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Deacetylase Activity Associates with Topoisomerase II and Is Necessary for Etoposide-induced Apoptosis

Deacetylase Activity Associates with Topoisomerase II and Is Necessary for Etoposide-induced... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 7, Issue of February 16, pp. 4539 –4542, 2001 Accelerated Publication © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. thought to have a more general role in DNA metabolism, with Deacetylase Activity Associates expression levels that remain relatively constant during cell with Topoisomerase II and Is and growth cycles (5). Both isoforms interact with the C-termi- nal region of the tumor suppressor protein, p53 (6). p53 is a Necessary for Etoposide-induced component of a multiprotein complex that contains the histone Apoptosis* deacetylase HDAC1 and the corepressor Sin3a (7–11). HDAC1, and the closely related HDAC2, are both compo- Received for publication, November 20, 2000, and in revised nents of two separate multiprotein complexes. The NuRD/Mi-2 form, December 22, 2000 repression complex contains both nucleosome remodeling and Published, JBC Papers in Press, January 2, 2001, histone deacetylase activities (12), whereas the Sin3 complex DOI 10.1074/jbc.C000824200 contains only the latter (9). Both complexes contain the Rb- Colin A. Johnson, Kay Padget‡, associated proteins RbAp46 and RbAp48 and associate with Caroline A. Austin‡, and Bryan M. Turner§ various, sometimes DNA-binding, transcriptional repressor From the Chromatin and Gene Expression Group, and corepressor proteins (11). The Xenopus NuRD complex Department of Anatomy, University of Birmingham (which contains homologues of mammalian HDAC1, RbAp48, Medical School, Birmingham B15 2TT, United and the methyl-CpG-binding protein MBD3) copurifies with Kingdom and the ‡School of Biochemistry and Genetics, DNA topoisomerase II (13), raising the possibility that mam- The Medical School, University of Newcastle, Newcastle-upon-Tyne NE2 4HH, United Kingdom malian topo II isoforms and HDAC1 may interact in a multi- protein complex. DNA topoisomerase II (topo II) is a ubiquitous nu- Here we show that HDAC1 and DNA topoisomerase II iso- clear enzyme that is involved in DNA replication, tran- forms physically interact both in vivo and in vitro. We also scription, chromosome segregation, and apoptosis. show that the HDAC inhibitor, TSA, suppresses apoptosis in- Here we show by immunoprecipitation, pull down with duced by the topo II poison etoposide, but not by the topo I glutathione S-transferase fusion proteins, and yeast inhibitor camptothecin. Our results raise the interesting pos- a and -b physi- two-hybrid analysis that both topo II sibility that chromatin remodeling by a topo II-HDAC-contain- cally interact with the histone deacetylase HDAC1. ing complex is involved in topo II-catalyzed DNA rearrange- The in vitro DNA decatenation activity of recombinant ments and/or generation of etoposide-induced DNA strand a and -b is inhibited by association with cata- topo II breaks in vivo. lytically inactive, recombinant HDAC1. We provide ev- idence for the in vivo significance of the topo II-HDAC1 MATERIALS AND METHODS association, showing that inhibition of HDAC activity Cells, Reagents, and Materials—The human cell lines HL-60 (promy- with trichostatin A suppresses apoptosis induced by elocytic leukemia; p53 null) and HeLa were grown in RPMI 1640 the topo II poison etoposide, but not by the topoi- medium containing 8% fetal calf serum. Regions of HDAC1 cDNA were somerase I inhibitor camptothecin. We suggest that subcloned into the pGEX3T-4 family of vectors (Amersham Pharmacia chromatin remodeling by an HDAC-containing com- Biotech) and verified by sequencing. GST fusion proteins were purified plex facilitates both topo II-catalyzed DNA rearrange- essentially as described previously (6). Recombinant human DNA to- ment and etoposide-induced DNA damage in vivo. poisomerase IIa and -b were made in a yeast system and purified as described previously (14). Characterization and use of rabbit polyclonal antibodies against topo IIa (18511a) and topo IIb (18513b) are de- For completion of cell division, the DNA of replicated chro- scribed elsewhere (15). A polyclonal rabbit antibody against mamma- lian HDAC1 was raised against a synthetic peptide corresponding to mosomes must be disentangled to allow the segregation of amino acid residues 467– 482 and affinity-purified as described previ- sister chromatids. In humans, this is achieved by the unique ously (16). Antibody against topo I was obtained commercially (Topo- decatenation activity of DNA topoisomerase II (topo II). Topo Gen, number 2012). II is essential for normal and neoplastic cellular proliferation, Immunoprecipitations, in Vitro Binding Assays, and Western Blot and several common anti-cancer drugs exert their cytotoxic Analysis—HeLa whole cell extract was prepared by lysing cells in effects through this enzyme (1, 2). incubation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10% (v/v) glycerol) containing 1.0% (v/v) Nonidet P-40, 0.5% Topoisomerase II activity in mammalian cells has been at- (w/v) sodium deoxycholate, 0.1% (w/v) SDS, protease inhibitors (0.1 mM tributed to at least two isoforms. Topo IIa (p170) associates TM phenylmethylsulfonyl fluoride and “Complete Mini ” tablets, Roche with chromosomes during prophase and throughout mitosis Molecular Biochemicals) and 50 units of DNase I (Amersham Pharma- and is thought to be a major component of the nuclear scaffold 8 cia Biotech) per 10 cells. The lysate was incubated in