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• W2147791710 abstract "DNA topoisomerase II (topo II) is a ubiquitous nuclear enzyme that is involved in DNA replication, transcription, chromosome segregation, and apoptosis. Here we show by immunoprecipitation, pull down with glutathioneS-transferase fusion proteins, and yeast two-hybrid analysis that both topo IIα and -β physically interact with the histone deacetylase HDAC1. The in vitro DNA decatenation activity of recombinant topo IIα and -β is inhibited by association with catalytically inactive, recombinant HDAC1. We provide evidence for the in vivo significance of the topo II-HDAC1 association, showing that inhibition of HDAC activity with trichostatin A suppresses apoptosis induced by the topo II poison etoposide, but not by the topoisomerase I inhibitor camptothecin. We suggest that chromatin remodeling by an HDAC-containing complex facilitates both topo II-catalyzed DNA rearrangement and etoposide-induced DNA damage in vivo. DNA topoisomerase II (topo II) is a ubiquitous nuclear enzyme that is involved in DNA replication, transcription, chromosome segregation, and apoptosis. Here we show by immunoprecipitation, pull down with glutathioneS-transferase fusion proteins, and yeast two-hybrid analysis that both topo IIα and -β physically interact with the histone deacetylase HDAC1. The in vitro DNA decatenation activity of recombinant topo IIα and -β is inhibited by association with catalytically inactive, recombinant HDAC1. We provide evidence for the in vivo significance of the topo II-HDAC1 association, showing that inhibition of HDAC activity with trichostatin A suppresses apoptosis induced by the topo II poison etoposide, but not by the topoisomerase I inhibitor camptothecin. We suggest that chromatin remodeling by an HDAC-containing complex facilitates both topo II-catalyzed DNA rearrangement and etoposide-induced DNA damage in vivo. topoisomerase trichostatin A glutathioneS-transferase fluorescein isothiocyanate fluorescence-activated cell sorter C-terminal domain retinoblastoma histone deacetylase For completion of cell division, the DNA of replicated chromosomes must be disentangled to allow the segregation of sister chromatids. In humans, this is achieved by the unique decatenation activity of DNA topoisomerase II (topo1 II). Topo II is essential for normal and neoplastic cellular proliferation, and several common anti-cancer drugs exert their cytotoxic effects through this enzyme (1Froelich-Ammon S.J. Osheroff N. J. Biol. Chem. 1995; 270: 21429-21432Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1071) Google Scholar). Topoisomerase II activity in mammalian cells has been attributed to at least two isoforms. Topo IIα (p170) associates with chromosomes during prophase and throughout mitosis and is thought to be a major component of the nuclear scaffold (3Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar, 4Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (501) Google Scholar). It has a peak of expression during G2/M of the cell cycle (5Austin C.A. Marsh K.L. Bioessays. 1998; 20: 215-226Crossref PubMed Scopus (231) Google Scholar). In contrast, the closely related topo IIβ (p180) isoform is thought to have a more general role in DNA metabolism, with expression levels that remain relatively constant during cell and growth cycles (5Austin C.A. Marsh K.L. Bioessays. 1998; 20: 215-226Crossref PubMed Scopus (231) Google Scholar). Both isoforms interact with the C-terminal region of the tumor suppressor protein, p53 (6Cowell I.G. Okorokov A.L. Cutts S.A. Padget K. Bell M. Milner J. Austin C.A. Exp. Cell Res. 2000; 255: 86-94Crossref PubMed Scopus (54) Google Scholar). p53 is a component of a multiprotein complex that contains the histone deacetylase HDAC1 and the corepressor Sin3a (7Murphy M. Ahn J. Walker K.K. Hoffman W.H. Evans R.M. Levine A.J. George D.L. Genes Dev. 1999; 13: 2490-2501Crossref PubMed Scopus (394) Google Scholar, 8Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1542) Google Scholar, 9Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1557) Google Scholar, 10Johnson C.A. Turner B.M. Semin. Cell Dev. Biol. 1999; 10: 179-188Crossref PubMed Scopus (93) Google Scholar, 11Ng H.H. Bird A. Trends Biochem. Sci. 2000; 25: 121-126Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). HDAC1, and the closely related HDAC2, are both components of two separate multiprotein complexes. The NuRD/Mi-2 repression complex contains both nucleosome remodeling and histone deacetylase activities (12Zhang Y. LeRoy G. Seelig H.