi, in which RNAi-resistant substitutions were introduced into cDNA, is highlighted in blue. Immunoprecipitation with myc antibodies from mitotic extract prepared from WT or ΔHP INCENP expressing HeLa cells (right). Note that the ΔHP mutant can assemble the complex but largely lacks the ability to bind to all three HP1 subtypes.

(B) Mitotic HeLa cell extracts expressing myc-tagged WT, ΔHP or 3A INCENP (left panel) were used to immunoprecipitate the CPC using antibodies to myc (middle panel) and assayed for Aurora B kinase activity toward GST-Hec1 (1-80) (right panel). The graph shows mean kinase activity of six independent experiments (error bars indicate SD). *P < 0.01, two-tailed Student’s t-test. Note that the ΔHP mutant seemed otherwise functional because the generation of multinuclear cells after INCENP depletion (Hauf et al., 2003; Ditchfield et al., 2003) was rescued by ΔHP INCENP expression (Figure S4F). An in vitro kinase assay with recombinant ΔHP INCENP-Aurora B verifies that Aurora B activity is not compromised (Figure S4L).

(C) Kinase reaction using recombinant proteins. Co-purified INCENP (full length)−Aurora B was used as an enzyme source, in the presence or absence of preincubation with HP1α, and phosphate incorporation on GST-Hec1(1-80) was measured after indicated times of reaction.

(D) INCENP−Aurora B kinase assay was performed following preincubation with all three subtypes of HP1 proteins. Note that HP1α and HP1γ, but not HP1β, are phosphorylated. Enhancement of kinase activity was similarly seen toward histone H3 (Figure S4I).

(E) INCENP−Aurora B preincubated with indicated HP1α proteins (upper panel) were assayed for their kinase activity as in (C) (lower panel). Enhancement of kinase activity was also seen toward histone H3 (Figure S4J).

(F) Two CPC populations, i.e., with or without enrichment of HP1-bound, were analyzed for Aurora B and INCENP phosphorylation status by Phos-tag gel electrophoresis and phosphospecific antibodies (INCENP-pTSS), respectively. (G) Kinetic profile of Aurora B kinase activity in the presence or absence of HP1α with different concentrations of substrate. Phosphate incorporation to GST-Hec1(1-80) was measured and plotted. Experiments were performed in triplicate and data represent mean values ± SEM. Note that the kcat value for the GST-Hec1(1-80) substrates in the presence of HP1α is significantly higher than that in the absence of HP1α.

(H) Binding assay between the kinase and substrate. Co-purified recombinant INCENP full-length and GST-Aurora B were incubated with His-tagged Hec1(1-80) in the presence or absence of indicated HP1α proteins (input), and resulting precipitates with GST-tag were analyzed by immunoblotting (GST pulldown). Graph shows the relative amount of Hec1(1-80)-His in the precipitate from five independent experiments (bars indicate SD). *P < 0.01, two-tailed Student’s t-test.

An allosteric activation of Aurora B by INCENP can be probed by the phosphorylation of Aurora B and INCENP (Bishop and Schumacher, 2002; Honda et al., 2003; Kelly et al., 2007). However, HP1 seems to contribute to the enhancement of kinase activity through different means because HP1 did not alter the phosphorylation levels on either Aurora B and INCENP (Figure 4F). To investigate how HP1 might promote Aurora B phosphorylation of its substrates, we conducted further kinetic analyses to characterize the enzyme. In the presence of HP1α, the Km value, a parameter typically reflecting affinity for the enzyme, revealed a modest decrease for Hec1 (Figure 4G and Supplemental Figure S4M, N), suggesting that HP1α improves substrate affinity for Aurora B. Consistent with this possibility, binding assays detected an increased amount of Hec1 fragment co-precipitated with GST-Aurora B when HP1α is bound to INCENP (Figures 4H and S4O–Q). More dramatically, we found the kcat value, which reflects the number of substrate molecules processed by the enzyme every second, was two-fold higher in the presence of HP1α than its absence (Figure 4G). These kinetic properties allow us to predict that HP1 allosterically enhances the activity of Aurora B/INCENP complex by increasing the rate of reaction, and to a lesser extent, by increasing the affinity of Aurora B/INCENP for its substrates. Notably, we found HP1 alters Aurora B activity toward multiple substrates including Dsn1 as well as nucleosomal histone H3 (Figures S4I, J, R–T), suggesting that this HP1-mediated allosteric regulation of the CPC is not restricted to Hec1 but applies more widely to other Aurora B substrates.