ice for 10 min, (3, 4). It has a peak of expression during G /M of the cell cycle and the clarified supernatant was used in standard immunoprecipita- (5). In contrast, the closely related topo IIb (p180) isoform is tions as described previously (6, 17). To confirm specificity, cognate blocking peptide (10 mg) was incubated with the antibody for 30 min before the addition of extract. Preimmune serum and irrelevant anti- * This work was supported by Wellcome Trust grants (to B. M. T. and sera were used as controls. GST pull down experiments used equivalent C. A. A.). The costs of publication of this article were defrayed in part by amounts of GST fusion proteins prebound to glutathione-Sepharose the payment of page charges. This article must therefore be hereby beads (Amersham Pharmacia Biotech) as described previously (6). In- marked “advertisement” in accordance with 18 U.S.C. Section 1734 teractions with recombinant topo IIa were performed in incubation solely to indicate this fact. buffer containing 0.1% (v/v) Nonidet P-40. § To whom correspondence should be addressed. Tel.: 44-121-414- Yeast Two-hybrid Assays—Yeast strains CG-1945 from a Match- 6824; Fax: 44-121-4146815; E-mail: [email protected]. maker Two-Hybrid System II kit (CLONTECH) were transformed with The abbreviations used are: topo, topoisomerase; TSA, trichostatin appropriate binary combinations of constructs containing the GAL4 A; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; DNA-binding domain and the GAL4 activation domain, as recom- FACS, fluorescence-activated cell sorter; CTD, C-terminal domain; Rb, retinoblastoma; HDAC, histone deacetylase. mended by the manufacturers. HIS3 reporter gene expression was This paper is available on line at http://www.jbc.org 4539 This is an Open Access article under the CC BY license. 4540 HDAC1 Interacts with DNA Topoisomerase II FIG.2. Pull down experiments of endogenous topo II and HDAC1. A, pull down of endogenous topo IIa by 5 mg of immobilized (bound) GST-HDAC1, but not by immobilized GST. Topo IIa is also depleted from the supernatant (unbound) by GST-HDAC1. B, pull down of endogenous HDAC1 (molecular mass, 62 kDa) by immobilized GST- topo IIa and topo IIb, containing the CTD of each isoform. C, pull down of deacetylase activity with immobilized GST-topo IIa CTD and topo IIb CTD. Bars represent ranges of two experiments. and topo IIb immunoprecipitate 6 –9% of total deacetylase ac- tivity from HeLa whole cell extract (Fig. 1B). The activity is fully inhibited by TSA. Negative control immunoprecipitations with preimmune serum, an irrelevant antibody (anti-CDK7), or FIG.1. Evidence for the physical association of topo IIa and HDAC1 proteins. A, Immunoprecipitation of endogenous topo IIa an antibody against DNA topoisomerase I (topo I) did not bring (molecular mass, 170 kDa), with the indicated antibodies (10 mg of total down activity above that of the no-antibody control. IgG), from 100 mg of HeLa whole cell extract (WCE). B, immunoprecipi- We performed in vitro pull down experiments of endogenous tation of deacetylase activity by antisera against topo IIa and topo IIb protein with GST fusion proteins. Full-length mammalian (18511a and 18513b) from HeLa WCE. Deacetylase activity is inhibited by TSA (10 ng/ml). Activity immunoprecipitated is expressed as a HDAC1, tagged with a GST moiety, but not GST itself, bound percentage of total input deacetylase activity, less a background of endogenous topo IIa in whole cell extract (Fig. 2A). In the nonenzymatic release of [ H]acetate of 1.94%. Percentages are averages converse experiment, a GST fusion protein containing the C- of three separate experiments, with bars representing S.D. terminal domain (CTD) of topo IIa was able to pull down endogenous HDAC1 (Fig. 2B). The fusion protein of the CTD of assayed on plates (6), in the presence of 25 mM 3-amino-1,2,4-triazole to topo IIb was also able to pull down small amounts of HDAC1 suppress background growth (18). (Fig. 2B). Fusion proteins of the CTD of topo IIa and topo IIb Enzyme Assays—Histone deacetylase activity was assayed as de- were able to pull down between 9 and 11% of deacetylase scribed previously (17). DNA strand-passage assays were performed on kinetoplast DNA (kDNA) as described previously (6). activity (Fig. 2C), comparable with the amounts brought down Detection of Apoptosis—HL-60 cells were grown until in mid-log by immunoprecipitation (Fig. 1B). Whereas GST-topo IIb pulls phase, then treated with 100 nM (30 ng/ml) TSA for 0.5 h before down less HDAC1, as detected on Western blots, than compa- additional treatments with either 100 mM (59 mg/ml) etoposide or 5.8 mM rable amounts of GST-topo IIa (Fig. 2B), the two different (2 mg/ml) camptothecin for 1.5 h. Control samples were treated with the fusion proteins bring down similar amounts of deacetylase dilution vehicles (0.1% Me SO and 0.1% ethanol). All cells were ob- activity (Fig. 2C). A possible explanation for this quantitative served in situ with phase-contrast microscopy to count cells with an apoptotic morphology, after staining with 10 mM Hoechst 33342 with discrepancy is that other deacetylases, in addition to HDAC1, the addition of 0.1 mM propidium iodide to visualize necrotic cells. Cells are preferentially associated with topo IIb. were also labeled with either FITC-annexin V conjugate (PharMingen) GST fusion proteins containing the C-terminal domain of TM or with the FAM-VAD-FMK reagent provided in the CaspaTag fluo- HDAC1 interact with recombinant topo IIa (Fig. 3). This do- rescein caspase activity kit (Intergen). Labeled cells were detected by main