-P. Lane W.S. Reinberg D. Cell. 1998; 95: 279-289Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar), whereas the Sin3 complex contains only the latter (9Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1557) Google Scholar). Both complexes contain the Rb-associated proteins RbAp46 and RbAp48 and associate with various, sometimes DNA-binding, transcriptional repressor and corepressor proteins (11Ng H.H. Bird A. Trends Biochem. Sci. 2000; 25: 121-126Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The Xenopus NuRD complex (which contains homologues of mammalian HDAC1, RbAp48, and the methyl-CpG-binding protein MBD3) copurifies with DNA topoisomerase II (13Wade P.A. Gegonne A. Jones P.L. Ballestar E. Aubry F. Wolffe A.P. Nat. Genet. 1999; 23: 62-66Crossref PubMed Scopus (713) Google Scholar), raising the possibility that mammalian topo II isoforms and HDAC1 may interact in a multiprotein complex. Here we show that HDAC1 and DNA topoisomerase II isoforms physically interact both in vivo and in vitro. We also show that the HDAC inhibitor, TSA, suppresses apoptosis induced by the topo II poison etoposide, but not by the topo I inhibitor camptothecin. Our results raise the interesting possibility that chromatin remodeling by a topo II-HDAC-containing complex is involved in topo II-catalyzed DNA rearrangements and/or generation of etoposide-induced DNA strand breaksin vivo. The human cell lines HL-60 (promyelocytic leukemia; p53 null) and HeLa were grown in RPMI 1640 medium containing 8% fetal calf serum. Regions of HDAC1 cDNA were subcloned into the pGEX3T-4 family of vectors (Amersham Pharmacia Biotech) and verified by sequencing. GST fusion proteins were purified essentially as described previously (6Cowell I.G. Okorokov A.L. Cutts S.A. Padget K. Bell M. Milner J. Austin C.A. Exp. Cell Res. 2000; 255: 86-94Crossref PubMed Scopus (54) Google Scholar). Recombinant human DNA topoisomerase IIα and -β were made in a yeast system and purified as described previously (14Austin C.A. Marsh K.L. Wasserman R.A. Willmore E. Sayer P.J. Wang J.C. Fisher L.M. J. Biol. Chem. 1995; 270: 15739-15746Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Characterization and use of rabbit polyclonal antibodies against topo IIα (18511α) and topo IIβ (18513β) are described elsewhere (15Cowell I.G. Willmore E. Chalton D. Marsh K.L. Jazrawi E. Fisher L.M. Austin C.A. Exp. Cell Res. 1998; 243: 232-240Crossref PubMed Scopus (38) Google Scholar). A polyclonal rabbit antibody against mammalian HDAC1 was raised against a synthetic peptide corresponding to amino acid residues 467–482 and affinity-purified as described previously (16White D.A. Belyaev N.D. Turner B.M. Methods Companion Methods Enzymol. 1999; 10: 417-424Crossref Scopus (61) Google Scholar). Antibody against topo I was obtained commercially (TopoGen, number 2012). HeLa whole cell extract was prepared by lysing cells in incubation buffer (50 mm Tris, pH 7.5, 150 mmNaCl, 5 mm EDTA, 5 mm EGTA, 10% (v/v) glycerol) containing 1.0% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, protease inhibitors (0.1 mmphenylmethylsulfonyl fluoride and “Complete MiniTM” tablets, Roche Molecular Biochemicals) and 50 units of DNase I (Amersham Pharmacia Biotech) per 108cells. The lysate was incubated in ice for 10 min, and the clarified supernatant was used in standard immunoprecipitations as described previously (6Cowell I.G. Okorokov A.L. Cutts S.A. Padget K. Bell M. Milner J. Austin C.A. Exp. Cell Res. 2000; 255: 86-94Crossref PubMed Scopus (54) Google Scholar, 17Nan X. Ng H.