QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 17, Issue 7, 3232-3241, July 2006

This Article Abstract Full Text (PDF) Supplemental Material All Versions of this Article: E05-09-0906v1 17/7/3232    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Terada, Y. Search for Related Content PubMed PubMed Citation Articles by Terada, Y.

Aurora-B/AIM-1 Regulates the Dynamic Behavior of HP1 at the G₂–M Transition

Yasuhiko Terada^*,

*Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455; and Department of Molecular Biology, Louis Pasteur Center for Medical Research, Kyoto 606, Japan

Submitted October 3, 2005; Revised April 11, 2006; Accepted April 27, 2006
Monitoring Editor: Kerry Bloom

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation and undergoes large-scale, progressive dissociation from heterochromatin in prophase cells. However, the mechanisms regulating the dynamic behavior of HP1 are poorly understood. In this study, the role of Aurora-B was investigated with respect to the dynamic behavior of HP1. Mammalian Aurora-B, AIM-1, colocalizes with HP1 to the heterochromatin in G₂. Depletion of Aurora-B/AIM-1 inhibited dissociation of HP1 from the chromosome arms at the G₂–M transition. In addition, depletion of INCENP led to aberrant cellular localization of Aurora-B/AIM-1, but it did not affect heterochromatin targeting of HP1. It was proposed in the binary switch hypothesis that phosphorylation of histone H3 at Ser-10 negatively regulates the binding of HP1 to the adjacent methylated Lys-9. However, Aurora-B/AIM-1-mediated phosphorylation of H3 induced dissociation of the HP1 chromodomain but not of the intact protein in vitro, indicating that the center and/or C-terminal domain of HP1 interferes with the effect of H3 phosphorylation on HP1 dissociation. Interestingly, Lys-9 methyltransferase SUV39H1 is abnormally localized together along the metaphase chromosome arms in Aurora-B/AIM-1–depleted cells. In conclusion, these results showed that Aurora-B/AIM-1 is necessary for regulated histone modifications involved in binding of HP1 by the N terminus of histone H3 during mitosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chromosomal passenger proteins are characterized by the unique property of translocation from the chromosome to the central spindle at the onset of anaphase (reviewed in Adams et al., 2001a; Terada 2001). In G₂ and prophase, chromosomal passengers are localized on the chromosome, whereas during prometaphase and metaphase they translocate to the inner centromeres. During anaphase the chromosomal passengers transfer to the spindle midzone where they eventually become part of the midbody (Earnshaw and Cooke, 1991). At the onset of anaphase, separase cleaves the cohesin protein into separate sister chromatids and activates at the same time the phosphatase Cdc14, which dephosphorylates Inner Centromere Protein (INCENP) and triggers its relocalization to the spindle midzone (Pereira and Schiebel, 2003). Recent results show that Aurora-B, together with INCENP and Survivin/BIR, is part of a chromosomal passenger complex. Aurora-B binds to Survivin and the C-terminal domain of INCENP (reviewed in Adams et al., 2001b; Terada 2001). It is of particular interest that all metazoan INCENPs as well as the INCENP homologue of Saccharomyces cerevisiae (Sli5p) share a highly conserved motif near the C terminus, the so-called IN-box (INCENP conserved box) (Adams et al., 2000), which is required for interaction with Aurora-B. Disruption of Aurora-B, INCENP, or Survivin/BIR function leads in several species to severe defects in chromosome segregation and cytokinesis (Tatsuka et al., 1998, Terada et al., 1998, Giet and Glover, 2001). In Drosophila cells and. elegans embryos that lack INCENP or Survivin, Aurora-B cannot properly localize to the kinetochores and midbody (Speliotes et al., 2000; Adams et al., 2001b). Similarly, in Drosophila cells, depletion of Aurora-B by double-stranded RNA-mediated interference (RNAi) leads to aberrant localization of INCENP (Adams et al., 2001b). These data indicate that INCENP is required for the proper localization of Aurora-B. Conversely, Aurora-B regulates the proper localization of INCENP by an unknown mechanism. Aurora-B is also responsible for the phosphorylation of histone H3 at Serine 10 (Hsu et al., 2000, Murnion et al., 2001). In addition, in some eukaryotes, the N-terminal tail of histone H3 is also phosphorylated during mitosis and meiosis. Phosphorylation at Serine 10 has been reported to be involved in proper chromosome condensation and segregation (Wei et al., 1999). In Tetrahymena, mutation of Serine 10 has been reported to lead to defects in condensation, thus indicating a correlation between phosphorylation at Serine 10 and chromosome condensation and segregation (Hsu et al., 2000). However, recent reports suggest a poor correlation between H3 phosphorylation and condensation levels in Drosophila S2 cells, Xenopus, and budding yeast (Adams et al., 2001b; Lavoie et al., 2002, MacCallum et al., 2002). Thus, the exact role of H3 phosphorylation in mitotic progression remains elusive (Hans and Dimitrov, 2001).

Heterochromatin protein 1 (HP1), a chromosomal protein originally identified because of its localization in the heterochromatin in the polytene nuclei of Drosophila, is composed of two related functional domains, an amino-terminal chromodomain (CD) (chromatin organization modifier) and a carboxy-terminal chromo shadow domain (CSD) (Aasland and Stewart, 1995, Bannister et al., 2001). HP1 functions as a chromatin organizer. Some of the molecular events leading to HP1-induced heterochromatin formation have recently been resolved due to the discovery of histone methyltansferase activity associated with the Su(var)3–9 in Drosophila, and in homologues from fission yeast (Clr4) to humans (SUV39HI). Notably, the H3 Lys-9 methyl epitope induced an affinity for heterochromatin in HP1 proteins. This epigenetic signal was recognized through the CD of the protein (Jenuwein and Allis, 2001). In addition, Fischle and Allis have proposed a binary switch hypothesis, which suggests that phosphorylation of Serine 10 negatively regulates the binding of HP1 to the adjacent methylated Lys-9 of histone H3 (Fischle et al., 2003). Indeed, very recently it was shown that inhibition of H3 phosphorylation at Serine 10 inhibited the dissociation of HP1 (Hirota et al., 2005, Fischle et al., 2005). These observations suggest that H3 S10 phosphorylation causes displacement of HP1 from the heterochromatin at the onset of mitosis. However, it was also reported that H3 phosphorylation alone is insufficient for HP1 dissociation and that acetylation of Lysine 14 is required to prevent binding of HP1 to H3 (Fass et al., 2002. Mateescu et al., 2004). These data suggest that in addition to H3 phosphorylation another factor may regulate HP1 dissociation in vivo.