has previously been shown to contain the LXCXE motif indirect fluorescence microscopy (all cells) or by FACS analysis on a (residues 414 – 418), that appears to mediate interactions with Coulter Epics flow cytometer. the retinoblastoma protein pRb (19). In contrast, an N-terminal RESULTS HDAC1 fusion protein, containing the catalytic site, showed Interaction of HDAC1 and DNA Topoisomerase II—HeLa minimal interaction with recombinant topo IIa (Fig. 3). whole cell extract was immunoprecipitated with affinity-puri- A yeast two-hybrid system (18) was used to test for direct in fied antibody against mammalian HDAC1 and precipitated vivo interaction between topo II and HDAC1. Inserts were material tested for the presence of topo IIa by Western blotting constructed to express the topo IIa and topo IIb C-terminal (Fig. 1A). Anti-HDAC1 brought down easily detectable domains (6) and the HDAC1 region 220 – 482 (Fig. 3). Expres- amounts of topo IIa. There was no detectable immunoprecipi- sion of the integrated, GAL4-dependent HIS3 reporter gene tation of topo IIa with preimmune serum, and immunoprecipi- was used to detect interactions between “bait” and “prey” pro- tation was completely abolished by inclusion in the incubation teins in vivo. Topo IIa CTD or topo IIb CTD as bait, together mix of the peptide used to raise the anti-HDAC1 antibody (Fig. with HDAC1 as prey, allowed growth of large colonies (over 2 1A). The anti-HDAC1 antibody did not immunoprecipitate de- mm diameter) on His-selective medium. All three proteins were tectable levels of topo IIb (data not shown), and antibody to ineffective when expressed individually (Fig. 4). topo IIb brought down only a comparatively small amount of To explore the biological significance of the topo II-HDAC1 the a isoform (Fig. 1A). However, antisera against both topo IIa interaction, we tested the ability of full-length recombinant HDAC1 Interacts with DNA Topoisomerase II 4541 FIG.5. Functional association of topo II and histone deacety- lase. Decatentation of 1.4 mg of kinetoplast DNA concatemer (concat.) to monomers of minicircle DNA by recombinant topo IIa (0.60 pmol) and topo IIb (0.22 pmol) is inhibited by increasing amounts of GST-HDAC1 FIG.3. In vitro interaction of recombinant topo IIa and (1.4 –13.9 pmol), but not by GST alone (4.2–13.9 pmol). HDAC1. Regions of HDAC1 cDNA were subcloned and expressed as GST fusion proteins. Immobilized fusion proteins (5 mg) were tested for interaction with 1 mg of recombinant topo IIa. FIG.6. TSA suppresses apoptosis induced by topo II-mediated DNA damage, but not by topo I inhibition, in HL-60 cells. A, cells were treated as indicated (see “Materials and Methods” for details). Nuclear changes were visualized by indirect fluorescence microscopy after staining with Hoechst 33342 with the addition of propidium iodide (PI) to visualize necrotic cells. The nuclear morphology of cells were scored for apoptotic cells (PI2; clear bars) and necrotic cells (PI1; gray bars) and expressed as percentages of total cells in the field of view. Values are averages of two separate experiments. B, plasma membrane changes were detected by the binding of annexin V conjugated to FITC, FIG.4. Yeast two-hybrid assay showing that the CTDs of topo after treatment with TSA, etoposide, or camptothecin as indicated (see IIa and topo IIb can interact with HDAC1 CTD (amino acids “Materials and Methods”). Staining was visualized by indirect fluores- 220 – 482) in vivo. Expression of the reporter gene HIS3 in yeast stain cence microscopy and expressed as percentages of total cells. Bars CG-1945 (CLONTECH) was determined by two parallel series of spot represent S.D. values for three separate experiments. C, broad-spec- assays on selective medium plates lacking tryptophan, leucine, and trum caspase activity was detected by labeling live cells with the FAM- histidine (2Trp, 2Leu, 2His), but in the presence of 25 mM 3-amino- VAD-FMK fluorescein-conjugated caspase substrate (see “Materials 1,2,4-triazole to suppress background growth (6, 18). Colony size was and Methods”) after treatment with inhibitors, as indicated. Labeled compared with that on plates lacking tryptophan and leucine (2Trp cells were detected by FACS analysis, and activity is expressed as the 2Leu) as control. Strong growth in 2His medium occurs only in cells in percentage of the viable cell subpopulation that stains positive for the which the bait and prey proteins physically interact. substrate. Values are averages of two separate experiments. HDAC1 to modulate the functional properties of recombinant environment (22). This change was measured by binding of topo IIa and -b. Both of these enzymes can decatenate kineto- FITC-conjungated annexin V and counting of labeled cells by plast DNA (kDNA) to minicircle monomers, a process that fluorescence microscopy (Fig. 6B). Activation of cysteine as- requires a double-stranded break in the kDNA to allow strand partyl proteases (caspases) (21) during the apoptosis of passage. The addition of increasing amounts of HDAC1 to the HL-60 cells was assayed with a fluorescent substrate and reaction decreases the decatenation of kDNA by topo IIa and -b FACS analysis of viable cells (Fig. 6C). Chromatin condensa- (Fig. 5). Addition of GST alone did not affect decatenation by tion, membrane changes, and caspase activation all demon- either topo IIa and -b. strated that prior treatment with 100 nM TSA suppresses the Suppression of Etoposide-mediated Apoptosis by the HDAC apoptotic effect of etoposide (Fig. 6, A–C). In contrast, TSA Inhibitor Trichostatin A—We tested the effect of the HDAC did not