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Tomoko Matsui,2,
Haruhiko Takisawa,2* and
M. Mitchell Smith1*
Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia 22908,1 Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan2
Received 26 July 2005/ Returned for modification 20 August 2005/ Accepted 14 November 2005
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ABSTRACT |
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INTRODUCTION |
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In addition to factors directly involved in the formation of the pre-RC, recent studies suggest a potential role of chromatin structure in the control of initiation of DNA replication. Chromatin structure has been linked to the initiation of DNA replication in several studies (9, 25, 40), and histone modification has been implicated in regulating replication timing in budding yeast (4, 46). Recent studies further suggest that histone acetylation is involved in origin activation at the chorion gene loci in Drosophila follicle cells (1) and in Xenopus early development (12). In addition, the histone deacetylase Sir2 has been shown to negatively regulate pre-RC assembly in budding yeast (38). These results suggest that histone acetylation is potentially involved in the control of pre-RC assembly; however, the molecular mechanism of this role is unknown.
The human protein Hbo1 (hHbo1), a MYST family histone acetyltransferase (HAT) (45), was originally identified by Iizuka and Stillman (20) through its binding to the human Orc1 protein; subsequently, Hbo1 was found to bind mouse Mcm2 as well (10). More recently, Hbo1 was found to associate with the latent replication origin of Kaposi's sarcoma-associated herpes virus, and siRNA inhibition of Hbo1 expression partially impaired Kaposi's sarcoma-associated herpes virus replication (41). These physical and functional interactions suggest that the acetylase activity of Hbo1 might participate in pre-RC formation and replication licensing.
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MATERIALS AND METHODS |
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Recombinant proteins. Plasmids for the expression of human His-tagged geminin (39) and glutathione S-transferase (GST)-Orc2 (16), were generous gifts from A Dutta and B. Stillman, respectively. Human His6-Hbo1 (amino acids 1 to 611) and His6-geminin proteins were expressed in bacteria and purified using Ni-NTA agarose (QIAGEN). Human GST-Orc2 protein was expressed and purified with glutathione-agarose (Sigma). His6-Mcm2 and His6-Cdc6 proteins were the generous gift of M. Alexandrow (3). For recombinant Xenopus Hbo1, the cDNA (XHbo1a) containing the full-length open reading frame was subcloned into pGEX6P-1 (GST-XHbo1) and was expressed in bacteria (strain BL21). The cleared lysates were incubated with glutathione-Sepharose 4B (Amersham Biosciences) at 4°C for 3 h. After being washed with phosphate-buffered saline (PBS), the GST moiety was cleaved with Precision Protease (Amersham Biosciences).
Antibodies and peptides. Rabbit polyclonal antibodies to hOrc2 and hMcm2 were a generous gift from B. Stillman. A rabbit antibody selective for the phosphorylated form of histone H1 was a gift from C. D. Allis (26). An affinity-purified rabbit polyclonal antibody to human Cdt1 was a gift from Hideo Nishitani. Commercial antibodies were purchased for antiMEK2 (N-20), anti-cyclin B1, anti-Cdc6, anti-cyclin E (Santa Cruz Biotechnology), anti-tetra-acetyl-histone H4 (Upstate Biotechnology), and antiactin (Sigma). Antibodies against human Hbo1 were raised in rabbits using peptide L74-1 (residues 158 to 172 of human Hbo1) synthesized by Sawady Technology (Tokyo, Japan). An affinity-purified fraction was used for immunofluorescence microscopy (IMF). Polyclonal antibodies against Xenopus Hbo1 were generated with recombinant Hbo1 protein expressed in bacteria, as well as two synthetic peptides. Peptide 1 (SLKDSGSDLSHRPKR) corresponds to residues 158 to 172 of human Hbo1; peptide 2 (CKVRAQSRDKQEDER) corresponds to residues 220 to 234 of human Hbo1. Affinity-purified antibody against peptide 1 was used for immunoprecipitation and immunodepletion. All antisera were affinity purified using full-length recombinant Xenopus Hbo1 and used for immunoblotting.