The HP1-mediated allosteric regulation is essential for Aurora B-mediated phosphorylation of kinetochore substrates

To address the relevance of HP1-mediated allosteric regulation of the CPC in cells, we examined phosphorylation levels of Aurora B substrates in the presence or absence of HP1 binding. In ΔHP INCENP-expressing cells, there was a marked reduction in phosphorylation levels of Hec1 (Figure 5A) and Dsn1 (shown in Figure 6D). We found the efficiency of H3 Ser10 phosphorylation was also impaired in an experimental condition where mitotic cells were released from Aurora B inhibitor treatment (Figure S5A), but was not detectably affected in unperturbed mitoses (as in HP1αβγ-depleted cells; Figure 3B). These results suggest that deprivation of HP1 from CPC affects kinase activity of Aurora B, which significantly perturbs phosphorylation of Aurora B substrates at kinetochores.

Figure 5. The HP1-mediated allosteric regulation is essential for Aurora B-mediated phosphorylation of kinetochore substrates.

Figure 5

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(A) Fluorescence intensities of Hec1-pS44 in WT or ΔHP INCENP-expressing HeLa cells. Relative fluorescence intensities are summarized as in Figure 3C. n >150 kinetochores from 10 cells for each condition were analyzed; error bars indicate SEM.

(B) The centromere localization of all three subtypes of HP1 proteins depends on their binding to INCENP. Chromosome spreads from mitotic INCENP WT or ΔHP expressing HeLa cells, with or without INCENP depletion, were stained with antibodies to HP1α, HP1β and HP1γ. Scale bar indicates 10 μm.

(C) Immunofluorescence microscopy of Hec1-pS44 in HeLa cells expressing CENP-B-fused INCENP (CB-INCENP-EGFP), either WT or ΔHP mutant, with or without RNAi treatment to deplete endogenous INCENP. Fluorescence intensities of Hec1-pS44 are summarized in the histogram. n >150 kinetochores from more than 8 cells were analyzed; error bars indicate SEM; scale bar, 5 μm.

Figure 6. Sufficient levels of Aurora B activity in non-transformed cells depends largely on HP1-mediated allosteric regulation of the CPC.

Figure 6

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(A) INCENP-associated kinase activity in mitotic RPE1, HT-1080, and U2OS cells. Kinase activity is analyzed for equivalent amounts of INCENP immunoprecipitated from three cell lines. Graph shows mean kinase activity from six independent experiments (error bars indicate SD). *P < 0.01, two-tailed Student’s t-test.

(B and C) Dsn1 phosphorylation levels are elevated in non-transformed cells than in cancer cells. Immunofluorescence microscopy of Dsn1-pS100 and Hec1 (B) or Dsn1 and Hec1 (C) in cancer (HeLa and U2OS) or non-transformed (RPE1 and TIG-3) cell lines. Relative fluorescence intensities of Dsn1-pS100 or Dsn1, both being normalized to Hec1, are shown in the histogram. n > 250 kinetochores from more than 10 cells for each condition were analyzed. Error bars indicate SEM; scale bar, 5 μm.

(D) Immunofluorescence microscopy of Dsn1-pS100 in WT or ΔHP INCENP-expressing HeLa and RPE1 cells, in the presence or absence of endogenous INCENP. Relative fluorescence intensities are summarized as in (B). n > 300 kinetochores from 14 cells for each condition; error bars indicate SEM; scale bar 5μm.