affect apoptosis induced by the topo I inhibitor camp- inhibitor trichostatin A (20) on apoptosis induced by the tothecin (Fig. 6, A–C). Note that topo I does not associate with chemotherapeutic agent etoposide (VP-16). Etoposide causes detectable amounts of deacetylase activity (Fig. 1B). An iden- topoisomerase II-mediated DNA damage by increasing the tical anti-apoptotic effect of TSA treatment was also observed steady-state concentration of covalent DNA cleavage com- for the human lung adenocarcinoma cell line H1299 and plexes (1, 2, 4). Cells treated with etoposide acquire an apo- HeLa cells (data not shown). ptotic morphology, notably the condensation of chromatin at DISCUSSION the nuclear periphery and blebbing of the plasma membrane (2, 21). HL-60 cells displayed apoptotic chromatin condensa- The results presented show that the histone deacetylase tion after only 1.5-h treatment with either 100 mM etoposide HDAC1 is physically associated with each of the two isoforms or 5.8 mM camptothecin, an inhibitor of topo I (Fig. 6A). of human topoisomerase II, topo IIa and topo IIb. The associ- Plasma membrane changes during early apoptosis include ation occurs in vivo, being detectable by coimmunoprecipitation the exposure of phosphatidylserine to the external cellular from human cell extracts and by yeast two-hybrid assay. It also 4542 HDAC1 Interacts with DNA Topoisomerase II occurs in vitro. GST-coupled recombinant topo IIa and topo IIb associated with HDAC1. Further support for these ideas comes pull down significant amounts of HDAC activity from cell ex- from the recent results of Tsai et al. (23), who show that topo II tracts, while recombinant HDAC1 inhibits the in vitro decat- is associated not only with HDAC1/2, but also with MTA2, a enation activity of recombinant topo IIa. Since completion of protein that is part of the NuRD chromatin remodeling com- the work reported here, Tsai et al. (23) have reported essen- plex. The NuRD complex contains both HDAC1/2 and Mi-2, a tially the same findings for the two very similar deacetylases protein with ATPase/helicase activity (12). HDAC1 and HDAC2. Interestingly, whereas Tsai et al. (23) We and others (23) find that HDAC1 can inhibit the catalytic find evidence for an interaction between topo IIa and various activity of topo II in vitro. This is an important confirmation of regions of HDAC2, including N-terminal residues 1–57, we find the ability of topo II and HDAC1 to interact, but is not, at first that only the C-terminal region of HDAC1 (residues 220 – 482) sight, consistent with the proposition outlined above that interacts with topo II in vitro. These two deacetylases seem to HDAC activity facilitates topo II catalysis, or its consequences. differ in their mode of interaction with topo II. This can be resolved by noting that the GST-HDAC1 construct In experiments to assess the biological significance of the used to inhibit topo II in vitro is catalytically inactive, presum- topo II-HDAC interaction, we analyzed the effect of the ably because it lacks essential protein partners such as deacetylase inhibitor TSA on processes known to require topo RbAp46/48 (8, 11). It would be wrong to assume that catalyti- II activity. The most striking effect so far has been on the cally active HDAC1, in the context of a multiprotein complex ability of the topo II poison etoposide to drive cells into apo- that includes both HDAC and chromatin remodeling activities ptosis. We show that treatment with TSA prior to the addition (23), is also inhibitory. It might even be the case that, in vivo, of etoposide suppresses apoptosis in a variety of cell lines. The topo II activity is inhibited only by association with HDAC effect is seen even with HL60 cells, in which apoptosis is rendered catalytically inactive by inhibitors such as TSA. Such detectable within less than 1 h, a finding that minimizes the an effect would complement the suppression of topo II activity probability that inhibition of apoptosis is due to pleiotropic brought about by inhibition of chromatin remodeling. effects of TSA, such as its ability to alter cell cycle progression. The inhibitory effect of TSA was detected in several p53-null Acknowledgments—We thank Dr. G. Anderson for help with the FACS analysis, Dr. J. Shuttleworth for the gift of anti-CDK7 antibody cell lines, so the interaction between HDAC1 and p53 (7) can- and GST-cdc2 fusion protein construct, Darren A. White and Jayne S. not be responsible. Microscopically detectable chromatin re- Lavender for technical support. modeling is a diagnostic characteristic of cells in the later stages of apoptosis, and recent reports indicate that both topo REFERENCES II and histone acetylation play a role in this process (24). 1. Froelich-Ammon, S. J., and Osheroff, N. (1995) J. Biol. Chem. 270, However, our results indicate that this is not the stage at which 21429 –21432 2. Kaufmann, S. H., and Earnshaw, W. C. (2000) Exp. Cell Res. 256, 42– 49 TSA exerts the inhibitory effect reported here. We have shown 3. Wang, J. C. (1996) Annu. Rev. Biochem. 65, 635– 692 that TSA inhibition is detectable even when using an assay 4. Burden, D. A., and Osheroff, N. (1998) Biochim. Biophys. Acta 1400, 139 –154 5. Austin, C. A., and Marsh, K. L. (1998) Bioessays 20, 215–226 that measures one of the earliest changes of apoptosis, namely 6. Cowell, I. G., Okorokov, A. L., Cutts, S. 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Deacetylase Activity Associates with Topoisomerase II and Is Necessary for Etoposide-induced Apoptosis

Johnson, Colin A.; Padget, Kay; Austin, Caroline A.; Turner, Bryan M.