-H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar). To confirm specificity, cognate blocking peptide (10 μg) was incubated with the antibody for 30 min before the addition of extract. Preimmune serum and irrelevant antisera were used as controls. GST pull down experiments used equivalent amounts of GST fusion proteins prebound to glutathione-Sepharose beads (Amersham Pharmacia Biotech) as described previously (6Cowell I.G. Okorokov A.L. Cutts S.A. Padget K. Bell M. Milner J. Austin C.A. Exp. Cell Res. 2000; 255: 86-94Crossref PubMed Scopus (54) Google Scholar). Interactions with recombinant topo IIα were performed in incubation buffer containing 0.1% (v/v) Nonidet P-40. Yeast strains CG-1945 from a Matchmaker Two-Hybrid System II kit (CLONTECH) were transformed with appropriate binary combinations of constructs containing the GAL4 DNA-binding domain and the GAL4 activation domain, as recommended by the manufacturers.HIS3 reporter gene expression was assayed on plates (6Cowell I.G. Okorokov A.L. Cutts S.A. Padget K. Bell M. Milner J. Austin C.A. Exp. Cell Res. 2000; 255: 86-94Crossref PubMed Scopus (54) Google Scholar), in the presence of 25 mm 3-amino-1,2,4-triazole to suppress background growth (18Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar). Histone deacetylase activity was assayed as described previously (17Nan X. Ng H.-H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar). DNA strand-passage assays were performed on kinetoplast DNA (kDNA) as described previously (6Cowell I.G. Okorokov A.L. Cutts S.A. Padget K. Bell M. Milner J. Austin C.A. Exp. Cell Res. 2000; 255: 86-94Crossref PubMed Scopus (54) Google Scholar). HL-60 cells were grown until in mid-log phase, then treated with 100 nm (30 ng/ml) TSA for 0.5 h before additional treatments with either 100 μm (59 μg/ml) etoposide or 5.8 μm (2 μg/ml) camptothecin for 1.5 h. Control samples were treated with the dilution vehicles (0.1% Me2SO and 0.1% ethanol). All cells were observed in situ with phase-contrast microscopy to count cells with an apoptotic morphology, after staining with 10 μm Hoechst 33342 with the addition of 0.1 μm propidium iodide to visualize necrotic cells. Cells were also labeled with either FITC-annexin V conjugate (PharMingen) or with the FAM-VAD-FMK reagent provided in the CaspaTagTM fluorescein caspase activity kit (Intergen). Labeled cells were detected by indirect fluorescence microscopy (all cells) or by FACS analysis on a Coulter Epics flow cytometer. HeLa whole cell extract was immunoprecipitated with affinity-purified antibody against mammalian HDAC1 and precipitated material tested for the presence of topo IIα by Western blotting (Fig.1 A). Anti-HDAC1 brought down easily detectable amounts of topo IIα. There was no detectable immunoprecipitation of topo IIα with preimmune serum, and immunoprecipitation was completely abolished by inclusion in the incubation mix of the peptide used to raise the anti-HDAC1 antibody (Fig. 1 A). The anti-HDAC1 antibody did not immunoprecipitate detectable levels of topo IIβ (data not shown), and antibody to topo IIβ brought down only a comparatively small amount of the α isoform (Fig. 1 A). However, antisera against both topo IIα and topo IIβ immunoprecipitate 6–9% of total deacetylase activity from HeLa whole cell extract (Fig. 1 B). The activity is fully inhibited by TSA. Negative control immunoprecipitations with preimmune serum, an irrelevant antibody (anti-CDK7), or an antibody against DNA topoisomerase I (topo I) did not bring down activity above that of the no-antibody control. We performed in vitro pull down experiments of endogenous protein with GST fusion proteins. Full-length mammalian HDAC1, tagged with a GST moiety, but not GST itself, bound endogenous topo IIα in whole cell extract (Fig. 2 A). In the converse experiment, a GST fusion protein containing the C-terminal domain (CTD) of topo IIα was able to pull down endogenous HDAC1 (Fig. 2 B). The fusion protein of the CTD of topo IIβ was also able to pull down small amounts of HDAC1 (Fig. 2 B). Fusion proteins of the CTD of topo IIα and topo IIβ were able to pull down between 9 and 11% of deacetylase activity (Fig.2 C), comparable with the amounts brought down by immunoprecipitation (Fig. 1 B). Whereas GST-topo IIβ pulls down less HDAC1, as detected on Western blots, than comparable amounts of GST-topo IIα (Fig. 2 B), the two