In metaphase cells, HP1 is localized at the centromeric heterochromatin that contains histone H3 protein highly methylated at Lysine 9. Loss-of-function mutations in the HP1 gene will lead to a number of structural defects in mitotic chromosomes, including lack of condensation, and segregation and telomere fusions (reviewed in Eissenberg and Elgin, 2000). This suggests that HP1 is also essential for mitotic progression. In the G₂ phase, HP1 is associated with heterochromatin, but it progressively dissociates from it at the G₂–M boundary, concomitant with histone H3 phosphorylation (Hendzel et al., 1997; Murzina et al., 1999; Sugimoto et al., 2001; Fass et al., 2002). Currently, the mechanisms contributing to the dynamic behavior of heterochromatin during mitosis are poorly understood. Many of the proteins interacting with HP1, the CAF-1 subunit p150 (Murzina et al., 1999), Ku70 (Song et al., 2001), and the lamin B receptor (Ye and Worman, 1996), bind to CSD of HP1, but interaction of lysine 9-methylated histone H3 with the CD region (Lachner et al., 2001) and INCENP with the hinge region (Ainsztein et al., 1998) have also been documented.

The objective of the present study was to elucidate the mechanism of HP1 protein dissociation from the chromosome arms at the G₂–M transition. Transfection experiments using Aurora-B/AIM-1 small interfering RNA (siRNA) or the kinase-negative form of Aurora-B/AIM-1 revealed that the kinase activity of Aurora-B/AIM-1 is required for the dissociation of HP1 from the chromosome arms in mitotic cells. This indicates that histone H3 phosphorylation is required for HP1 dissociation from the chromosome arms. In addition, it was shown that INCENP contributes to the proper localization of Aurora-B/AIM-1 in heterochromatin. Last, it was shown that Aurora-B/AIM-1 regulates the localization of SUV39H1. The implications of these findings for heterochromatin organization are discussed.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmid Construction
FLAG-tagged expression vectors (pEFhyg I carrying the EF1 promoter; Terada et al., 1995) for wild-type rat Aurora-B/AIM-1 (WT) and for its dominant negative form (K106R) have been described previously (Terada et al., 1998). The entire coding sequence of human histone H3 cDNA was isolated by reverse transcription-PCR using HeLa mRNA as template. The (S10A) mutation in histone H3 was introduced by site-directed mutagenesis of pCMV C-terminal hemagglutinin (HA)-H3 wild type, using QuikChange (Stratagene, La Jolla, CA). Histone H3 S10A was generated using the following primers: sense, 5'-GCAGACTGCCCGCAAAGCGACCGGTGGTAAAG-3' and antisense, 5'-CTTTACCACCGGTCGCTTTGCGGGCAGTCTGC. Human T7-HP1 (pCDNA3) and full-length His₆-HP1 was generously provided by I. Lee (Department of Biochemistry, University of Ulsan College of Medicine, Ulsan, Korea). The chromodomain of HP1 (residue 15–72) was also expressed using the pRSET-A-His₆ vector. Green fluorescent protein (GFP)-HP1 was generated from full-length human HP1 cDNA, which was cloned into the BamHI and XbaI sites of the pEGFP-C1 vector (Clontech, Mountain View, CA) for expression of HP1 as a GFP fusion protein in mammalian cells. FLAG-SUV39H1 (kindly provided by M. L. Cleary, Department of Pathology, Stanford University School of Medicine, Stanford, CA) was cloned into the pGEX vector (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) to obtain the glutathione-Sepharose fusion protein encoding glutathione S-transferase (GST)-SUV39H1 (3–412) and GST-SUV39H1 delta N (82–412).

Generation and Purification of GST and His₆ Fusion Proteins
Recombinant proteins were expressed in 1-l cultures of Escherichia coli strain BL21 and solubilized in 10 ml of radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 5 mM EDTA, 1% NP-40, and 0.5% sodium deoxycholate) containing a full set of protease inhibitors (Roche Diagnostics, Indianapolis, IN) and lysozyme (Sigma-Aldrich, St. Louis, MO) by freeze-thawing in liquid N₂, followed by sonication. Soluble proteins were cleared by centrifugation, purified with 400 µl of glutathione-Sepharose beads (GE Healthcare) or Ni/NTA beads (QIAGEN, Valencia, CA), and washed in RIPA buffer. Coomassie staining of SDS-PAGE gels was used to determine protein concentration.

Antibodies
The following antibodies were used: polyclonal and monoclonal anti-AIM-1 (used at a dilution of 1:200; N12 at a dilution of 1:500; BD Biosciences Transduction Laboratories, Lexington, KY); monoclonal Aurora-A (used at a dilution of 1:200; Cell Signaling Technology, Beverly, MA); polyclonal anti-INCENP (used at 1:100; gift from W. C. Earnshaw, Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland); polyclonal anti-H3, dimethyl K9, and pSer-10 histone H3 antibodies (Upstate Biotechnology, Lake Placid, NY); HP1 (Mab3446, Chemicon International, Temecula, CA); polyclonal and monoclonal mouse anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, and BAbCO, Richmond, CA), polyclonal and monoclonal mouse anti-FLAG (Sigma-Aldrich); and anti-SUV39H1 (MG44) monoclonal antibody (gift from M. L. Cleary).