Journal of Biological Chemistry – Feb 1, 2001
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 7, Issue of February 16, pp. 4539 –4542, 2001 Accelerated Publication © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. thought to have a more general role in DNA metabolism, with Deacetylase Activity Associates expression levels that remain relatively constant during cell with Topoisomerase II and Is and growth cycles (5). Both isoforms interact with the C-termi- nal region of the tumor suppressor protein, p53 (6). p53 is a Necessary for Etoposide-induced component of a multiprotein complex that contains the histone Apoptosis* deacetylase HDAC1 and the corepressor Sin3a (7–11). HDAC1, and the closely related HDAC2, are both compo- Received for publication, November 20, 2000, and in revised nents of two separate multiprotein complexes. The NuRD/Mi-2 form, December 22, 2000 repression complex contains both nucleosome remodeling and Published, JBC Papers in Press, January 2, 2001, histone deacetylase activities (12), whereas the Sin3 complex DOI 10.1074/jbc.C000824200 contains only the latter (9). Both complexes contain the Rb- Colin A. Johnson, Kay Padget‡, associated proteins RbAp46 and RbAp48 and associate with Caroline A. Austin‡, and Bryan M. Turner§ various, sometimes DNA-binding, transcriptional repressor From the Chromatin and Gene Expression Group, and corepressor proteins (11). The Xenopus NuRD complex Department of Anatomy, University of Birmingham (which contains homologues of mammalian HDAC1, RbAp48, Medical School, Birmingham B15 2TT, United and the methyl-CpG-binding protein MBD3) copurifies with Kingdom and the ‡School of Biochemistry and Genetics, DNA topoisomerase II (13), raising the possibility that mam- The Medical School, University of Newcastle, Newcastle-upon-Tyne NE2 4HH, United Kingdom malian topo II isoforms and HDAC1 may interact in a multi- protein complex. DNA topoisomerase II (topo II) is a ubiquitous nu- Here we show that HDAC1 and DNA topoisomerase II iso- clear enzyme that is involved in DNA replication, tran- forms physically interact both in vivo and in vitro. We also scription, chromosome segregation, and apoptosis. show that the HDAC inhibitor, TSA, suppresses apoptosis in- Here we show by immunoprecipitation, pull down with duced by the topo II poison etoposide, but not by the topo I glutathione S-transferase fusion proteins, and yeast inhibitor camptothecin. Our results raise the interesting pos- a and -b physi- two-hybrid analysis that both topo II sibility that chromatin remodeling by a topo II-HDAC-contain- cally interact with the histone deacetylase HDAC1. ing complex is involved in topo II-catalyzed DNA rearrange- The in vitro DNA decatenation activity of recombinant ments and/or generation of etoposide-induced DNA strand a and -b is inhibited by association with cata- topo II breaks in vivo. lytically inactive, recombinant HDAC1. We provide ev- idence for the in vivo significance of the topo II-HDAC1 MATERIALS AND METHODS association, showing that inhibition of HDAC activity Cells, Reagents, and Materials—The human cell lines HL-60 (promy- with trichostatin A suppresses apoptosis induced by elocytic leukemia; p53 null) and HeLa were grown in RPMI 1640 the topo II poison etoposide, but not by the topoi- medium containing 8% fetal calf serum. Regions of HDAC1 cDNA were somerase I inhibitor camptothecin. We suggest that subcloned into the pGEX3T-4 family of vectors (Amersham Pharmacia chromatin remodeling by an HDAC-containing com- Biotech) and verified by sequencing. GST fusion proteins were purified plex facilitates both topo II-catalyzed DNA rearrange- essentially as described previously (6). Recombinant human DNA to- ment and etoposide-induced DNA damage in vivo. poisomerase IIa and -b were made in a yeast system and purified as described previously (14). Characterization and use of rabbit polyclonal antibodies against topo IIa (18511a) and topo IIb (18513b) are de- For completion of cell division, the DNA of replicated chro- scribed elsewhere (15). A polyclonal rabbit antibody against mamma- lian HDAC1 was raised against a synthetic peptide corresponding to mosomes must be disentangled to allow the segregation of amino acid residues 467– 482 and affinity-purified as described previ- sister chromatids. In humans, this is achieved by the unique ously (16). Antibody against topo I was obtained commercially (Topo- decatenation activity of DNA topoisomerase II (topo II). Topo Gen, number 2012). II is essential for normal and neoplastic cellular proliferation, Immunoprecipitations, in Vitro Binding Assays, and Western Blot and several common anti-cancer drugs exert their cytotoxic Analysis—HeLa whole cell extract was prepared by lysing cells in effects through this enzyme (1, 2). incubation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10% (v/v) glycerol) containing 1.0% (v/v) Nonidet P-40, 0.5% Topoisomerase II activity in mammalian cells has been at- (w/v) sodium deoxycholate, 0.1% (w/v) SDS, protease inhibitors (0.1 mM tributed to at least two isoforms. Topo IIa (p170) associates TM phenylmethylsulfonyl fluoride and “Complete Mini ” tablets, Roche with chromosomes during prophase and throughout mitosis Molecular Biochemicals) and 50 units of DNase I (Amersham Pharma- and is thought to