Correspondence to: Philippe Collas, Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway. Tel:+47-22851013 Fax:+47-22851497 E-mail:philippe.collas{at}basalmed.uio.no.
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Abstract |
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Association of the condensin multiprotein complex with chromatin is required for chromosome condensation at mitosis. What regulates condensin targeting to chromatin is largely unknown. We previously showed that the nuclear A kinaseanchoring protein, AKAP95, is implicated in chromosome condensation. We demonstrate here that AKAP95 acts as a targeting protein for human chromosome-associated protein (hCAP)-D2/Eg7, a component of the human condensin complex, to chromosomes. In HeLa cell mitotic extract, AKAP95 redistributes from the nuclear matrix to chromatin. When association of AKAP95 with chromatin is prevented, the chromatin does not condense. Condensation is rescued by a recombinant AKAP95 peptide containing the 306 COOH-terminal amino acids of AKAP95. Recombinant AKAP95 binds chromatin and elicits recruitment of Eg7 to chromosomes in a concentration-dependent manner. Amount of Eg7 recruited correlates with extent of chromosome condensation: resolution into distinct chromosomes is obtained only when near-endogenous levels of Eg7 are recruited. Eg7 and AKAP95 immunofluorescently colocalize to the central region of methanol-fixed metaphase chromosomes. GST pull-down data also suggest that AKAP95 recruits several condensin subunits. The results implicate AKAP95 as a receptor that assists condensin targeting to chromosomes.
Key Words: chromosome, condensin, AKAP, hCAP, mitosis
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Introduction |
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The eukaryotic genome must compact and resolve into distinct chromosomes for proper segregation at mitosis. Chromosome condensation requires a family of highly conserved ATPases called structural maintenance of chromosome (SMC)1 proteins (
SMC homologues exist in a variety of organisms ranging from yeast to mammals (
Recent antibody-blocking and rescue experiments have identified a role of the A kinaseanchoring protein, AKAP95, in chromatin condensation and maintenance of condensed chromosomes during mitosis and in mitotic extract (
We demonstrate here that chromatin-bound AKAP95 acts as a targeting protein for hCAP-D2/Eg7 in a mitotic extract. The results suggest that AKAP95 provides an additional level of regulation for the association of the condensin complex with chromosomes.
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Materials and Methods |
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Antibodies and Peptides
Affinity-purified polyclonal antibodies against AKAP95 were from Upstate Biotechnology (1-386) was described previously (
Nuclei, Nuclear Matrices and Chromatin
Interphase HeLa nuclei were isolated from confluent cells as described (
High salt-extracted nuclear matrices were prepared from purified nuclei essentially as described (
Metaphase Chromosome Spreads
Metaphase chromosome spreads were prepared from HeLa cells synchronized with Colcemid as described previously (
Mitotic Extracts and Chromatin Condensation Assay
HeLa cells synchronized in mitosis with 1 µM nocodazole were homogenized by sonication in lysis buffer, and the lysate cleared at 10,000 g for 10 min and at 200,000 g for 3 h to produce a mitotic cytosolic extract (
Nuclear breakdown and chromatin condensation were carried out at 30°C for up to 2 h in mitotic extract containing an ATP generating system (
Immunological Procedures and GST Pull-down
Immunofluorescence and immunoblotting were performed as described earlier (
For GST pull-downs, chromatin essentially devoid of endogenous AKAP95 (AKAP95-free chromatin; see Results) was incubated for 2 h in mitotic extract containing 500 ng/ml GST-AKAP951-386 or 500 ng/ml GST as a control. At the end of incubation, the chromatin was sedimented through 1 M sucrose and solubilized in 500 µl TKM buffer (50 mM Tris, pH 7.5, 25 mM KCl, and 5 mM MgCl2) containing 5 U micrococcal nuclease. After 45 min at room temperature, insoluble material was removed by sedimentation, and 50 µl of a 50% slurry of glutathione-agarose beads was added to the soluble chromatin supernatant. After 2 h at 4°C, beads were sedimented, washed three times in TKM buffer, and proteins were eluted in boiling SDS sample buffer.
