Cytoplasmic Sequestration of HDAC7 from Mitochondrial and Nuclear Compartments upon Initiation of Apoptosis

Control of global histone acetylation status is largely governed by the opposing enzymatic activities of histone acetyltransferases and deacetylases (HDACs). HDACs were originally identified as modulators of nuclear histone acetylation status and have been linked to chromosomal condensation and subsequent gene repression. Accumulating evidence highlights HDAC modification of non-histone targets. Mitochondria were first characterized as intracellular organelles responsible for energy production through the coupling of oxidative phosphorylation to respiration. More recently, mitochondria have been implicated in programmed cell death whereby release of pro-apoptotic inner membrane space factors facilitates apoptotic progression. Here we describe the novel discovery that the nuclear encoded Class II human histone deacetylase HDAC7 localizes to the mitochondrial inner membrane space of prostate epithelial cells and exhibits cytoplasmic relocalization in response to initiation of the apoptotic cascade. These results highlight a previously unrecognized link between HDACs, mitochondria, and programmed cell death. Control of global histone acetylation status is largely governed by the opposing enzymatic activities of histone acetyltransferases and deacetylases (HDACs). HDACs were originally identified as modulators of nuclear histone acetylation status and have been linked to chromosomal condensation and subsequent gene repression. Accumulating evidence highlights HDAC modification of non-histone targets. Mitochondria were first characterized as intracellular organelles responsible for energy production through the coupling of oxidative phosphorylation to respiration. More recently, mitochondria have been implicated in programmed cell death whereby release of pro-apoptotic inner membrane space factors facilitates apoptotic progression. Here we describe the novel discovery that the nuclear encoded Class II human histone deacetylase HDAC7 localizes to the mitochondrial inner membrane space of prostate epithelial cells and exhibits cytoplasmic relocalization in response to initiation of the apoptotic cascade. These results highlight a previously unrecognized link between HDACs, mitochondria, and programmed cell death. Originally identified as negative regulators of nuclear histone acetylation, HDACs 1The abbreviations used are: HDAC, histone deacetylase; IMS, inner membrane space; GFP, green fluorescent protein; FBS, fetal bovine serum; Tricine, N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine; AIF, apoptosis-inducing factor; PARP, poly(ADP-ribose) polymerase; NLS, nuclear localization sequence. have been intimately linked to chromatin condensation and subsequent gene repression (1Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6679) Google Scholar). More recently, increasing evidence has demonstrated HDAC modification of non-histone substrates (2North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1251) Google Scholar, 3Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1902) Google Scholar, 4Zhang Y. Li N. Caron C. Matthias G. Hess D. Khochbin S. Matthias P. EMBO J. 2003; 22: 1168-1179Crossref PubMed Scopus (583) Google Scholar) and an involvement in a broader array of biological events including apoptosis (5Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6Liu F. Dowling M. Yang X.J. Kao G.D. J. Biol. Chem. 2004; 279: 34537-34546Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 7Paroni G. Mizzau M. Henderson C. Del Sal G. Schneider C. Brancolini C. Mol. Biol. Cell. 2004; 15: 2804-2818Crossref PubMed Scopus (113) Google Scholar, 8Verdin E. Dequiedt F. Kasler H. Novartis Found. Symp. 2004; 259 (163-169): 115-131Crossref PubMed Google Scholar) and radiation sensitivity (9Zhang Y. Jung M. Dritschilo A. Radiat. Res. 2004; 161: 667-674Crossref PubMed Scopus (127) Google Scholar). Human Class I HDACs are generally nuclear proteins homologous to the yeast protein Rpd3 (HDAC1, -2, -3, and -8). Class II HDACs (HDAC4, -5, -6, -7, and -9) are related to HDA1 and often demonstrate regulated nucleocytoplasmic flux. Class III HDACs are structurally and phylogenetically distinct, being most similar to the NAD+-dependent yeast SIR2 proteins. HDAC7 is a nuclear encoded Class II HDAC having a conserved C-terminal catalytic domain and a large, highly divergent N-terminal domain implicated in muscle differentiation (10Dressel U. Bailey P.J. Wang S.C. Downes M. Evans R.M. Muscat G.E. J. Biol. Chem. 2001; 276: 17007-17013Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Cytoplasmic sequestration of HDAC7 can be enhanced by 14-3-3 protein interactions (11Kao H.Y. Verdel A. Tsai C.C. Simon C. Juguilon H. Khochbin S. J. Biol. Chem. 2001; 276: 47496-47507Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and observed during T cell receptor-mediated apoptosis (8Verdin E. Dequiedt F. Kasler H. Novartis Found. Symp. 2004; 259 (163-169): 115-131Crossref PubMed Google Scholar). Mitochondria were first characterized as intracellular organelles responsible for energy production through the coupling of oxidative phosphorylation to respiration. More recently, mitochondria have been implicated in genetically programmed cell death (12Desagher S. Martinou J.C. Trends Cell Biol. 2000; 10: 369-377Abstract Full Text Full Text PDF PubMed Scopus (1696) Google Scholar) whereby release of pro-apoptotic mitochondrial inner membrane space factors (13Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar) facilitates the progression of the apoptotic cascade. Dysregulation of the mitochondrial apoptotic program has been linked to both enhanced cell death (14Jordan J. Cena V. Prehn J.H. J. Physiol. Biochem. 