be a major component of the nuclear scaffold 8 cia Biotech) per 10 cells. The lysate was incubated in ice for 10 min, (3, 4). It has a peak of expression during G /M of the cell cycle and the clarified supernatant was used in standard immunoprecipita- (5). In contrast, the closely related topo IIb (p180) isoform is tions as described previously (6, 17). To confirm specificity, cognate blocking peptide (10 mg) was incubated with the antibody for 30 min before the addition of extract. Preimmune serum and irrelevant anti- * This work was supported by Wellcome Trust grants (to B. M. T. and sera were used as controls. GST pull down experiments used equivalent C. A. A.). The costs of publication of this article were defrayed in part by amounts of GST fusion proteins prebound to glutathione-Sepharose the payment of page charges. This article must therefore be hereby beads (Amersham Pharmacia Biotech) as described previously (6). In- marked “advertisement” in accordance with 18 U.S.C. Section 1734 teractions with recombinant topo IIa were performed in incubation solely to indicate this fact. buffer containing 0.1% (v/v) Nonidet P-40. § To whom correspondence should be addressed. Tel.: 44-121-414- Yeast Two-hybrid Assays—Yeast strains CG-1945 from a Match- 6824; Fax: 44-121-4146815; E-mail: [email protected]. maker Two-Hybrid System II kit (CLONTECH) were transformed with The abbreviations used are: topo, topoisomerase; TSA, trichostatin appropriate binary combinations of constructs containing the GAL4 A; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; DNA-binding domain and the GAL4 activation domain, as recom- FACS, fluorescence-activated cell sorter; CTD, C-terminal domain; Rb, retinoblastoma; HDAC, histone deacetylase. mended by the manufacturers. HIS3 reporter gene expression was This paper is available on line at http://www.jbc.org 4539 This is an Open Access article under the CC BY license. 4540 HDAC1 Interacts with DNA Topoisomerase II FIG.2. Pull down experiments of endogenous topo II and HDAC1. A, pull down of endogenous topo IIa by 5 mg of immobilized (bound) GST-HDAC1, but not by immobilized GST. Topo IIa is also depleted from the supernatant (unbound) by GST-HDAC1. B, pull down of endogenous HDAC1 (molecular mass, 62 kDa) by immobilized GST- topo IIa and topo IIb, containing the CTD of each isoform. C, pull down of deacetylase activity with immobilized GST-topo IIa CTD and topo IIb CTD. Bars represent ranges of two experiments. and topo IIb immunoprecipitate 6 –9% of total deacetylase ac- tivity from HeLa whole cell extract (Fig. 1B). The activity is fully inhibited by TSA. Negative control immunoprecipitations with preimmune serum, an irrelevant antibody (anti-CDK7), or FIG.1. Evidence for the physical association of topo IIa and HDAC1 proteins. A, Immunoprecipitation of endogenous topo IIa an antibody against DNA topoisomerase I (topo I) did not bring (molecular mass, 170 kDa), with the indicated antibodies (10 mg of total down activity above that of the no-antibody control. IgG), from 100 mg of HeLa whole cell extract (WCE). B, immunoprecipi- We performed in vitro pull down experiments of endogenous tation of deacetylase activity by antisera against topo IIa and topo IIb protein with GST fusion proteins. Full-length mammalian (18511a and 18513b) from HeLa WCE. Deacetylase activity is inhibited by TSA (10 ng/ml). Activity immunoprecipitated is expressed as a HDAC1, tagged with a GST moiety, but not GST itself, bound percentage of total input deacetylase activity, less a background of endogenous topo IIa in whole cell extract (Fig. 2A). In the nonenzymatic release of [ H]acetate of 1.94%. Percentages are averages converse experiment, a GST fusion protein containing the C- of three separate experiments, with bars representing S.D. terminal domain (CTD) of topo IIa was able to pull down endogenous HDAC1 (Fig. 2B). The fusion protein of the CTD of assayed on plates (6), in the presence of 25 mM 3-amino-1,2,4-triazole to topo IIb was also able to pull down small amounts of HDAC1 suppress background growth (18). (Fig. 2B). Fusion proteins of the CTD of topo IIa and topo IIb Enzyme Assays—Histone deacetylase activity was assayed as de- were able to pull down between 9 and 11% of deacetylase scribed previously (17). DNA strand-passage assays were performed on kinetoplast DNA (kDNA) as described previously (6). activity (Fig. 2C), comparable with the amounts brought down Detection of Apoptosis—HL-60 cells were grown until in mid-log by immunoprecipitation (Fig. 1B). Whereas GST-topo IIb pulls phase, then treated with 100 nM (30 ng/ml) TSA for 0.5 h before down less HDAC1, as detected on Western blots, than compa- additional treatments with either 100 mM (59 mg/ml) etoposide or 5.8 mM rable amounts of GST-topo IIa (Fig. 2B), the two different (2 mg/ml) camptothecin for 1.5 h. Control samples were treated with the fusion proteins bring down similar amounts of deacetylase dilution vehicles (0.1% Me SO and 0.1% ethanol). All cells were ob- activity (Fig. 2C). A possible explanation for this quantitative served in situ with phase-contrast microscopy to count cells with an apoptotic morphology, after staining with 10 mM Hoechst 33342 with discrepancy is that other deacetylases, in addition to HDAC1, the addition of 0.1 mM propidium iodide to