The centromeric localization of HP1 was undetectable in cells expressing ΔHP1 INCENP (Figure 5B). This indicates that, contrary to the regulation in interphase in which HP1 anchors CPC to chromatin (Kang et al., 2011), CPC anchors HP1 to centromeres in mitosis, and thus the complex containing ΔHP INCENP could indeed well associate to chromosomes (Figure S4D). Nonetheless, reflecting impaired activity of Aurora B, the centromeric localization of CPC becomes less defined when HP1 cannot bind to INCENP (as seen in low-dose Aurora B inhibitor treatment; see Figure S3G). Accordingly, reduced phosphorylation levels of kinetochore substrates could be caused as much by diffuse localization of Aurora B as by impaired activity of Aurora B per se. To distinguish between these two possibilities, comparable amounts of CPC were forced to localize to centromeres by fusing INCENP to CENP-B, and we then asked if the existence of HP1 in the complex affects the level of phosphorylation. We found that tethering of the CPC does not enhance Aurora B-mediated phosphorylation of kinetochore substrates unless the complex contains HP1, indicating that lack of HP1 primarily impairs CPC activity in cells as it did in vitro (Figures 5C and S5B–D).

Reduced levels of Aurora B activity in cancer cells

The finding that HP1 binding to CPC is required to convey the activity of Aurora B in full raises the possibility that cancer cells have lower Aurora B activity than diploid, non-transformed cells do. Consistent with this idea, cancer cell lines HT-1080 and U2OS, with reduced levels of HP1-bound CPC, revealed lower Aurora B kinase activity than non-transformed RPE1 cells when comparable amount of CPC is assessed biochemically (Figure 6A). To ask if this difference in Aurora B activity affects phosphorylation of its kinetochore substrates, we made a comparison of phosphorylation levels between non-transformed and cancer cell lines. While the difference was not absolutely clear for Hec1, phosphorylation of Dsn1 in prometaphase revealed higher levels in non-transformed cells than in cancer cells (Figures 6B, C).

If the difference in Aurora B-mediated phosphorylation of Dsn1 between non-transformed versus cancer cells is attributable to the difference in the amount of HP1-bound CPC, deprivation of HP1 from the complex must cause greater changes in the former than the latter. Consistent with these predictions, we found that phosphorylation levels were significantly reduced in RPE1 cells when they express ΔHP1 mutant version of INCENP, whereas the change was modest in HeLa cells (Figure 6D). Importantly, the result that deprivation of HP1 resulted in a decline of phosphorylation to comparable levels in both cell lines is consistent with the idea that sufficient phospho-regulation of Dsn1 depends primarily on the HP1-mediated allosteric regulation of Aurora B (Figure 6D).

HP1 binding to CPC is required to prevent chromosome missegregation in non-transformed cells but not in cancer cells

Finally, to address the impact of HP1 binding to CPC on mitotic fidelity, we examined the emergence of lagging chromosomes when HP1-CPC binding is inhibited. For this, endogenous INCENP was depleted in subclones that stably express either WT or ΔHP INCENP derived from various cell lines (Figures S6A–G), and anaphase cells were examined for chromosome missegregation. In spontaneously progressing (unperturbed) mitotic RPE1 cells, as well as in non-transformed TIG-3 diploid fibroblasts, the missegregation rate was ~5% in control or WT INCENP-expressing cells. Under these conditions, ΔHP INCENP expression resulted in missegregation in more than 10% of cases (> twofold increase over controls; Figure 7A). In anaphase following monopolar spindle formation induced by an Eg5 inhibitor Monastrol, the basal missegregation rate was ~10%, and that of ΔHP expressing cells was increased to ~20% (Figure S7A).

Figure 7. HP1 binding to the CPC is required to prevent chromosome segregation errors in non-transformed cells.

Figure 7

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(A) Cells stably expressing WT or ΔHP INCENP were generated in non-transformed (RPE1 and TIG-3; Figures S6A, B) and cancer (HeLa, U2OS, HT-1080, A549, HCT 116 and DLD-1; Figures S6C–G) cell lines. Chromosome missegregation rates in anaphase were calculated with or without knockdown of endogenous INCENP from more than 200 cells in unperturbed mitoses or in cells that had been released from Monastrol treatment (Figure S7A). The histogram shows the mean ± SD of three independent experiments. Similar results were obtained in two other independent subclones of RPE1 cells expressing ΔHP INCENP (Figure S7B–D), and also in subclones of RPE1 (Figure S7E–G) and HeLa (Figure S7H; characterized in Figure 4B) expressing 3A INCENP.

(B) CPCs were immunoprecipitated using antibodies to INCENP from mitotic extracts of parental HeLa cells and from cells engineered to conditionally overexpress GFP-tagged HP1α, and blotted for HP1 proteins. Graph shows the relative amount of HP1 referenced to INCENP in the precipitates. The amount of endogenous HP1 proteins in parental cells was set to 1.0.