Antibody Staining and Microscopy
HeLa or Chinese hamster ovary (CHO) cells expressing enhanced green fluorescent protein (EGFP)-HP1 were washed with phosphate-buffered saline (PBS), followed by swelling in a hypotonic solution of 37.5 mM KCl containing proteinase inhibitors, and fixed in 4% (wt/vol) paraformaldehyde in PHEM buffer (Cimini et al., 2003). Cells were subsequently permeabilized for 10 min with 0.5% Triton X-100 in PHEM buffer and blocked for 1 h with 5% goat serum. Methanol-fixed cells were rehydrated with 0.05% Tween 20-containing PBS and treated with a mixture of primary antibodies. GFP fluorescence was directly observed by epifluorescence microscopy. Coverslips were incubated with 2–5 µg/ml 4,6-diamidino-2-phenylindole (DAPI). Cells were analyzed using a Nikon Eclipse microscope. To determine HP1 or SUV39HI fluorescence at the centromeres or chromosome arms, the same camera settings were used for all digital images. The best in-focus image of a centromere was determined visually, and, based on the CREST signal, a region corresponding to the kinetochore was generated using Adobe Photoshop software (Adobe Systems, Mountain View, CA). The mean fluorescence intensity of the region was then recorded together with the mean fluorescence intensity of a randomly selected area around the chromosome arm outside the kinetochore and was quantified using MetaMorph software version 6.1 (Molecular Devices, Sunnyvale, CA). To localize either HP1 or SUV39HI at the centromere or the chromosomal arm, the average intensity values (at least 5 areas) of HP1 or SUV39HI around the centromeres or chromosome arms were calculated for each experimental condition. Cells with centromeric HP1 or SUV39HI were identified as those that displayed 5 times higher fluorescence intensity at the kinetochores than at the chromosome arms. Results were compared using a statistical t test.

Cell Line Establishment and Live Cell Microscopy
HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). CHO cells were cultured in Ham’s F-10 supplemented with 10% FBS. PEGFP-C1-HP1 and/or FLAG-tagged SUV39H1 and puromycin-resistant plasmid (10:1) was transfected into CHO1 or HeLa with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The cells were selected with G418 or puromycin added to the growth medium 48 h after transfection. Resistant cells were selected. Selection was over a period of 2 wk with regular medium changes. Several G418- or puromycin-resistant colonies were picked and expanded for immunostaining. For live cell microscopy, after transfection with FLAG- Aurora-B/AIM-1 KR or control vector into cells expressing GFP-HP1, the cells were grown directly on glass coverslips. Cells were maintained during imaging in DMEM plus 10 mM HEPES buffer in a Rose chamber on a heated stage at 37°C (Bionomic Controller BC-100; 20/20 Technologies, Wilmington, NC). Cells were imaged at 5-min intervals on a Nikon TE300.

Cell Cycle Synchronization and Transfection
For cell cycle synchronization, G₁/S phase HeLa cells were obtained from a double-thymidine block as described previously. Under these conditions, HeLa cells will arrest in early S phase (Heintz et al., 1983, Pines and Hunter, 1989); S-phase HeLa cells were obtained by releasing cells synchronized by a double-thymidine block into fresh medium for a period of 2 h; M-phase HeLa cells were obtained from a thymidine/nocodazole block.

RNAi in HeLa Cells
HeLa cells were synchronized in S phase using the double-thymidine block protocol (2 cycles of 15-h culture with thymidine and 9-h culture free of thymidine); enrichment of mitotic cells was achieved by nocodazole treatment after release from the thymidine block. When HeLa cells were transfected during the second thymidine block, the cells were washed 9 h after the initiation of the second thymidine block, transfected for 2 h in Opti-MEM using Lipofectamine 2000 (Invitrogen), and cultured again for 4 h in the original culture medium in the continued presence of thymidine before thymidine was removed. siRNA for human Aurora-B/AIM-1 was purchased from Cell Signaling Technology, and annealed siRNAs for human Aurora-B/AIM-1 (5'-CAG AAG AGC TGC ACA TTT G-3', 5'-GCA GAG AGA TCG AAA TCC A-3', 5'-CCA AAC TGC TCA GGC ATA A-3', and 5'-TGG GAC ACC CGA CAT CTT A-3') and for human Aurora-A (5'-AAC ACC CAA AAG AGC AAG CAG) were kindly provided by Z. Chen (Eisai Research Institute). siRNA for the scrambled sequence was used as a control. For INCENP RNAi, cDNA fragments corresponding to the N-terminal 600-base pair region of human INCENP were amplified by PCR and inserted into the pTOPO vector (Invitrogen). INCENP cDNA flanked by T7-promotor binding sites was amplified by PCR, using the M13F and T7-TOPO primers (5'-GCGTAATACGACTCACTATAGGTAACGGCCGCCAGTGTGCTG) and the pTOPO-INCENP plasmids as templates. RNA strands were synthesized using the MEGAscript kit (Ambion. Austin, TX) from PCR-derived linear template carrying a T7 promoter at both ends; double-stranded RNA (dsRNA) was formed by annealing as described previously (Yang D et al., 2002). To prepare siRNAs for INCENP and E. coli endoribonuclease (RNase III) as a control RNAi, 100 µg of dsRNAs was digested with 0.5 µg of recombinant RNase III for 2 h in a 100-µl reaction buffer at 25°C. The siRNAs were purified using Q-Sepharose column (GE Healthcare).

Chromatin Isolation
Chromatin fractions were prepared using a modified method described by Remboutsika et al. (1999). Briefly, HeLa cells were harvested, washed twice with ice-cold PBS, and resuspended in 15 mM Tris-HCl buffer, pH 7.5, containing 5 mM MgCl₂, 60 mM KCl, 15 mM NaCl, 5 mM MgCl₂, 1 mM CaCl₂, 1 mM dithiothreitol (DTT), 2 mM sodium vanadate, 250 mM sucrose, protease inhibitor cocktail (Sigma-Aldrich), and 1 mM phenylmethylsulfonyl fluoride (buffer N). An equal volume of buffer N containing 0.6% NP-40 was added to the cells, and the suspension was gently mixed and incubated on ice for 5 min. Nuclei were pelleted at 2000 x g for 5 min at 4°C in a microfuge. The cytoplasmic fraction (C) was recovered and the nuclei washed three times with buffer N before lysis in 10 mM PIPES buffer, pH 6.5. After centrifugation at 6000 x g for 20 min at 4°C in a microfuge, the chromatin pellet was washed once in the same buffer and recovered in SDS-PAGE loading buffer (Ch). After centrifugation at 6000 x g for 10 min at 4°C in a microfuge, equivalent samples (in terms of initial nuclei) from the C and Ch fractions were subjected to 10% SDS-PAGE and imunoblot analysis.