Acetylation, immunoprecipitation, and H1 kinase assay. Acetylation assays were performed in 20-µl reaction mixtures (50 mM HEPES [pH 8.0], 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium butyrate, 1 µl of [3H]acetyl-coenzyme A [CoA] [2.1 Ci/mmol; Amersham]), 1 to 3 µg of highly purified substrate proteins or 2 µg of GST fusion proteins, and 2 µg of His-Hbo1 and were incubated at 30°C for 1 h. The reaction mixture was subjected to electrophoresis with a 10% polyacrylamide gel electrophoresis (PAGE) gel and autoradiography. Gels containing [3H]acetate-labeled proteins were fixed with 10% glacial acetic acid and 40% methanol for 1 h and were enhanced by impregnation with a commercial fluorography enhancing solution (Amplify; Perkin-Elmer) for 30 min. Gels were then dried, and autoradiography was performed at –70°C for 7 to 21 days. Immunoprecipitation and histone acetyltransferase assays were as described previously (20), except that chicken core histones were used as substrates. H1 kinase assays were carried out as described previously (2).
Cell lines, RNAi, and Ads. A549 and HeLa cell lines were purchased from the American Type Culture Collection. For synchronization at the G1/S boundary, cells were incubated in medium containing 2 mM thymidine (Sigma) for 12 h and then in thymidine-free medium for 6 h, followed by an additional 12-h incubation in medium containing 2 mM thymidine. To synchronize HeLa cells at mitosis, cultures were treated with 50-ng/ml nocodazole (Sigma) for 16 h, and rounded mitotic cells were then detached from the dishes by manual shaking. After several washes with PBS, prewarmed regular medium was added to release the cells from the arrest. For analyses of cell cycle progression, ethanol-fixed cells were stained with propidium iodide and analyzed by flow cytometry (FACS Core Facility, University of Virginia). RNA interference (RNAi) was accomplished as described previously (15) by transfection with duplex RNA (Dharmacon) corresponding to nucleotides 92 to 112 of the Hbo1 coding region. Replication-deficient recombinant viruses were created as described previously (17). Adenovirus (Ad) stocks were maintained as described previously (34) and purified by cesium chloride density gradient centrifugation. Viruses were used at a multiplicity of infection of 900. Details of the construction of the green fluorescent protein (GFP) and Hbo1 antisense adenoviruses are available upon request.
Indirect IMF. Cells were treated with hypotonic buffer (10 mM HEPES-KOH [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.1% Triton X-100) (37). Cells were fixed with 4% paraformaldehyde and permeablized with 0.2% Triton X-100. After being blocked with PBS containing 2% goat serum, the fixed cells were stained with the appropriate primary antibody for 1 h. After several PBS washes, cells were incubated for 1 h with Cy3-labeled goat anti-rabbit immunoglobulin G (IgG) (1:1,000 dilution) and/or fluorescein isothiocyanate-labeled donkey anti-mouse IgG (1:1,000 dilution) (both from Jackson ImmunoResearch Laboratories). For the Xenopus experiments, interphase egg extracts were supplemented with sperm chromatin (4,000 sperm heads per µl) and 1 µM Cy3-dCTP. At appropriate times after incubation, extracts were diluted 10 fold with extraction buffer (EB; 50 mM HEPES-KOH [pH 7.5], 100 mM KCl, 2.5 mM MgCl2) containing 0.25% NP-40, and then formaldehyde was added to a final concentration of 3.7%. Samples were recovered by centrifugation though EB containing 30% sucrose onto a cover glass. The cover glass was washed with Tris-buffered saline plus 0.05% Tween 20 and incubated overnight at 4°C with primary rabbit antibody in Tris-buffered saline plus 0.05% Tween 20 containing 5% skim milk and then with a second antibody of Alexa Fluor 488-labeled anti-rabbit goat IgG (Invitrogen).
Preparation of egg extracts and immunodepletion. Interphase Xenopus egg extracts and sperm chromatin were prepared as previously described (22). The extracts were supplemented with 40-µg/ml cycloheximide, 30 mM phosphocreatine, and 15-µg/ml creatine phosphokinase. Immunodepletion of Hbo1 from the extracts was performed as follows. Affinity-purified anti-Hbo1 peptide antibody or control IgG was bound to protein A-Sepharose Fast Flow (Amersham Bioscience) at 5 µg of antibody per µl of beads and washed with EB. Interphase egg extract was mixed with a 0.1 volume of the beads for 30 min at 4°C. After removal of the beads, the extracts were similarly treated twice with freshly prepared antibody beads, and the obtained extracts were used for further experiments.