(C) A diagram depicting composition of the CPC in non-transformed and cancer cell lines. Non-transformed cells contain more HP1-bound CPC than cancer cells do, and thus the former has higher Aurora B kinase activity than the latter. The HP1-bound CPC is particularly important to phosphorylate kinetochore substrates and thus to destabilize incorrect microtubule attachments in non-transformed cells. In wide range of cancer cells, however, proportion of HP1-bound CPC and Aurora kinase B activity are reduced beyond the threshold required to prevent chromosome segregation errors.

(D) Generation of transformed RPE1-67R cell lines (upper panel). Mitotic extracts from parental RPE1 cells and transformed RPE1-67R cells (input) were analyzed as in (B). Phospho-H3 Ser10 bands confirm the mitotic state. The amount of HP1 in INCENP complexes in RPE1 cells is set to 1.0 in the histogram.

Strikingly, however, ΔHP INCENP expression had no detectable effect on mitotic fidelity in a series of cancer cell lines: missegregation was seen in 8–25% of unperturbed mitoses and in 20–35% after monopolar configuration in all conditions examined (Figure 7A). In light of above results in Figure 6, we reasoned that the amount of HP1-bound CPC is already reduced below the threshold required to prevent segregation errors in cancer cells, such that a further reduction has no effect. To test this idea, we asked whether missegregation could be alleviated by HP1 overexpression and increasing the amount of HP1-bound CPC. Although overexpressed HP1α was efficiently incorporated into the CPC, this in turn caused compensatory dissociation of endogenous HP1 proteins from the complex (Figures 7B and S7I, J), and therefore failed to increase the amount of HP1-bound CPC and rescue segregation errors (Figure S7K). These observations imply that cancer cells have an inherent reduction in HP1-binding capacity of CPC (Figure 7C). To address if malignant transformation affects the HP1-binding capacity, we engineered transformed-RPE1, or RPE1-67R cells. In these cells, the amounts of immunoprecipitated HP1 proteins were decidedly decreased, suggesting that reduced amount of HP1-bound CPC is acquired during cellular transformation (Figure 7D).

Discussion

Aurora B yields higher activity when the CPC is bound to HP1

Our data indicate that, instead of facilitating the activation process, HP1–CPC interaction contributes to Aurora B activity by increasing the rate of reaction (Figure 4G). To explain how mechanistically HP1 alters the rate of Aurora B substrate processing will require structural studies of the complex. As the region of INCENP covering the HP1-binding site is predicted to be intrinsically unstructured according to DICHOT database (Fukuchi et al., 2009), a possible speculation is that HP1 binding affects INCENP conformation in a way that accelerates Aurora B’s substrate processing.

The HP1-mediated allosteric regulation of the CPC seems to apply to multiple Aurora B substrates. When assessed in vitro using recombinant proteins, Aurora B’s affinity to Hec1, Dsn1 and nucleosomal histone H3 were all comparably elevated by HP1 (Figures 4H, S4R and S4T), and thus HP1 conferred additional activity on Aurora B to similar extent (Figures 4E and S4J). However, disrupting HP1-mediated regulation in cells revealed diverse effects; phosphorylation of kinetochore proteins, Hec1 and Dsn1, were severely impaired (Figures 3C and S3C), whereas that of histone H3 were not detectably affected in unperturbed mitoses (Figure 3B). What might be possible explanations for these different sensitivities to reduced Aurora B activity? Phosphorylation of kinetochore substrates is in general unstable presumably due to their low abundance, distance-dependent accessibility of kinase (Liu et al., 2009; Wang 2011a) and existence of counteracting phosphatase (Wurzenberger and Gerlich, 2011; Funabiki and Wynne, 2013). These conditions presumably explain why higher reaction rates achieved by HP1 are required to ensure phosphorylation of kinetochore substrates.