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Results |
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AKAP95 Redistributes from the Nuclear Matrix to Chromatin upon Nuclear Disassembly in Mitotic Extract
We have previously reported that AKAP95 primarily cofractionates with detergent-, high salt-, DNase-, and RNase-resistant matrices of purified HeLa cell nuclei (
Redistribution of AKAP95 from the nuclear matrix to chromatin was demonstrated upon nuclear disassembly in a cell-free extract derived from mitotic HeLa cells. The extract supports disassembly of purified interphase HeLa nuclei, including nuclear envelope breakdown and chromatin condensation (Fig 1 a). Western blotting analysis of nuclear matrix and chromatin fractions prepared at successive stages of nuclear disassembly indicated that AKAP95 was redistributed from the matrix to the chromatin within 30 min (Fig 1 b). Release of AKAP95 from the nuclear matrix was further illustrated by solubilization of AKAP95 upon incubation of purified matrices for 1 h in mitotic extract, but not in interphase extract (Fig 1 c). This assay demonstrates the redistribution of AKAP95 from the nuclear matrix to chromatin upon mitotic nuclear disassembly.
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Association of AKAP95 with Chromatin Is a Prerequisite for Chromosome Condensation
To investigate the functional significance of the association of AKAP95 with chromatin during nuclear disassembly, we produced chromatin that was essentially devoid of endogenous AKAP95. To this end, monoclonal antibodies against the nuclear matrix protein NuMA, or control pre-immune mouse IgGs were introduced into purified nuclei (-NuMA). In either situation, NuMA was solubilized (data not shown), suggesting that the lack of chromosome condensation resulted from the absence of targeting of AKAP95 to chromatin rather than from persistence of the nuclear matrix. The mechanism of inhibition of AKAP95 targeting to chromatin by anti-NuMA antibodies is under investigation.
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A recombinant AKAP95 fragment comprising the COOH-terminal 306 amino acids of AKAP95 (GST-AKAP951-386) was capable of rescuing chromosome condensation. When AKAP95-free chromatin was incubated into mitotic extract containing 500 ng/ml GST-AKAP95
1-386, or as a control 500 ng/ml GST, we found that GST-AKAP95
1-386 readily bound chromatin, as shown by immunoblotting (Fig 2 b, 67-kD band). The amount of GST-AKAP95
1-386 detected on blots of chromatin was similar to, or lower than, that of endogenous AKAP95 (data not shown), suggesting that levels of recombinant AKAP95 peptide bound to chromatin were near physiological. Remarkably, GST-AKAP95
1-386 also restored condensation of the chromatin into distinct chromosomes over 1.5 h while GST alone was ineffective (Fig 2 c). Thus, GST-AKAP95
1-386 was sufficient to bind chromatin and restore chromosome condensation. We concluded that association of AKAP95 with chromatin was necessary for chromosome condensation.
AKAP95 Recruits hCAP-D2/Eg7, a Component of the Human Condensin Complex
We have previously shown that immunoblocking of AKAP95 in HeLa cell nuclei hinders chromatin condensation and correlates with the absence on chromatin of hCAP-D2/Eg7, the human homologue of Xenopus XCAP-D2/pEg7 (
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Whether AKAP95 played a direct role in recruiting Eg7 to chromatin was determined by producing chromatin either devoid of AKAP95, or harboring GST-AKAP951-386 as described in the previous section. Control chromatin containing endogenous AKAP95 was obtained from nuclei condensed in mitotic extract. Chromatin contents in AKAP95 or GST-AKAP95
1-386 were verified on Western blot (Fig 4 a, top). Remarkably, Eg7 was selectively recruited to chromatin harboring either endogenous or recombinant AKAP95 but was absent from AKAP95-free chromatin (Fig 4 a, bottom). Note that traces of endogenous AKAP95 detectable on blot of chromatin were not sufficient to elicit Eg7 recruitment (Fig 4 a). This AKAP95 probably originated from the minor chromatin-associated fraction of AKAP95 within interphase nuclei (Fig 1 b, 0 min;
1-386 added to the extract was raised (Fig 4 b). Moreover, a pull-down of GST-AKAP95
1-386 with glutathione-agarose beads from chromatin condensed in mitotic extract containing 500 ng/ml GST-AKAP95
1-386 brought down Eg7, while no Eg7 was associated with GST alone (Fig 4 c, Blot). Analysis of proteins of the GST pull-down complexes by Coomassie blue staining revealed several polypeptides, including major bands of 165, 150, 135, 100, and 67 kD (Fig 4 c), which corresponded in size to those of hCAP-C, hCAP-D2/Eg7, hCAP-E, the putative human homologue of XCAP-H (100 kD) and GST-AKAP95
1-386, respectively. This raises the possibility that subunits of the human condensin complex were present.