In Vitro Histone Methyltransferase Assay
In vitro histone methyltransferase reactions were performed with GST-SUV39H1 (82–412) as described previously (Rea et al., 2000) Briefly, reactions were carried out in a final volume of 50 µl in methylase buffer (50 mM Tris-HCl, pH 8.5, 20 mM KCl, 10 mM MgCl₂, 10 mM -mercaptoethanol, and 250 mM sucrose) containing as substrates histone H3 (Roche Diagnostics) and S-adenosyl-L-methionine (Sigma-Aldrich) as the methyl donor. After incubation at 37°C for 60 min, SDS loading buffer was added reaction, and the reactions boiled for 5 min.

In Vitro Kinase Assay
Aurora-B/AIM-1 kinase protein extracted from adenovirus-infected cells was incubated for 30 min at 30°C in a total volume of 20 µl containing 0.5 mg/ml histone H3, 50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT, 5 mM NaF, 1 mM DTT, 0.05 mM sodium vanadate, and 0.1 mM ATP.

HP1 Binding Assays
His₆-HP1 Ni/NTA beads were incubated for 1 h with methylated histone H3 peptide in binding buffer in a total volume of 100 µl. The bound and unbound (supernatant) proteins were separated by 14% SDS-PAGE and visualized after immunoblotting with anti-H3 antibodies. For kinase reactions, the beads were equilibrated with a kinase buffer and incubated for 30 min at 30°C in a total volume of 20 µl supplemented with 0.6 µM purified Aurora-B/AIM-1. Beads were then washed extensively with washing buffer.

Purification of Kinase-Active Aurora-B/AIM-1
The adenovirus encoding Aurora-B/AIM-1 was generated by ligating a rat N-terminal FLAG-tagged Aurora-B/AIM-1 cDNA (Terada et al., 1998) into pACCMV.pLpA and amplified as described previously (Albrecht and Hansen, 1999). E1-transformed human 293 cells were infected with the adenovirus, and mitotic cells were enriched after treatment with nocodazole for 12 h. The cells were collected 60 h after infection and were lysed for 20 min on ice in TBSN buffer consisting of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 5 mM EGTA, 1.5 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.5 mM Na₃VO₄, and a protease inhibitor cocktail (Roche Diagnostics). FLAG-tagged Aurora-B/AIM-1 protein was purified using a FLAG purification kit (Sigma-Aldrich).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In G₂ Cells, HP1 Colocalizes with Aurora-B/AIM-1 to Heterochromatin and Dissociates from It at the G₂–M Transition Concurrent with Histone H3 Phosphorylation
To clarify the molecular behavior of HP1 during G₂ phase and mitosis, stable CHO and HeLa cells expressing EGFP-HP1 were established. The localization of HP1 in these cells was analyzed by conventional immunofluorescence microscopy. In G₁/S-phase cells that usually contain unphosphorylated H3, HP1 was distributed in discrete, dispersed nuclear foci and at the nucleolar periphery (Supplemental Figure 1A, G₁/S). In these cells, a similar nuclear distribution of large foci representing condensed chromatin masses as revealed by DAPI staining was observed for HP1. Global histone H3 phosphorylation begins in the pericentromeric heterochromatin in late G₂ and subsequently progresses to cover the entire heterochromatin before the onset of mitosis (Hendzel et al., 1997). During interphase, Aurora-B/AIM-1, a mitotic H3 kinase, is not localized at G₁/S heterochromatin sites (Supplemental Figure 1B, G₁/S), but in G₂-phase cells, HP1 is clearly localized in condensed DNA regions as shown by DAPI staining (Supplemental Figure 1B, G₂). During prophase, most of the HP1 protein has dissociated from the heterochromatin, the starting point for H3 phosphorylation (Supplemental Figure 1A, prophase). In prophase cells, HP1 underwent large-scale but progressive dissociation from heterochromatin into the cytoplasm. This dissociation correlated with H3 phosphorylation and Aurora-B/AIM-1 localization (Supplemental Figure 1A, prophase).

Although in prometaphase cells most of the HP1 protein occurred as a diffuse signal in the cytoplasm, when cells were extracted with the nonionic detergent Triton X-100, which removed cytoplasmic HP1, before fixation, the centromeric localization of HP1 and Aurora-B/AIM-1 became obvious (Supplemental Figure 1B, prometaphase). During anaphase, HP1 began to target again the chromosome, and upon dissociation of Aurora-B/AIM-1 from the centromeres to the midspindle, H3 was dephosphorylated (Supplemental Figure 1, A and B, anaphase). During telophase, HP1 was almost exclusively directed to the nuclei, concurrent with the translocation of Aurora-B/AIM-1 to the midbody and the nearly complete dephosphorylation of histone H3. Virtually identical results were obtained when HeLa cells were used (see below).

Aurora-B/AIM-1 Is Required for Dissociation of HP1 from Heterochromatin during the G₂–M Transition
Because Aurora-B/AIM-1 colocalizes with HP1 to the heterochromatin during G₂, and dissociation of HP1 protein occurs in parallel with histone H3 phosphorylation, it is likely that Aurora-B/AIM-1 regulates the dissociation of HP1 from heterochromatin during G₂–M transition. To elucidate the functional relationship between Aurora-B/AIM-1 and HP1, the effect of Aurora-B/AIM-1 siRNA on mitotic progression was examined. HeLa cells treated for 48 h with either Aurora-B/AIM-1 or Aurora-A siRNA were substantially, albeit incompletely, depleted of the protein (Figure 1B). Depletion of Aurora-B/AIM-1 led to a significant decrease of histone H3 phosphorylation (H3P), and in mitotic cells HP1 remained associated with the chromosome arms (Figure 1, A and C). In contrast, both HP1 dissociation and phosphorylation status of H3 in cells expressing either Aurora-A (related to centrosome function; Glover et al., 1995, Terada et al., 2003) or control siRNA were not affected (Figure 1C). In addition, in HeLa cells expression of kinase-negative Aurora-B/AIM-1 K106R (KR) showed similar results, as did Aurora-B/AIM-1 RNAi (Figure 1A), thus indicating that the kinase activity of Aurora-B/AIM-1 is required for HP1 dissociation. Moreover, videomicroscopy of CHO cells expressing Aurora-B/AIM-1 KR demonstrated clearly that HP1 failed to dissociate from the chromosome arms and that the chromosomes failed to align on the metaphase plate (Supplemental Movies 1 and 2). Eventually, both chromosome segregation and cytokinesis failed and could not be completed, leading to multinucleated cells as was shown previously (Terada et al., 1998). To conclude, dissociation of HP1 from the chromosomal arm depends on the kinase activity of Aurora-B/AIM-1 but not of Aurora-A.