different fusion proteins bring down similar amounts of deacetylase activity (Fig.2 C). A possible explanation for this quantitative discrepancy is that other deacetylases, in addition to HDAC1, are preferentially associated with topo IIβ. GST fusion proteins containing the C-terminal domain of HDAC1 interact with recombinant topo IIα (Fig.3). This domain has previously been shown to contain the LXCXE motif (residues 414–418), that appears to mediate interactions with the retinoblastoma protein pRb (19Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1080) Google Scholar). In contrast, an N-terminal HDAC1 fusion protein, containing the catalytic site, showed minimal interaction with recombinant topo IIα (Fig. 3). A yeast two-hybrid system (18Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar) was used to test for direct in vivo interaction between topo II and HDAC1. Inserts were constructed to express the topo IIα and topo IIβ C-terminal domains (6Cowell I.G. Okorokov A.L. Cutts S.A. Padget K. Bell M. Milner J. Austin C.A. Exp. Cell Res. 2000; 255: 86-94Crossref PubMed Scopus (54) Google Scholar) and the HDAC1 region 220–482 (Fig. 3). Expression of the integrated, GAL4-dependent HIS3 reporter gene was used to detect interactions between “bait” and “prey” proteins in vivo. Topo IIα CTD or topo IIβ CTD as bait, together with HDAC1 as prey, allowed growth of large colonies (over 2 mm diameter) on His-selective medium. All three proteins were ineffective when expressed individually (Fig. 4). To explore the biological significance of the topo II-HDAC1 interaction, we tested the ability of full-length recombinant HDAC1 to modulate the functional properties of recombinant topo IIα and -β. Both of these enzymes can decatenate kinetoplast DNA (kDNA) to minicircle monomers, a process that requires a double-stranded break in the kDNA to allow strand passage. The addition of increasing amounts of HDAC1 to the reaction decreases the decatenation of kDNA by topo IIα and -β (Fig. 5). Addition of GST alone did not affect decatenation by either topo IIα and -β. We tested the effect of the HDAC inhibitor trichostatin A (20Yoshida M. Horinouchi S. Beppu T. Bioessays. 1995; 17: 423-430Crossref PubMed Scopus (667) Google Scholar) on apoptosis induced by the chemotherapeutic agent etoposide (VP-16). Etoposide causes topoisomerase II-mediated DNA damage by increasing the steady-state concentration of covalent DNA cleavage complexes (1Froelich-Ammon S.J. Osheroff N. J. Biol. Chem. 1995; 270: 21429-21432Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1071) Google Scholar, 4Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (501) Google Scholar). Cells treated with etoposide acquire an apoptotic morphology, notably the condensation of chromatin at the nuclear periphery and blebbing of the plasma membrane (2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1071) Google Scholar, 21Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 69: 383-424Crossref Scopus (2450) Google Scholar). HL-60 cells displayed apoptotic chromatin condensation after only 1.5-h treatment with either 100 μm etoposide or 5.8 μm camptothecin, an inhibitor of topo I (Fig. 6 A). Plasma membrane changes during early apoptosis include the exposure of phosphatidylserine to the external cellular environment (22Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995; 184: 39-51Crossref PubMed Scopus (4627) Google Scholar). This change was measured by binding of FITC-conjungated annexin V and counting of labeled cells by fluorescence microscopy (Fig.6 B). Activation of cysteine aspartyl proteases (caspases) (21Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 69: 383-424Crossref Scopus (2450) Google Scholar) during the apoptosis of HL-60 cells was assayed with a fluorescent substrate and FACS analysis of viable cells (Fig. 6 C). Chromatin condensation, membrane changes, and caspase activation all demonstrated that prior treatment with 100 nm TSA suppresses the apoptotic effect of etoposide (Fig. 6, A–C). In contrast, TSA did not affect apoptosis induced by the topo I inhibitor camptothecin (Fig. 6, A–C). Note that topo I does not associate with detectable