2003; 59: 129-141Crossref PubMed Google Scholar) as well as hyperproliferative growth (15Hu W. Kavanagh J.J. Lancet Oncol. 2003; 4: 721-729Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). Here we describe the novel discovery that the nuclear encoded Class II human histone deacetylase HDAC7 localizes to the mitochondrial inner membrane space (IMS) of several human cell lines, in particular, prostate cancer epithelial cells. Upon induction of the apoptotic cascade, HDAC7 is released from mitochondria and, along with nuclear HDAC7, is redistributed to the cytoplasm. These results highlight a previously unrecognized link between mitochondria, histone deacetylases, and the initiation of apoptosis. Antibodies and Reagents—Mitochondrial lysate (M22430), ProLong® antifade reagent (P7481), and anti-oxidative complex V (3D5) were purchased from Molecular Probes. Antibodies to HDAC7 (H-273), Tom20 (F-10), AIF (E-1), cytochrome c (6H2), and Smac/DIABLO (C-20) were purchased from Santa Cruz Biotechnology. GFP-Bax was a kind gift from Dr. Tomas Vomastek, University of Virginia. HDAC7-FLAG was a kind gift from Dr. Eric Verdin, UCSF. C4-2 cells were originally obtained from the laboratory of Dr. Leyland Chung, University of Texas Southwestern. Other cell lines were obtained from laboratory frozen stocks and maintained as follows: MRC5CV1 (15% FBS in RPMI, 2 mm l-glutamine, penicillin/streptomycin, 1 mm sodium pyruvate, non-essential amino acids), AT5BIVA (20% FBS, penicillin/streptomycin, 2 mm l-glutamine, non-essential amino acids, 0.1% hydrocortisone), PC-3 (RPMI 1640, 10% FBS), and SQ20B (20% FBS, penicillin/streptomycin, 2 mm l-glutamine, non-essential amino acids, 0.1% hydrocortisone). Site-directed Mutagenesis—HDAC7-R8P site-directed mutagenesis was carried out using Stratagene QuikChange II XL site-directed mutagenesis kit according to the manufacturer's protocol using the following PAGE-purified primers: forward primer, 5′-GGTGGGCCAGCCGCCCCCAGTGG-3′; and reverse primer, 5′-CCACTGGGGGCGGCTGGCCCACC-3′. PCR cycling parameters were as follows: denaturing at 95° C for 50 s, annealing at 60° C for 50 s, and extension for 9 min at 68° C. Confocal and Non-confocal Immunofluorescent Microscopy—Confocal microscopy was carried out using an Olympus BX61 laser scanning confocal microscope using ×60 oil immersion objective with standard lasers and filter sets for fluorescein isothiocyanate and Texas Red analysis. 4′,6-diamidino-2-phenylindole (Sigma) staining was used for non-confocal identification of nucleic acid content. Subsequent confocal image acquisition and analysis were carried out using the Fluoview™ software package. Routine non-confocal indirect immunofluorescence was performed on a Nikon E600 microscope according to standard protocols using fluorescein isothiocyanate and Texas Red secondary antibodies (Jackson Immunologicals) and appropriate optical filter sets. Non-confocal image acquisition and analysis was performed using MetaVue™ (version 5.0.3) imaging analysis software (Universal Imaging Corp.). Computer Amino Acid Analysis—TopPredII and Kyte-Doolittle hydrophobicity plotting and mitochondrial targeting peptide prediction were performed online. 2R. Bakin and M. Jung, personal communication. Cell Transfections—Routine Lipofectin (Invitrogen) transfection of GFP-Bax was performed according to the manufacturer's protocols. GFP-HDAC7 stable C4-2 cells were selected for G418 and pooled as mass populations. Western Blotting—Protein samples were run on 10–20% gradient SDS-PAGE Tricine minigels (Invitrogen), blotted to nitrocellulose, and blocked in 5% blocking buffer (Bio-Rad). Primary antibodies were added for 1 h at room temperature, washed in 0.1% SDS, Tween. Secondary horseradish peroxidase-conjugated antibodies were added for 1 h at room temperature and washed in 0.1% SDS, Tween. Blots were developed in ECL reagent (Amersham Biosciences) and exposed to film (Amersham Biosciences). Mitochondria Isolation, Permeabilization, and Subfractionation— Mitochondria were isolated from 80% confluent C4-2 cell monolayers using a Pierce mitochondrial isolation kit according to the manufacturer's instructions with the addition of complete EDTA-free protease inhibitor mixture (Roche Applied Science). For permeabilization of the mitochondrial outer membrane (16Schulz I. Methods Enzymol. 