View larger version (37K): [in this window] [in a new window]   Figure 1. Aberrant localization of HP1 by depletion of Aurora-B/AIM-1. Immunostaining of HeLa cells expressing EGFP-HP1 with anti-H3 phos (H3P) after expression of Aurora-B/AIM-1 RNAi for 48 h. Scrambled siRNA was used as a control. After expression of Aurora-B/AIM-1 RNAi or kinase-negative Aurora-B/AIM-1 KR for 48 h, cells were swollen in a hypotonic solution, fixed in paraformaldehyde, and then permeabilized with 0.5 Triton X-100. During M phase, HP1 still remained associated with the chromosome arms in mitotic cells lacking H3 phosphorylation due to Aurora-B/AIM-1 RNAi or overexpression of kinase-negative Aurora-B/AIM-1 K106R (KR), whereas HP1 was associated with the centromeres in the control (control). Scale bar, 10 µm. (B) Immunoblotting of cell extracts of mitotic HeLa cell 48 h after expression of RNAi with different antibodies: anti-AIM-1 (Aur-B/AIM-1), Aurora-A (Aur-A), and -Tubulin antibodies as a loading control. (C) Quantification of HP1 associated with the chromosome arms 48 h after expression of control, Aurora-A, or Aurora-B/AIM-1 RNAi. HeLa cells expressing EGFP-HP1 were synchronized, and the mitotic cells were counted and classified as indicated. Results represent the average of three independent experiments (>200 cells each); bars indicate standard deviations as described in Materials and Methods.

Endogenous HP1 Dissociates from Chromatin during Mitosis and Is Inhibited by Depletion of Aurora-B/AIM-1

View larger version (28K): [in this window] [in a new window]   Figure 2. Aurora-B/AIM-1 inhibits its release of HP1 from chromatin in M-phase HeLa cells. (A) Samples of HeLa cells were synchronized, released at different time points, collected, and the DNA content analyzed by flow cytometry. (B) Synchronized cells were lysed by addition of 0.3% NP-40 in buffer N, containing 5 mM MgCl2. The Ch fraction was pelleted at 2000 x g for 5 min at 4°C in a microfuge. The C fraction was recovered. Equal amounts of proteins prepared from cytoplasmic and chromatin fractions were run on a 12% gel and blotted with anti-HP1. (C) HeLa cells were synchronized in S phase using the double-thymidine block protocol. Mitotic cells were enriched by nocodazole treatment after release from the thymidine block. Cells were transfected with Aurora-B/AIM-1 (+) or control (–) RNAi during the second thymidine block, washed 9 h after initiation of the second thymidine block, and cultured again in the original culture medium for 4 h in the continued presence of thymidine. After removal of the drug, cells were cultured in fresh medium for 5 h and treated with nocodazole for 4–5 h. Both the Ch and C fractions were prepared from the exponentially growing cells (L) or mitotic cells (M). Immunoblots with anti-HP1 antibody was performed as shown in C.

INCENP Is Required for Aurora-B/AIM-1 Recruitment to Heterochromatin but Not for HP1 Recruitment

View larger version (40K): [in this window] [in a new window]   Figure 3. Depletion of INCENP by RNAi inhibits association of Aurora-B/AIM-1 but not HP1, with heterochromatin in the G2 phase. HeLa cells expressing EGFP-HP1 were transfected with control or INCENP siRNA. G2 phase cells were obtained by releasing cells synchronized by a double-thymidine block into fresh medium for a period of 10–12 h. M-phase cells were obtained from a thymidine/nocodazole block as shown in Figure 2C. (A) Cells treated with INCENP (INCENP RNAi) or control (RNase III siRNA) siRNAs were immunostained with anti-AIM-1 or anti-phos H3 (H3P) antibodies. DNA was stained with 50 ng/ml Hoechst 33258 to monitor the location of heterochromatin. Aurora-B/AIM-1 colocalizes with HP1 in heterochromatin. In INCENP-depleted cells, Aurora-B/AIM-1 does not associate with heterochromatin in G2 phase. During M phase, HP1 still remains associated with the chromosome arms. Bar, 10 µm. (B) For an immunoblot analysis, equal amounts of proteins prepared from HeLa cells transfected with INCENP (+) or control siRNA (–) were run on a 10% gel, blotted with anti-INCENP, and with anti--tubulin antibodies as a loading control. (C) Quantification of HP1 associated with interphase heterochromatin or the chromosome arms 48 h after transfection of control, or INCENP siRNAs. HeLa cells expressing EGFP-HP1 were synchronized, and the mitotic cells were counted and classified. Results are the average of three independent experiments (>200 cells each), and bars indicate standard deviations as described in Materials and Methods. Quantification of HP1 associated with the chromosome arms after expression of control, Aurora-A, or Aurora-B/AIM-1 RNAi for 48 h.

Phosphorylation of H3 Induces the Dissociation of the HP1 Chromodomain but Not of the Intact Protein from H3 Methylated at Lysine 9