Chromatin preparation from frog eggs. Egg extracts were supplemented with sperm chromatin at a concentration of 4,000 sperm heads per µl and 40-µg/ml aphidicolin. At appropriate times after incubation at 23°C, extracts were diluted 10 fold with EB containing 0.25% NP-40, and the same buffer containing 10% (wt/wt) and 30% (wt/wt) sucrose were underlayered. The extracts were then centrifuged at 5,500 x g in a swinging-bucket rotor at 4°C for 5 min, and only the top layer was removed. Next, EB containing 0.25% NP-40 was layered on top of the samples, and the samples were centrifuged at 8,500 x g in a swinging-bucket rotor for 5 min. The top and second layers were then removed, leaving the bottom layer containing 30% sucrose, and the bottom layer diluted with EB was further centrifuged at 8,500 x g to pellet the chromatin fractions.
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RESULTS |
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Hbo1 is required for pre-RC assembly during G1 phase. Since normal pre-RC formation takes place during G1 phase, we next examined the chromatin loading of Mcm2-7 and ORC in synchronously dividing cultures. Our strategy was to inhibit Hbo1 expression with antisense adenovirus in cultures that were synchronized in mitosis by inhibiting microtubule function with the drug nocodazole. Since microtubule function is required for the directed targeting of adenovirus particles to the nucleus (42), adenovirus infection had to be done prior to nocodazole treatment. Different times of infection were tested and optimized at 10 h (Fig. 2A). Shorter times did not achieve adequate Hbo1 inhibition, while longer times affected the release of antisense-treated cells from mitosis, presumably due to checkpoint activation as a result of damage in the previous cell cycle. Thus, cells were first infected with either control GFP adenovirus or Hbo1 antisense adenovirus and allowed to grow for 10 h to begin expression of the transcripts. Infected cells were then synchronized in M phase by treatment with nocodazole for 16 h and finally released into G1 phase by washing out the nocodazole. Following release from nocodazole, we assayed the expression of Hbo1 and the chromatin association of Mcm6 and Orc2 by IMF. Using this regimen, Hbo1 inhibition was timed to occur during the course of G1 phase. As shown in Fig. 2B and C, the levels of Hbo1 protein in control and antisense cells were initially similar at 4 h postrelease. However, in cells treated with antisense adenovirus, Hbo1 protein began to be preferentially depleted starting at 6 to 8 h and was significantly diminished by 10 and 12 h postrelease, compared to controls. In the Hbo1 antisense cells, the chromatin association of Mcm6 increased for 4 to 6 h postrelease, but by 8 h it lagged behind that of control cells; by 10 to 12 h, its association was markedly decreased. In contrast, the chromatin association of Orc2 was essentially unaffected by Hbo1 antisense expression in these experiments. To complement these IMF experiments, we also carried out biochemical fractionation experiments on synchronously dividing populations (Fig. 3). At time intervals after nocodazole release, cells were sampled and fractionated into cytoplasmic and chromatin-bound fractions (Fig. 1A). The amounts of Hbo1, Mcm6, and Orc2 in the S3 chromatin fractions then were assayed by immunoblotting (Fig. 3B and C). For cells treated with antisense adenovirus, but not control adenovirus, the amount of chromatin-bound Hbo1 was substantially lost by 8 h. In control cells, the amount of Mcm6 was found to increase during the course of the experiment. Consistent with the IMF results, the chromatin association of Mcm6 in Hbo1as-treated cells began to increase normally but then decreased significantly between 4 to 8 h, coincident with the loss of Hbo1. As before, the chromatin association of Orc2 was unaffected by Hbo1 inhibition.