The finding that HP1 proteins are displaced from centromeres beyond detectable levels when cells express otherwise functional CPC but for HP1 binding, ΔHP1-INCENP, arguably indicates that anchoring of HP1 to CPC is the prime mechanism to enrich HP1 to centromeres (Kang et al., 2011; Figure 5B). However this is not to exclude the possibility that CPC components other than INCENP may interact with HP1, including Borealin (Liu et al., 2014). Consistent with this, we found that a minor amount of HP1 co-immunoprecipitated with the CPC in those cells expressing ΔHP1-INCENP mutant (Figures 4A, B).

The HP1-binding capacity is impaired in a wide range of cancer cells

Despite the fact that loss of chromosome segregation fidelity is widespread in malignancies, surprisingly few gene alterations associated with mitotic control have been identified (Wood et al., 2007; Jones et al., 2008; Kandoth et al., 2013). The findings that the decrease of HP1-bound CPC recapitulates the pathological condition in cancer cells importantly provide a molecular basis for chromosome segregation errors in cancers. Components of the CPC are reported to be overexpressed in a number of solid tumors (for reviews see Hindriksen et al., 2015), and as opposed to what one might assume from those observations, the activity of Aurora B, especially for that required to eliminate error-prone microtubule attachment, is reduced in cancer cells.

What would be the relevant Aurora B substrate at kinetochores whose hypo-phosphorylation impairs error correction function in cancer cells? Remarkably, the difference of Aurora B activity between non-transformed and cancer cells was mirrored by the level of Dsn1 phosphorylation (Figure 6B). Given that phosphorylation of Dsn1 has a decisive role in Aurora B-mediated control of kinetochore-microtubule interaction (Welburn et al., 2010), reduced levels of Dsn1 phosphorylation must have a major impact on chromosome segregation defect in cancers. By contrast, the difference was not visibly reflected by phosphorylation on Hec1 N-terminal tail when one out of nine phospho-sites was probed (data not shown). This was unexpected as Hec1 phosphorylation of the N-terminal tail will have a great effect on microtubule binding activity. Nevertheless, the current model suggests that only a few residues are transiently phosphorylated at any given time during error correction (Zaytsev et al., 2014), and this mode of regulation seems to be sufficiently mediated by the activity of Aurora B with reduced proportion of HP1-bound CPC in cancer cells.

Surprisingly, unlike in non-transformed cells, eliminating the HP1 binding did not affect missegregation rates in cancer cells (Figure 7). In fact, partial inhibition of Aurora B with ZM447439 inhibitor increased missegregation rates in non-transformed cells, but not in cancer cells (data not shown). These observations in turn allow us to hypothesize a threshold level of Aurora B function is required to prevent segregation errors. How cells ensure this threshold level and what condition leads to an inherent reduction in the HP1-binding capacity of CPC below the threshold are important following questions for the future. In light of the multiple positive-feedback mechanisms proposed to regulate CPC loading onto centromeres (Salimian et al., 2011; Wang et al., 2011b; Qian et al., 2013; van der Horst and Lens 2014), we consider it plausible that an inherent limitation in HP1-binding capacity in cancers is the consequence of a system-level deficiency in Aurora B control.

Experimental Procedures

Cell culture, siRNA transfection, immunoblot, and immunofluorescence were performed using standard methods. Stable cell lines were generated by plasmid transfection or lentivirus infection and selection by antibiotics. For other details, see Supplemental Experimental Procedures.

Recombinant Proteins

To prepare INCENP full-length and GST-Aurora B, coexpression vector pGEX6P2-human Aurora B-rbs-human INCENP was generated (modified from pGEX6P2-Xenopus Aurora B 60-361-rbs-Xenopus INCENP 790-856; Sessa et al., 2005). All complexes were expressed in E. coli strain Rosetta-gami B (DE3) and purified identically. Detailed methods were described in Supplemental Experimental Procedures.