View larger version (26K): [in this window] [in a new window]   Figure 4. H3 phosphorylation is required but not sufficient to trigger dissociation of HP1 from H3 methylated at Lysine 9. (A) HA-tagged wild-type histone H3 (WT) or HA-tagged nonphosphorylatable H3 (S10A) were transfected into an EGFP-HP1-expressing HeLa cell line. Cells were synchronized and fixed as shown in Figure 2C and visualized by GFP signal for HP1, DAPI for DNA, and immunostaining with anti-HA (H3). Overexpression of H3 S10A, but not H3 WT, inhibited HP1 dissociation from the chromosome arms. (B) Quantification of HP1 associated with the chromosome arms 48 h after transfection of HA-H3 WT or S10A. HeLa cells expressing EGFP-HP1 were synchronized as shown in Figure 2C, stained with anti-HA antibody, and classified as indicated. The mitotic cells expressing HA-H3 were counted. Results are the average from three independent experiments (>200 cells each), and bars indicate standard deviations as described in Materials and Methods. (C) Quantitative analysis of the induction of chromosome segregation (lagging chromosomes and chromatin bridges) and cytokinesis defects (multinucleated cells) after HA-histone H3 WT or S10A mutant expression. The graph represents HA-positive cells expressing histone H3 72 h after transfection. Results are the average from three independent experiments (>200 cells each), and bars indicate standard deviations. (D) Effect of Aurora-B/AIM-1–mediated histone H3 phosphorylation on in vitro HP1 binding to histone H3 methylated at Lysine 9. Histone H3 peptide was subjected to an in vitro histone methyltransferase assay as a methyl donor. In vitro histone methyltransferase reactions were performed with human histone methyltransferase (HMTase) SUV39H1 (lanes 2, 4, 5, 7, and 8), as detected with anti-dmLys-9 H3 antibody (top, lanes 1 and 2) and H3 antibody (bottom). The methylated H3 peptides were collected and used in an His6-HP1 whole protein (lanes 3–5) or CD (lanes 6–8) pull-down assay after kinase assay in the presence or absence of FLAG-Aurora-B/AIM-1 protein. H3 phosphorylation was detected with anti-phos H3 (H3P) antibody (middle and bottom, lanes 3–8). Bound proteins were resolved by 14% SDS-PAGE and immunoblotted with anti-H3 (top).

Abnormal Localization of Histone Methyltansferase SUV39H1 in Cells Lacking Aurora-B/AIM-1
It was recently reported that histone methyltansferase SUV39H1 is dynamically distributed in mitotic chromatin. It displays dispersed staining at prophase, gives highly specific centromeric signals at prometaphase and metaphase, and again shows significantly reduced staining at the onset of anaphase (Aagaard et al., 2000). SUV39H1 has been shown to form a complex with HP1, and its enzymatic activity is required for proper targeting of HP1 to the heterochromatin (Aagaard et al., 2000). To investigate whether the abnormal localization of HP1 by Aurora-B/AIM-1 RNAi could be correlated with impaired targeting of SUV39HI during mitosis, HeLa cells that stably express FLAG-tagged SUV39H1 were immunostained. These experiments showed that in cells depleted of Aurora-B/AIM-1 where the dissociation of HP1 was affected, SUV39H1 was localized together with HP1 along the entire metaphase chromosome arms, whereas in control cells SUV39H1 protein was concentrated at the centromere regions (Figure 5, A and B). These data were further verified by immunoblots. Depletion of Aurora-B/AIM-1 significantly inhibited the dissociation of SUV39HI from the chromatin-bound fraction in mitotic cells, whereas it decreased in control cells (Figure 5C). These observations suggest that Aurora-B/AIM-1 is involved in the dissociation of both HP1 and SUV39HI from chromosome arms.

View larger version (51K): [in this window] [in a new window]   Figure 5. Aberrant localization of SUV39H1 in mitotic chromosomes lacking Aurora-B/AIM-1. Immunofluorescence of mitotic chromosomes in control and Aurora-B/AIM-1–depleted HeLa cells stably expressing EGFP-HP1 and FLAG-tagged SUV39H1 with anti-FLAG antibody. (A) After expression of Aurora-B/AIM-1 RNAi for 48 h, cells were treated for 5 h with nocodazole, fixed, and stained with anti-FLAG antibody (SUV39HI). In cells depleted of Aurora-B/AIM-1, HP1 and SUV39H1 were localized at the metaphase chromosome arms. (B) Quantification of SUV39HI associated with the chromosome arms 48 h after transfection of control or Aurora-B/AIM-1 siRNAs. HeLa cells were synchronized. The mitotic cells were counted and classified as indicated. Results are the average of three independent experiments (>200 cells each), and bars indicate standard deviations as described in Materials and Methods. (C) HeLa cells were synchronized in S phase using the double-thymidine block protocol as shown in Figure 2C. Mitotic cells were enriched using nocodazole after release from the thymidine block. After transfection with Aurora-B/AIM-1 (+) or control (–) RNAi during the second thymidine block, HeLa cells were washed 9 h after the initiation of the second thymidine block and cultured again in the original culture medium for 4 h in the continued presence of thymidine. After removal of the drug, cells were further cultured for 5 h in fresh medium and treated with nocodazole for 4–5 h. Ch and C fractions were prepared from the exponentially growing cells (L) or mitotic cells (M). Immunoblot was performed with anti-SUV39HI antibody.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Trimethylation of histone H3 at Lysine 9 is important for targeting HP1 to heterochromatin region. As proposed in the binary switch hypothesis, H3 phosphorylation negatively regulates the binding of HP1 to the adjacent methylated Lysine 9 (Fischle et al., 2003). Furthermore, inhibition or depletion of Aurora-B/AIM-1 triggered the retention of HP1 in chromatin during mitosis (Figures 1 and 2). In addition, an in vitro-binding assay revealed that Aurora-B/AIM-1–mediated phosphorylation of H3 induced the dissociation of the HP1 chromodomain but not of the intact protein (Figure 4D). This observation concurs with that of previous studies showing that in GST pull-down assays, phosphorylation of H3 at Serine 10 does not prevent binding of plant HP1 (Fass et al., 2002) and that phosphomethylated H3 peptide stably interacts with the intact HP1 protein (Mateescu et al., 2004). While this manuscript was in review, similar findings on the regulation of HP1-chromatin binding were reported by others (Fischle et al., 2005; Hirota et al., 2005). However, these observations suggest that H3 phosphorylation at Serine 10 is both necessary and sufficient for the dissociation of HP1 from the mitotic chromosomes. One reason for the different results could be that Aurora-B/AIM-1 protein purified from HeLa cells in this study may exhibit little enzymatic activity to induce dissociation of the intact HP1 Nonetheless, results presented here suggest that the center and/or C-terminal domain of HP1 interferes with or reduces the effect of H3 phosphorylation on the dissociation of HP1. It was recently reported that the combined effects of H3 phosphorylation at Serine 10 and Lysine 14 acetylation are responsible for the dissociation of HP1 from chromatin during mitosis and that an H3 peptide carrying a triple MetLys-9-pSer-10-AcLys-14 modification no longer binds to HP1 (Mateescu et al., 2004). In addition to H3 phosphorylation, the decrease of Lysine 9 methylation and/or Lysine 14 acetylation may be required for the dissociation of HP1 from chromatin during mitosis in mammalian cells.