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Cell cycle regulation of Hbo1 HAT activity. Since the previous experiments showed that Hbo1 is required for pre-RC assembly during G1 phase, we asked whether its HAT activity is regulated during the cell cycle. A549 cells were synchronized at the G1/S boundary by a double-thymidine block, released into the cell cycle, and then assayed for the expression of Hbo1 by immunoblotting and Hbo1 HAT activity in anti-Hbo1 immunoprecipitates (Fig. 4A). HAT activity was assayed as the transfer of [3H]acetate from acetyl-CoA to purified chicken histones as substrate (Materials and Methods). Cell cycle progression of the cultures was monitored by flow cytometry (Fig. 4B) and by assays of the H1 kinase activity of anti-cyclin B1 immunoprecipitates (Fig. 4C). The levels of total Hbo1 protein did not change significantly during the cell cycle (Fig. 4A). In contrast, fluorograms of the HAT assays revealed that the activity of Hbo1 was low during S phase, rose during G2/M, and remained active through G1 (Fig. 4A). Thus, Hbo1 HAT activity is cell cycle regulated and is most active during the time of pre-RC assembly and licensing.
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An authentic Xenopus Hbo1 protein would also be predicted to coimmunoprecipitate with Orc1 or other components of the pre-RC, since human Hbo1 coimmunoprecipitates with Orc1 and Mcm2. Consistent with this prediction, we found that Orc1 and Orc2 are coimmunoprecipitated with XHbo1 antibody (Fig. 6D and data not shown). In addition, Cdt1 and geminin are also coimmunoprecipitated with p124 (Fig. 6D). Thus, the composition of the immunoprecipitates is also consistent with p124 representing XHbo1.
Taken together, these results argue that our antibodies against XHbo1 are detecting and immunodepleting a Xenopus Hbo1-like protein with histone H4 acetyltransferase activity. The predicted amino acid sequence of XHbo1a would express a protein of about 70 kDa, which is smaller than 124 kDa. Thus, based on the immunoreactivity of the different antibodies and the properties of the immunoprecipitates, we presume p124 to be a variant or modified form of Xenopus Hbo1.
Depletion of XHbo1 prevents pre-RC assembly. We next examined the chromatin binding of pre-RC components in extracts depleted of XHbo1, compared with extracts that were mock depleted (Fig. 7). Consistent with the results of inhibiting Hbo1 expression in human cells, we found that the chromatin binding of Mcm2 was diminished in XHbo1-depleted extracts. But the binding of Orc2 was slightly increased in the depleted extracts, and the amounts of histones bound to the chromatin fractions were essentially unchanged by the depletion of XHbo1 (Fig. 6C). In addition, there was an increase in the binding of Cdc6 (Fig. 7A). The lack of Mcm2 binding was not due to a decrease in pre-RC components, since the amounts of Mcm2, Orc2, Cdc6, and Cdt1 remained similar in both mock- and XHbo1-depleted extracts (Fig. 7B). Interestingly, we also found that the chromatin binding of Cdt1 was specifically decreased in XHbo1-depleted extracts (Fig. 7A), suggesting that Hbo1 might act through Cdt1 to regulate Mcm2-7 loading in Xenopus eggs. This differs from the mammalian cell results, where we observed a strong inhibition of Mcm2 and Mcm6 loading without a significant decrease in chromatin-bound Cdt1 (Fig. 1B; see Discussion).
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Cdt1 and geminin, but not Mcm3 or Cdc6, were coimmunoprecipitated with XHbo1 in the extracts (Fig. 6D). Reasoning that this might affect the activity of these proteins in the extracts, we tested whether an excess amount of recombinant Cdt1 could rescue the deficiency in the licensing activity of Hbo1-depleted extracts. Figure 8 demonstrates that the addition of recombinant Cdt1 rescued licensing activity, as measured by the chromatin binding of Mcm2 and Mcm3. At the same time, the chromatin binding of Cdc6 was suppressed by the loading of Mcm2-7 onto chromatin. These results suggest that Cdt1 by itself, or together with other interacting proteins, may be inactivated in Hbo1-depleted extracts.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initiation of DNA replication in human tissue culture cells and Xenopus egg cells differs in several respects. For instance, because of their rapid replication cycle, there are a greater number of active origins in Xenopus eggs than in human somatic cells. Abundant geminin protein is stored in an inactive form in interphase Xenopus eggs, whereas most geminin is subjected to degradation at G1 phase in human cells (47). Despite these differences, it is striking that Hbo1 is required for Mcm2-7 binding to chromatin in both experimental systems. This argues that Hbo1 acetylation is involved in an evolutionarily conserved pathway in replication licensing. We also detected some differences between the two systems. In Xenopus extracts, the depletion of XHbo1 partially reduced the chromatin association of Cdt1 (Fig. 7A), while in mammalian cells Mcm2-7, loading was inhibited without a major change in Cdt1 association (Fig. 1B). Thus, the regulation of pre-RC assembly by Hbo1 likely involves additional targets. Our results suggest that potential targets include both histones, as well as nonhistone components of the pre-RC.