amounts of deacetylase activity (Fig.1 B). An identical anti-apoptotic effect of TSA treatment was also observed for the human lung adenocarcinoma cell line H1299 and HeLa cells (data not shown). The results presented show that the histone deacetylase HDAC1 is physically associated with each of the two isoforms of human topoisomerase II, topo IIα and topo IIβ. The association occursin vivo, being detectable by coimmunoprecipitation from human cell extracts and by yeast two-hybrid assay. It also occursin vitro. GST-coupled recombinant topo IIα and topo IIβ pull down significant amounts of HDAC activity from cell extracts, while recombinant HDAC1 inhibits the in vitro decatenation activity of recombinant topo IIα. Since completion of the work reported here, Tsai et al. (23Tsai S.C. Valkov N. Yang W.M. Gump J. Sullivan D. Seto E. Nat. Genet. 2000; 26: 349-353Crossref PubMed Scopus (134) Google Scholar) have reported essentially the same findings for the two very similar deacetylases HDAC1 and HDAC2. Interestingly, whereas Tsai et al. (23Tsai S.C. Valkov N. Yang W.M. Gump J. Sullivan D. Seto E. Nat. Genet. 2000; 26: 349-353Crossref PubMed Scopus (134) Google Scholar) find evidence for an interaction between topo IIα and various regions of HDAC2, including N-terminal residues 1–57, we find that only the C-terminal region of HDAC1 (residues 220–482) interacts with topo II in vitro. These two deacetylases seem to differ in their mode of interaction with topo II. In experiments to assess the biological significance of the topo II-HDAC interaction, we analyzed the effect of the deacetylase inhibitor TSA on processes known to require topo II activity. The most striking effect so far has been on the ability of the topo II poison etoposide to drive cells into apoptosis. We show that treatment with TSA prior to the addition of etoposide suppresses apoptosis in a variety of cell lines. The effect is seen even with HL60 cells, in which apoptosis is detectable within less than 1 h, a finding that minimizes the probability that inhibition of apoptosis is due to pleiotropic effects of TSA, such as its ability to alter cell cycle progression. The inhibitory effect of TSA was detected in several p53-null cell lines, so the interaction between HDAC1 and p53 (7Murphy M. Ahn J. Walker K.K. Hoffman W.H. Evans R.M. Levine A.J. George D.L. Genes Dev. 1999; 13: 2490-2501Crossref PubMed Scopus (394) Google Scholar) cannot be responsible. Microscopically detectable chromatin remodeling is a diagnostic characteristic of cells in the later stages of apoptosis, and recent reports indicate that both topo II and histone acetylation play a role in this process (24Durrieu F. Samejima K. Fortune J.M. Kandels-Lewis S. Osheroff N. Earnshaw W.C. Curr. Biol. 2000; 10: 923-926Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). However, our results indicate that this is not the stage at which TSA exerts the inhibitory effect reported here. We have shown that TSA inhibition is detectable even when using an assay that measures one of the earliest changes of apoptosis, namely the alteration in membrane phospholipids detected by binding of annexin V (22Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995; 184: 39-51Crossref PubMed Scopus (4627) Google Scholar). These findings argue that TSA is acting at a relatively early stage in apoptosis, prior to the onset of major changes in nuclear ultrastructure. It remains possible that TSA also effects more subtle chromatin changes, possibly those determining expression of genes required for progression through apoptosis (25Green D.R. Cell. 2000; 102: 1-4Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). These effects are not mutually exclusive. Indeed, recent results indicate that both topo II and changes in acetylation act at various stages in the pathways by which cells progress through apoptosis (24Durrieu F. Samejima K. Fortune J.M. Kandels-Lewis S. Osheroff N. Earnshaw W.C. Curr. Biol. 2000; 10: 923-926Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar,26Allera C. Lazzarini G. Patrone E. Alberti I. Barboro P. Sanna P. Melchiori A. Parodi S. Balbi C. J. Biol. Chem. 1997; 272: 