The histone methyltansferase SUV39H1 has been implicated to play, through selective methylation of H3 at Lysine 9, an essential role in the initial steps of heterochromatin formation, and to generate a binding site for HP1 (Rea et al., 2000. Bannister et al., 2001, Lachner et al., 2001, Nakayama et al., 2001). Furthermore, loss of methylated H3 tails was shown to be accompanied by delocalization of HP1 from heterochromatin (Maison et al., 2002). These findings underscore the essential role SUV39H1 plays in targeting of HP1 and in establishing heterochromatin. Interestingly, methylation of Lys-9 interferes with Aurora-B/AIM-1–dependent phosphorylation of Ser-10. It is also inhibited by the preexiting phosphorylation of Serine 10 (Jenuwein and Allis, 2001). Thus, Aurora-B/AIM-1–mediated phosphorylation of H3 around the heterochromatin may interfere with additional methylation by SUV39H1 and thereby trigger the sequential dissociation of the HP1 and SUV39H1 complex, which eventually leads to the disruption of the heterochromatin structure. Consistent with this model, HP1 and SUV39H1 are localized along the metaphase chromosome arms in mitotic cells lacking Aurora-B/AIM-1 (Figure 5, A and B). These observations suggest that Aurora-B/AIM-1 may decrease the methylation status of the mitotic chromosome arm by regulating the localization of SUV39H1 and thereby inducing the dissociation of HP1 from heterochromatin, although bulk trimethylation levels at Lysine 9 remain unchanged during the cell cycle (Fischle et al., 2005). However, the possibility that in Aurora-B/AIM-1–depleted cells the retention of SUV39HI at the chromatin is due to the direct binding of HP1 to SUV39HI cannot be excluded because SUV39HI can bind directly to HP1. To conclude the present studies established a correlation between H3 phosphorylation, heterochromatin dynamics, and chromosomal passenger proteins. Further studies are needed to establish the exact role the proteins play during dissociation from heterochromatin during mitosis.

	ACKNOWLEDGMENTS

 
I am grateful to R. Kuriyama, S. Sturm, W. C. Earnshaw, S. Watanabe, T. Hirano, and J. Harder for helpful comments, and to R. Link, M. Wessendorf, and S. Skjolaas for helpful advice. I am indebted to W. C. Earnshaw for INCENP antibody, I. Lee for HP1 plasmids, M. L. Cleary for SUV39HI plasmid and antibody, Z. Chen for siRNAs for Aurora-A and -B, C. J. Nelsen and J. H. Albrecht the adenovirus system, and J. Harder for technical help of MetaMorph. This study was supported by the Minnesota Medical Foundation and the Uehara Memorial Foundation.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-09-0906) on May 10, 2006.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Yasuhiko Terada ( terad002{at}tc.umn.edu)

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Aagaard, L., Schmid, M., Warburton, P., Jenuwein, T. (2000). Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres. J. Cell Sci 113, 817–829.[Abstract]

Aasland, R. and Stewart, A. F. (1995). The chromo shadow domain, a second chromo domain in heterochromatin-binding protein 1, HP1. Nucleic Acids Res 23, 3168–3173.[Abstract/Free Full Text]

Adams, R. R., Carmena, M., Earnshaw, W. C. (2001a). Chromosomal passengers and the (aurora) ABCs of mitosis. Trends Cell Biol 11, 49–54.[CrossRef][Medline]

Adams, R. R., Maiato, H., Earnshaw, W. C., Carmena, M. (2001b). Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol 153, 865–880.[Abstract/Free Full Text]

Adams, R. R., Wheatley, S. P., Gouldsworthy, A. M., Kandels-Lewis, S. E., Carmena, M., Smythe, C., Gerloff, D. L., Earnshaw, W. C. (2000). INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Curr. Biol 10, 1075–1078.[CrossRef][Medline]

Ainsztein, A. M., Kandels-Lewis, S. E., Mackay, A. M., Earnshaw, W. C. (1998). INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1. J. Cell Biol 143, 1763–1774.[Abstract/Free Full Text]

Albrecht, J. H. and Hansen, L. K. (1999). Cyclin D1 promotes mitogen-independent cell cycle progression in hepatocytes. Cell Growth Differ 6, 397–404.

Bannister, A. J., Zegerman, P., Partridge, J. F., Miska, E. A., Thomas, J. O., Allshire, R. C., Kouzarides, T. (2001). Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120–124.[CrossRef][Medline]

Cimini, D., Mattiuzzo, M., Torosantucci, L., Degrassi, F. (2003). Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects. Mol. Biol. Cell 14, 3821–3833.[Abstract/Free Full Text]

Christensen, T. W. and Tye, B. K. (2003). Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation. Mol. Biol. Cell 6, 2206–2215.

Earnshaw, W. C. and Cooke, C. A. (1991). Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation. J. Cell Sci 98, 443–461.[Abstract/Free Full Text]

Eissenberg, J. C. and Elgin, S. C. (2000). The HP1 protein family: getting a grip on chromatin. Curr. Opin. Genet. Dev 2, 204–210.

Fass, E., Shahar, S., Zhao, J., Zemach, A., Avivi, Y., Grafi, G. (2002). Phosphorylation of histone h3 at serine 10 cannot account directly for the detachment of human heterochromatin protein 1gamma from mitotic chromosomes in plant cells. J. Biol. Chem 774, 30921–30927.

Fischle, W., Tseng, B. S., Dormann, H. L., Ueberheide, B. M., Garcia, B. A., Shabanowitz, J., Hunt, D. F., Funabiki, H., Allis, C. D. (2005). Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438, 1116–1122.[CrossRef][Medline]

Fischle, W., Wang, Y., Allis, C. D. (2003). Binary switches and modification cassettes in histone biology and beyond. Nature 425, 475–479.[CrossRef][Medline]

Giet, R. and Glover, D. M. (2001). Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J. Cell Biol 152, 669–682.[Abstract/Free Full Text]

Glover, D. M., Leibowitz, M. H., McLean, D. A., Parry, H. (1995). Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81, 95–105.[CrossRef][Medline]

Hans, F. and Dimitrov, S. (2001). Histone H3 phosphorylation and cell division. Oncogene 20, 3021–3027.[CrossRef][Medline]

Heintz, N., Sive, H. L., Roeder, R. G. (1983). Regulation of human histone gene expression: kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle. Mol. Cell Biol 3, 539–550.[Abstract/Free Full Text]