visualize necrotic cells. Cells are preferentially associated with topo IIb. were also labeled with either FITC-annexin V conjugate (PharMingen) GST fusion proteins containing the C-terminal domain of TM or with the FAM-VAD-FMK reagent provided in the CaspaTag fluo- HDAC1 interact with recombinant topo IIa (Fig. 3). This do- rescein caspase activity kit (Intergen). Labeled cells were detected by main has previously been shown to contain the LXCXE motif indirect fluorescence microscopy (all cells) or by FACS analysis on a (residues 414 – 418), that appears to mediate interactions with Coulter Epics flow cytometer. the retinoblastoma protein pRb (19). In contrast, an N-terminal RESULTS HDAC1 fusion protein, containing the catalytic site, showed Interaction of HDAC1 and DNA Topoisomerase II—HeLa minimal interaction with recombinant topo IIa (Fig. 3). whole cell extract was immunoprecipitated with affinity-puri- A yeast two-hybrid system (18) was used to test for direct in fied antibody against mammalian HDAC1 and precipitated vivo interaction between topo II and HDAC1. Inserts were material tested for the presence of topo IIa by Western blotting constructed to express the topo IIa and topo IIb C-terminal (Fig. 1A). Anti-HDAC1 brought down easily detectable domains (6) and the HDAC1 region 220 – 482 (Fig. 3). Expres- amounts of topo IIa. There was no detectable immunoprecipi- sion of the integrated, GAL4-dependent HIS3 reporter gene tation of topo IIa with preimmune serum, and immunoprecipi- was used to detect interactions between “bait” and “prey” pro- tation was completely abolished by inclusion in the incubation teins in vivo. Topo IIa CTD or topo IIb CTD as bait, together mix of the peptide used to raise the anti-HDAC1 antibody (Fig. with HDAC1 as prey, allowed growth of large colonies (over 2 1A). The anti-HDAC1 antibody did not immunoprecipitate de- mm diameter) on His-selective medium. All three proteins were tectable levels of topo IIb (data not shown), and antibody to ineffective when expressed individually (Fig. 4). topo IIb brought down only a comparatively small amount of To explore the biological significance of the topo II-HDAC1 the a isoform (Fig. 1A). However, antisera against both topo IIa interaction, we tested the ability of full-length recombinant HDAC1 Interacts with DNA Topoisomerase II 4541 FIG.5. Functional association of topo II and histone deacety- lase. Decatentation of 1.4 mg of kinetoplast DNA concatemer (concat.) to monomers of minicircle DNA by recombinant topo IIa (0.60 pmol) and topo IIb (0.22 pmol) is inhibited by increasing amounts of GST-HDAC1 FIG.3. In vitro interaction of recombinant topo IIa and (1.4 –13.9 pmol), but not by GST alone (4.2–13.9 pmol). HDAC1. Regions of HDAC1 cDNA were subcloned and expressed as GST fusion proteins. Immobilized fusion proteins (5 mg) were tested for interaction with 1 mg of recombinant topo IIa. FIG.6. TSA suppresses apoptosis induced by topo II-mediated DNA damage, but not by topo I inhibition, in HL-60 cells. A, cells were treated as indicated (see “Materials and Methods” for details). Nuclear changes were visualized by indirect fluorescence microscopy after staining with Hoechst 33342 with the addition of propidium iodide (PI) to visualize necrotic cells. The nuclear morphology of cells were scored for apoptotic cells (PI2; clear bars) and necrotic cells (PI1; gray bars) and expressed as percentages of total cells in the field of view. Values are averages of two separate experiments. B, plasma membrane changes were detected by the binding of annexin V conjugated to FITC, FIG.4. Yeast two-hybrid assay showing that the CTDs of topo after treatment with TSA, etoposide, or camptothecin as indicated (see IIa and topo IIb can interact with HDAC1 CTD (amino acids “Materials and Methods”). Staining was visualized by indirect fluores- 220 – 482) in vivo. Expression of the reporter gene HIS3 in yeast stain cence microscopy and expressed as percentages of total cells. Bars CG-1945 (CLONTECH) was determined by two parallel series of spot represent S.D. values for three separate experiments. C, broad-spec- assays on selective medium plates lacking tryptophan, leucine, and trum caspase activity was detected by labeling live cells with the FAM- histidine (2Trp, 2Leu, 2His), but in the presence of 25 mM 3-amino- VAD-FMK fluorescein-conjugated caspase substrate (see “Materials 1,2,4-triazole to suppress background growth (6, 18). Colony size was and Methods”) after treatment with inhibitors, as indicated. Labeled compared with that on plates lacking tryptophan and leucine (2Trp cells were detected by FACS analysis, and activity is expressed as the 2Leu) as control. Strong growth in 2His medium occurs only in cells in percentage of the viable cell subpopulation that stains positive for the which the bait and prey proteins physically interact. substrate. Values are averages of two separate experiments. HDAC1 to modulate the functional properties of recombinant environment (22). This change was measured by binding of topo IIa and -b. Both of these enzymes can decatenate kineto- FITC-conjungated annexin V and counting of labeled cells by plast DNA (kDNA) to minicircle monomers, a process that fluorescence microscopy (Fig. 6B). Activation of cysteine as- requires a double-stranded break in