Histone acetylation and pre-RC assembly. In vitro, histones are preferentially acetylated by Hbo1 compared with the other nonhistone substrates tested (M. Iizuka and M. M. Smith, unpublished data). Several lines of evidence support a role for histone acetylation in replication licensing. In budding yeast, shifting the balance of histone modification toward hyperacetylation by deleting the histone deacetylase Rpd3 accelerates the timing of replication initiation, consistent with a positive role for acetylation in licensing (4, 46). Conversely, mutation of the acetylated lysines in histone H4 results in a prolonged S phase, also consistent with a positive role for acetylation and a direct link with histone modification (30). Previous findings also link acetylation to origin activity (1, 38, 41). Here, we found that the depletion of XHbo1 from Xenopus egg extracts results in the deacetylation of histone H4 in the chromatin (Fig. 6C). Furthermore, downregulation of Hbo1 in mammalian cells by antisense RNA expression or siRNA also leads to hypoacetylation of histone H4 in mammalian cell lines (Iizuka and Smith, unpublished). There are several mechanisms by which histone acetylation could facilitate pre-RC assembly. First, acetylation could be important in establishing a chromatin conformation conducive to the recruitment of Mcm2-7 or its assembly into the pre-RC. Alternatively, acetylation could facilitate direct protein-protein interactions between Mcm2-7 or other pre-RC components and the histone tails. In either case, excess Cdt1 might drive pre-RC assembly in the face of an unfavorable chromatin context.
Nonhistone acetylation and pre-RC assembly. An increasing number of nonhistone proteins are being recognized as substrates for histone acetyltransferases (48). At present, the cellular nonhistone targets of Hbo1 have not been determined, but we found at least that in vitro, Hbo1 can acetylate Orc2, Mcm2, Cdc6, and geminin. The rescue of the licensing activity of XHbo1-depleted egg extracts by recombinant Cdt1 makes geminin a particularly attractive Hbo1 target candidate. In interphase extracts, geminin is present in excess of Cdt1, but geminin by itself is inactivated through ubiquitination and deubiquitination at the exit of M phase (23). Inactivated geminin in the extracts is reactivated only after its nuclear accumulation, and its inactivation does not accompany any changes in its molecular mass (19). It is not known how geminin is inactivated, but we speculate that inactivation may be mediated by acetylation of geminin itself or of other proteins involved in geminin inactivation. Depletion of XHbo1 would then lead to the deacetylation and activation of geminin, causing a block to licensing which could be overcome by the addition of excess recombinant Cdt1. We tested this hypothesis by depleting geminin before depleting Hbo1 from the extracts; in one experiment, this restored licensing in the Hbo1-depleted extracts. However, this experiment was difficult to reproduce, since the double depletion of geminin and XHbo1 from the extracts generally led to almost complete depletion of Cdt1 from the extracts as well.
While the molecular mechanisms remain to be determined, our findings with human and Xenopus egg extracts show that Hbo1-mediated acetylation is nevertheless an important mechanism for regulating the initiation of DNA replication in vertebrates. Protein phosphorylation is well known to play a central role in regulating DNA replication (18); however, relatively little is known about other protein modifications. Thus, the identification of Hbo1 as a protein acetyltransferase critical for pre-RC assembly defines a novel and previously unrecognized pathway in DNA replication licensing. Our results have significant implications for translational research as well, including the potential development of drugs to control cell proliferation by specifically blocking Hbo1 activity.
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ACKNOWLEDGMENTS |
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We thank Bruce Stillman, David Allis, Hideo Nishitani, Mark Alexandrow, Daniel Engel, and Anindya Dutta for reagents.
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FOOTNOTES |
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M.I. and T.M. contributed equally to this work.
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