Hendzel, M. J., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P., Allis, C. D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360.[CrossRef][Medline]

Hirota, T., Lipp, J. J., Toh, B. H., Peters, J. M. (2005). Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature 438, 1176–1180.[CrossRef][Medline]

Hsu, J. Y., Sun, Z. W., Li, X., Reuben, M., Tatchell, K., Bishop, D. K., Grushcow, J. M., Brame, C. J., Caldwell, J. A., Hunt, D. F., Lin, R., Smith, M. M., Allis, C. D. (2000). Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102, 279–291.[CrossRef][Medline]

Jenuwein, T. and Allis, C. D. (2001). Translating the histone code. Science 293, 1074–1080.[Abstract/Free Full Text]

Lachner, M., O’Carroll, D., Rea, S., Mechtler, K., Jenuwein, T. (2001). Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116–120.[CrossRef][Medline]

Lavoie, B. D., Hogan, E., Koshland, D. (2002). In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin. J. Cell Biol 156, 805–815.[Abstract/Free Full Text]

Maison, C., Bailly, D., Peters, A. H., Quivy, J. P., Roche, D., Taddei, A., Lachner, M., Jenuwein, T., Almouzni, G. (2002). Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component. Nat. Genet 30, 329–334.[CrossRef][Medline]

MacCallum, D. E., Losada, A., Kobayashi, R., Hirano, T. (2002). ISWI remodeling complexes in Xenopus egg extracts: identification as major chromosomal components that are regulated by INCENP-aurora B. Mol. Biol. Cell 13, 25–39.[Abstract/Free Full Text]

Mateescu, B., England, P., Halgand, F., Yaniv, M., Muchardt, C. (2004). Tethering of HP1 proteins to chromatin is relieved by phosphoacetylation of histone H3. EMBO Rep 5, 490–496.[CrossRef][Medline]

Murnion, M. E., Adams, R. R., Callister, D. M., Allis, C. D., Earnshaw, W. C., Swedlow, J. R. (2001). Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation. J. Biol. Chem 276, 26656–26665.[Abstract/Free Full Text]

Murzina, N., Verreault, A., Laue, E., Stillman, B. (1999). Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins. Mol. Cell 4, 529–540.[CrossRef][Medline]

Nakayama, J., Rice, J. C., Strahl, B. D., Allis, C. D., Grewal, S. I. (2001). Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110–113.[Abstract/Free Full Text]

Pak, D. T., Pflumm, M., Chesnokov, I., Huang, D. W., Kellum, R., Marr, J., Romanowski, P., Botchan, M. R. (1997). Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell 91, 311–323.[CrossRef][Medline]

Pereira, G. and Schiebel, E. (2003). Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14. Science 302, 2120–2124.[Abstract/Free Full Text]

Pines, J. and Hunter, T. (1989). Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell 58, 833–846.[CrossRef][Medline]

Remboutsika, E., Lutz, Y., Gansmuller, A., Vonesch, J. L., Losson, R., Chambon, P. (1999). The putative nuclear receptor mediator TIF1alpha is tightly associated with euchromatin. J. Cell Sci 112, 1671–1683.[Abstract]

Rea, S., et al. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593–599.[CrossRef][Medline]

Shibahara, K. and Stillman, B. (1999). Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 96, 575–585.[CrossRef][Medline]

Song, K., Jung, Y., Jung, D., Lee, I. (2001). Human Ku70 interacts with heterochromatin protein 1alpha. J. Biol. Chem 276, 8321–8327.[Abstract/Free Full Text]

Speliotes, E. K., Uren, A., Vaux, D., Horvitz, H. R. (2000). The survivin-like C. elegans BIR-1 protein acts with the Aurora-like kinase AIR-2 to affect chromosomes and the spindle midzone. Mol. Cell 2, 211–223.

Sugimoto, K., Tasaka, H., Dotsu, M. (2001). Molecular behavior in living mitotic cells of human centromere heterochromatin protein HP1-alpha ectopically expressed as a fusion to red fluorescent protein. Cell Struct. Funct 6, 705–718.

Tatsuka, M., Katayama, H., Ota, T., Tanaka, T., Odashima, S., Suzuki, F., Terada, Y. (1998). Multinuclearity and increased ploidy caused by overexpression of the aurora- and Ipl1-like midbody-associated protein mitotic kinase in human cancer cells. Cancer Res 58, 4811–4816.[Abstract/Free Full Text]

Terada, Y., Tatsuka, M., Jinno, S., Okayama, H. (1995). Requirement for tyrosine phosphorylation of Cdk4 in G1 arrest induced by ultraviolet irradiation. Nature 376, 358–362.[CrossRef][Medline]

Terada, Y., Tatsuka, M., Suzuki, F., Yasuda, Y., Fujita, S., Otsu, M. (1998). AIM-1: a mammalian midbody-associated protein required for cytokinesis. EMBO J 17, 667–676.[CrossRef][Medline]

Terada, Y. (2001). Role of chromosomal passenger complex in chromosome segregation and cytokinesis. Cell Struct. Funct 6, 653–657.

Terada, Y., Uetake, Y., Kuriyama, R. (2003). Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells. J. Cell Biol 163, 757–763.

Wei, Y., Yu, L., Bowen, J., Gorovsky, M. A., Allis, C. D. (1999). Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 97, 99–109.[CrossRef][Medline]

Yang, D., Buchholz, F., Huang, Z., Goga, A., Chen, C. Y., Brodsky, F. M., Bishop, J. M. (2002). Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 9942–9947.[Abstract/Free Full Text]

Ye, Q. and Worman, H. J. (1996). Interaction between an integral protein of the nuclear envelope inner membrane and human chromodomain proteins homologous to Drosophila HP1. J. Biol. Chem 271, 14653–14656.[Abstract/Free Full Text]

This article has been cited by other articles:

				Y. Terada and Y. Yasuda Human Immunodeficiency Virus Type 1 Vpr Induces G2 Checkpoint Activation by Interacting with the Splicing Factor SAP145 Mol. Cell. Biol., November 1, 2006; 26(21): 8149 - 8158. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Material All Versions of this Article: E05-09-0906v1 17/7/3232    most recent Alert me when this article is cited Alert me if a correction is posted