In Vitro Kinase Assays

Kinase assays were carried out in kinase buffer (20 mM Tris-HCl, 50 mM MgCl2, 100 mM NaCl, 20 μM ATP, pH7.5) containing 0.2 mCi/ml [γ-32P]-ATP with 0.25 mg/ml (8 μM) GST-Hec1 (1-80) (or with 1, 2, 4, 8, 16, 32 and 64 μM GST-Hec1 (1-80) in Figure 4G) or 0.1 mg/ml (6 μM) unmodified recombinant histone H3 (New England Biolabs) as substrates. Reactions were incubated for 20 min (or 5 min intervals in Figure 4C or 10 min in Figure 4G) at 30 °C with 20 nM INCENP and 120 nM GST-Aurora B proteins in the presence or absence of 1 μM HP1 proteins. In Figure S4M, 8 μM GST-Hec1(1-80) was combined with 25–800 μM ATP. The reaction was terminated by the addition of Laemmli sample buffer and boiling for 4 min. The phosphorylation of GST-Hec1 (1-80) or H3 were visualized with Typhoon scanner (GE Healthcare) and quantified with liquid scintillation counting. Values for the Michaelis-Menten constant (Km) were calculated using the Lineweaver-Burke double reciprocal plot. For kinase assay using immunoprecipitation samples, see Supplemental Experimental Procedures.

GST Pull-Down Assay

Hec1(1-80)-His, nucleosomal histone H3, or Dsn1(51-150)-His binding assays were carried out in Pull-down buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween-20, 1 mM DTT, 20 μM ZM447439, pH7.5) containing 50 nM INCENP full-length and 300 nM GST-Aurora B complexes (or 500 nM GST as control) and 1.8 μM Hec1(1-80)-His, 1.0 μM Dsn1(51-150)-His, or 0.2 μM Recombinant Nucleosomes (H3.1) (Active Motif) proteins in the presence or absence of 0.6 μM HP1α WT or I165E mutant proteins, and then glutathione sepharose 4B beads were incubated for 1h at 4°C. The beads were washed three times with Pull-down buffer. Samples were analyzed by immunoblot.

Immunofluorescence

Cells were plated onto coverslips. For Hec1-pS44 staining, cells were fixed through incubation with 4% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 7.0) for 20 s at room temperature (r.t.), followed by incubation in PHEM containing 0.5% Triton X-100 for 5 min at r.t.. Cells were fixed 20 min 4% paraformaldehyde in PHEM for 20 min at r.t., then treated with PHEM containing 1% Triton X-100 for 5 min at r.t.. Cell were incubated with 3% BSA in TBS containing 0.01% Triton X-100 (TBS-T) as a blocking agent for 1 h at r.t. and incubated with the appropriate antibodies diluted in 3% BSA in TBS-T. For Dsn1-pS100 staining, cells were incubated with 1% Triton X-100 and 100 nM microcystin in PHEM for 5 min at 37 °C, followed by fixed with 2% PFA in PHEM for 20 min at r.t.. After fixation, cells were incubated with 3% BSA in TBS-T for 1 h at r.t. and incubated with the appropriate antibodies diluted in 3% BSA in TBS-T. Alternatively cells were fixed through incubation with 4% paraformaldehyde in PBS for 10 min at r.t., permeabilized using 0.2% Triton X-100 in PBS for 5 min at r.t., then treated with 3% BSA in PBS containing 0.01% Triton X-100 (PBS-T) for 1 h at r.t. and incubated with the appropriate antibodies diluted in 3% BSA in PBS-T. Antibodies using immunofluorescence staining and a method for quantification of immunofluorescence signals, see Supplemental Experimental Procedures.

Highlights.

  • The HP1-binding capacity of the CPC is impaired in a wide range of cancer cells

  • HP1 binding to the CPC is required for full Aurora B activity

  • HP1-mediated full Aurora B activity ensures phosphorylation of kinetochore substrates

  • HP1 is an essential modulator of CPC preventing chromosome segregation errors

Acknowledgments

We are grateful to Masaki Inagaki for phospho-INCENP antibodies, to Andrea Musacchio for plasmids, to Eiji Hara for cell lines, to Hitoshi Kurumizaka and Yuji Tanno for technical help, to Kota Nagasaka and Kazuki Kumada for critical suggestions. Y.A. acknowledges a fellowship from Japanese Society for the Promotion of the Science (JSPS). This work was supported by research grants from JSPS and the Ministry of Education, Culture, Sports and Technology of Japan (MEXT) to T.H., and by National Institutes of Health grant R01GM088371 to J.G.D.

Footnotes

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Author Contributions

Y.A., J.G.D and T.H. were responsible for experimental design, data interpretation and writing the manuscript. Y.A., K. S., K.T., Y.H., K.S.K.U conducted the experiments. J.A.H. generated RPE1-67R cell lines.

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