the kDNA to allow strand partyl proteases (caspases) (21) during the apoptosis of passage. The addition of increasing amounts of HDAC1 to the HL-60 cells was assayed with a fluorescent substrate and reaction decreases the decatenation of kDNA by topo IIa and -b FACS analysis of viable cells (Fig. 6C). Chromatin condensa- (Fig. 5). Addition of GST alone did not affect decatenation by tion, membrane changes, and caspase activation all demon- either topo IIa and -b. strated that prior treatment with 100 nM TSA suppresses the Suppression of Etoposide-mediated Apoptosis by the HDAC apoptotic effect of etoposide (Fig. 6, A–C). In contrast, TSA Inhibitor Trichostatin A—We tested the effect of the HDAC did not affect apoptosis induced by the topo I inhibitor camp- inhibitor trichostatin A (20) on apoptosis induced by the tothecin (Fig. 6, A–C). Note that topo I does not associate with chemotherapeutic agent etoposide (VP-16). Etoposide causes detectable amounts of deacetylase activity (Fig. 1B). An iden- topoisomerase II-mediated DNA damage by increasing the tical anti-apoptotic effect of TSA treatment was also observed steady-state concentration of covalent DNA cleavage com- for the human lung adenocarcinoma cell line H1299 and plexes (1, 2, 4). Cells treated with etoposide acquire an apo- HeLa cells (data not shown). ptotic morphology, notably the condensation of chromatin at DISCUSSION the nuclear periphery and blebbing of the plasma membrane (2, 21). HL-60 cells displayed apoptotic chromatin condensa- The results presented show that the histone deacetylase tion after only 1.5-h treatment with either 100 mM etoposide HDAC1 is physically associated with each of the two isoforms or 5.8 mM camptothecin, an inhibitor of topo I (Fig. 6A). of human topoisomerase II, topo IIa and topo IIb. The associ- Plasma membrane changes during early apoptosis include ation occurs in vivo, being detectable by coimmunoprecipitation the exposure of phosphatidylserine to the external cellular from human cell extracts and by yeast two-hybrid assay. It also 4542 HDAC1 Interacts with DNA Topoisomerase II occurs in vitro. GST-coupled recombinant topo IIa and topo IIb associated with HDAC1. Further support for these ideas comes pull down significant amounts of HDAC activity from cell ex- from the recent results of Tsai et al. (23), who show that topo II tracts, while recombinant HDAC1 inhibits the in vitro decat- is associated not only with HDAC1/2, but also with MTA2, a enation activity of recombinant topo IIa. Since completion of protein that is part of the NuRD chromatin remodeling com- the work reported here, Tsai et al. (23) have reported essen- plex. The NuRD complex contains both HDAC1/2 and Mi-2, a tially the same findings for the two very similar deacetylases protein with ATPase/helicase activity (12). HDAC1 and HDAC2. Interestingly, whereas Tsai et al. (23) We and others (23) find that HDAC1 can inhibit the catalytic find evidence for an interaction between topo IIa and various activity of topo II in vitro. This is an important confirmation of regions of HDAC2, including N-terminal residues 1–57, we find the ability of topo II and HDAC1 to interact, but is not, at first that only the C-terminal region of HDAC1 (residues 220 – 482) sight, consistent with the proposition outlined above that interacts with topo II in vitro. These two deacetylases seem to HDAC activity facilitates topo II catalysis, or its consequences. differ in their mode of interaction with topo II. This can be resolved by noting that the GST-HDAC1 construct In experiments to assess the biological significance of the used to inhibit topo II in vitro is catalytically inactive, presum- topo II-HDAC interaction, we analyzed the effect of the ably because it lacks essential protein partners such as deacetylase inhibitor TSA on processes known to require topo RbAp46/48 (8, 11). It would be wrong to assume that catalyti- II activity. The most striking effect so far has been on the cally active HDAC1, in the context of a multiprotein complex ability of the topo II poison etoposide to drive cells into apo- that includes both HDAC and chromatin remodeling activities ptosis. We show that treatment with TSA prior to the addition (23), is also inhibitory. It might even be the case that, in vivo, of etoposide suppresses apoptosis in a variety of cell lines. The topo II activity is inhibited only by association with HDAC effect is seen even with HL60 cells, in which apoptosis is rendered catalytically inactive by inhibitors such as TSA. Such detectable within less than 1 h, a finding that minimizes the an effect would complement the suppression of topo II activity probability that inhibition of apoptosis is due to pleiotropic brought about by inhibition of chromatin remodeling. effects of TSA, such as its ability to alter cell cycle progression. The inhibitory effect of TSA was detected in several p53-null Acknowledgments—We thank Dr. G. Anderson for help with the FACS analysis, Dr. J. Shuttleworth for the gift of anti-CDK7 antibody cell lines, so the interaction between HDAC1 and p53 (7) can- and GST-cdc2 fusion protein construct, Darren A. White and Jayne S. not be responsible. 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Published: Feb 1, 2001

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