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Implications of AKAP95-mediated targeting of increasing amounts of Eg7 to chromatin were addressed by assessing the extent of chromosome condensation at each concentration of GST-AKAP951-386 used in the previous experiment. Clearly, higher degrees of condensation were consistently achieved with increasing concentrations of AKAP95 peptide (Fig 4 d). Whereas no significant condensation occurred with 05 ng/ml peptide (compare with Fig 2 c, +GST), some condensation was detected at 1050 ng/ml. Chromosome compaction was only seen at 100500 ng/ml peptide (Fig 4 d). However, as emphasized in photograph enlargements (Fig 4 d, bottom) resolution into distinct chromosomes only took place with
500 ng/ml AKAP95 peptide. At this level, chromosomes were resolved to a degree similar to that of control chromosomes harboring endogenous AKAP95 (Fig 4 d, left). This suggests that chromosome condensation is limited by the amount of Eg7 or Eg7-associated polypeptides recruited to chromatin. Thus, in the absence of the majority of endogenous AKAP95, the recombinant AKAP95 fragment (a) binds chromatin, (b) recruits Eg7 in a concentration-dependent manner, and (c) elicits chromosome condensation. Resolution into distinct chromosomes takes place only when nearly endogenous levels of Eg7 are recruited. We conclude from these results that AKAP95 acts as a targeting molecule for Eg7, alone or as part of the condensin complex, to chromatin.
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Discussion |
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Association of condensins with chromosomes is mitosis-specific. In Xenopus, all five 13S condensin subunits are found on chromosomes in mitotic extract (
Our results provide strong evidence for an additional regulatory mechanism of condensin association with chromosomes. (a) Depleting chromatin of AKAP95 before nuclear disassembly in mitotic extract prevents chromatin condensation; (b) a recombinant AKAP95 fragment is capable of binding chromatin; (c) the fragment elicits recruitment of Eg7 to chromatin in a concentration-dependent manner; (d) a GST pull-down of recombinant AKAP95 brings down a complex containing Eg7; (e) recombinant AKAP95 induces chromosome condensation to an extent proportional to the concentration of peptide added and to the amount of Eg7 recruited to chromatin. We have previously shown that antibody blocking of AKAP95 in the interphase nucleus also blocked chromatin condensation and inhibited Eg7 recruitment to chromatin (
Whether the entire condensin complex is recruited to chromosomes or whether individual subunits are targeted individually has not been, to our knowledge, specifically addressed. Xenopus egg extracts contain no free XCAP-C, although little XCAP-E is detected, such that condensins exist as complexes (
We propose a model for AKAP95-mediated recruitment of condensins to chromatin and chromosome condensation (Fig 5). In interphase, AKAP95 resides in a complex within the nuclear matrix, while Eg7, perhaps in association with other condensin subunits, is restricted to the cytoplasm. Upon entry into mitosis, AKAP95 is released from the matrix and associates with chromatin. Subsequent disassembly of the nuclear envelope allows the recruitment of Eg7/condensin to chromatin for chromosome condensation. Our results implicate AKAP95 as a receptor protein that assists condensin targeting to chromosomes.
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Footnotes |
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1 Abbreviations used in this paper: AKAP, A kinaseanchoring protein; hCAP, human chromosome-associated protein; SMC, structural maintenance of chromosome.
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Acknowledgements |
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We are grateful to K. Tasken for the AKAP95 peptide.
This work was supported by Association pour la Recherche contre le Cancer (K. Le Guellec), the Research Council of Norway (P. Collas) and the Norwegian Cancer Society (R. Steen and P. Collas).
Submitted: 16 February 2000
Revised: 13 March 2000
Accepted: 16 March 2000
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References |
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