10817-10822Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 27Ikura T. Ogryzko V.V. Grigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 28Kratzmeier M. Albig W. Hanecke K. Doenecke D. J. Biol. Chem. 2000; 275: 30478-30486Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In attempting to explain the effect of TSA on etoposide-induced apoptosis, it is important to note that etoposide is a topo II poison that blocks the enzyme after DNA cleavage but prior to strand passage (4Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (501) Google Scholar). Covalent topoisomerase-DNA cleavage complexes accumulate in the presence of such poisons. DNA replication, transcription, or helicase activity all disrupt these complexes, releasing the DNA double-strand breaks that can precipitate apoptosis (1Froelich-Ammon S.J. Osheroff N. J. Biol. Chem. 1995; 270: 21429-21432Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 4Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (501) Google Scholar). Reducing either the accumulation of topo II-DNA complexes, or their breakdown, will both reduce DNA damage and hence apoptosis. A possible explanation for the results presented here is that HDAC-dependent chromatin remodeling is necessary for the initiation of topo II-catalyzed DNA rearrangement or for dissociation of topo II-DNA complexes, or both. If this were the case, then HDAC inhibitors such as TSA would be expected to prevent the appearance of DNA damage in the presence of topo II poisons, and consequent progression into apoptosis, exactly as we have found. Crucially, TSA has no effect on apoptosis induced by the topo I inhibitor camptothecin. We show here that topo I is not associated with HDAC1. Further support for these ideas comes from the recent results of Tsai et al. (23Tsai S.C. Valkov N. Yang W.M. Gump J. Sullivan D. Seto E. Nat. Genet. 2000; 26: 349-353Crossref PubMed Scopus (134) Google Scholar), who show that topo II is associated not only with HDAC1/2, but also with MTA2, a protein that is part of the NuRD chromatin remodeling complex. The NuRD complex contains both HDAC1/2 and Mi-2, a protein with ATPase/helicase activity (12Zhang Y. LeRoy G. Seelig H.-P. Lane W.S. Reinberg D. Cell. 1998; 95: 279-289Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). We and others (23Tsai S.C. Valkov N. Yang W.M. Gump J. Sullivan D. Seto E. Nat. Genet. 2000; 26: 349-353Crossref PubMed Scopus (134) Google Scholar) find that HDAC1 can inhibit the catalytic activity of topo II in vitro. This is an important confirmation of the ability of topo II and HDAC1 to interact, but is not, at first sight, consistent with the proposition outlined above that HDAC activity facilitates topo II catalysis, or its consequences. This can be resolved by noting that the GST-HDAC1 construct used to inhibit topo II in vitro is catalytically inactive, presumably because it lacks essential protein partners such as RbAp46/48 (8Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1542) Google Scholar, 11Ng H.H. Bird A. Trends Biochem. Sci. 2000; 25: 121-126Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). It would be wrong to assume that catalytically active HDAC1, in the context of a multiprotein complex that includes both HDAC and chromatin remodeling activities (23Tsai S.C. Valkov N. Yang W.M. Gump J. Sullivan D. Seto E. Nat. Genet. 2000; 26: 349-353Crossref PubMed Scopus (134) Google Scholar), is also inhibitory. It might even be the case that, in vivo, topo II activity is inhibited only by association with HDAC rendered catalytically inactive by inhibitors such as TSA. Such an effect would complement the suppression of topo II activity brought about by inhibition of chromatin remodeling. We thank Dr. G. Anderson for help with the FACS analysis, Dr. J. Shuttleworth for the gift of anti-CDK7 antibody and GST-cdc2 fusion protein construct, Darren A. White and Jayne S. Lavender for technical support." @default.
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• W2147791710 title "Deacetylase Activity Associates with Topoisomerase II and Is Necessary for Etoposide-induced Apoptosis" @default.
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