1990; 192: 280-300Crossref PubMed Scopus (124) Google Scholar), cells were plated on glass coverslips for 3 days and then treated with either 0.01 or 0.5% saponin in 4% paraformaldehyde for 30 min at room temperature. Routine indirect immunofluorescence was subsequently carried out using the appropriate primary and secondary fluorescent antibodies. Mitochondrial subfractionation was carried out on freshly isolated mitochondria. Isolated mitochondria were immediately subfractionated according to protocols established by Greenawalt (17Greenawalt J.W. Methods Enzymol. 1974; 31: 310-323Crossref PubMed Scopus (245) Google Scholar). Briefly, purified mitochondria were resuspended in isolation medium (70 mm sucrose, 220 mm d-mannitol, 2 mm HEPES, 0.5 mg/ml bovine serum albumin adjusted to pH 7.4 with KOH) at a concentration of 100 mg/ml. An equal volume of stock 1.2% digitonin medium was then added and stirred gently on ice for 15 min. Three more volumes of isolation medium were then added and centrifuged at 10,000 × g for 10 min. The sediment consisted of the crude mitoplast (i.e. inner membrane and matrix components) fraction. The supernatant was removed and centrifuged at 144,000 × g for 60 min. Pelleted material contained mitochondrial outer membranes, whereas the remaining fluid consisted largely of soluble inner membrane proteins. Imaging of Live Cells—All live imaging was carried out using a Nikon TE300 live imaging system with ×60 oil immersion objective using MetaMorph 6.1 software (Universal Imaging Corporation). Mito-Tracker Red® was added to culture medium for 30 min according to the manufacturer's protocols. Mitochondrial Localization of Mammalian HDAC7—Nuclear encoded proteins destined for mitochondria contain cleavable N-terminal signaling peptides of degenerate amino acid length and composition that are necessarily removed after mitochondrial import (18Rapaport D. EMBO Rep. 2003; 4: 948-952Crossref PubMed Scopus (163) Google Scholar, 19Hartl F.U. Pfanner N. Nicholson D.W. Neupert W. Biochim. Biophys. Acta. 1989; 988: 1-45Crossref PubMed Scopus (547) Google Scholar). N-terminal primary amino acid analysis of human HDAC7 identified a novel mitochondrial targeting presequence (20Emanuelsson O. Nielsen H. Brunak S. von Heijne G. J. Mol. Biol. 2000; 300: 1005-1016Crossref PubMed Scopus (3638) Google Scholar) in both mammalian isoforms of HDAC7 that was not present in any other known human HDAC (Fig. 1A). This presequence was moderately conserved in both rat and mouse, albeit with divergent N-terminal amino acid additions of unknown biological significance. Mitochondrial targeting presequences often exhibit a conserved amphipathic α-helix containing clustered positively charged hydrophobic and hydroxylated amino acid residues (21Schwer B. North B.J. Frye R.A. Ott M. Verdin E. J. Cell Biol. 2002; 158: 647-657Crossref PubMed Scopus (465) Google Scholar). HDAC7 secondary structure analysis of the N terminus revealed a clustering of basic amino acids commonly observed in amphipathic α-helical structures, including the NAD+-dependent and mitochondrial localized human Class III deacetylase SIRT3 (Fig. 1B). Confocal laser microscopy of untreated human prostate epithelial C4-2 cells revealed robust and distinct colocalization of endogenous HDAC7 with the mitochondria-specific proteins Hsp60, Tom20, and AIF (Fig. 2A). Similar mitochondria-specific localization of HDAC7 was observed in other human cell lines including AT5BIVA and MR5CV1 fibroblasts as well as PC-3 epithelial cells (Fig. 2B), suggesting that mitochondrial HDAC7 localization may be a general biological phenomenon of human cells. Live imaging of stable expression of N-terminal GFP-tagged HDAC7 (GFP-HDAC7) similarly exhibited colocalization with the mitochondria-specific dye MitoTracker Red (Fig. 2C). HDAC7 Is N-terminally Processed in Mitochondria—By an incompletely understood mechanism, three peptidases mediate a physiologically necessary endoproteolytic cleavage of both nuclear and mitochondria-encoded precursor polypeptides destined for mitochondrial residence (22Gakh O. Cavadini P. Isaya G. Biochim. Biophys. Acta. 2002; 1592: 63-77Crossref PubMed Scopus (321) Google Scholar, 23Ito A. Biochem. Biophys. Res. Commun. 1999; 265: 611-616Crossref PubMed Scopus (60) Google Scholar). Failure to remove such targeting presequences has been implicated in human disease including the pathophysiology of Friedreich ataxia (24Patel P.I. Isaya G. Am. J. Hum. Genet. 2001; 69: 15-24Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 25Branda S.S. Cavadini P. Adamec J. Kalousek F. Taroni F. Isaya G. J. Biol. Chem. 1999; 274: 22763-22769Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Mitochondrial processing peptidase initially cleaves the vast majority of N-terminal mitochondrial targeting presequences. Based on additional uncharacterized protein targeting motifs downstream of the mitochondria-processing peptidase inner membrane peptidase and mitochondrial peptidase subsequently of precursor polypeptides destined for mitochondrial Amino acid an mass of 100 and for and of HDAC7 protein expression in C4-2 cells to an HDAC7 of that the majority of HDAC7 in C4-2 cells is in mitochondria, we a of HDAC7 in both C4-2 mitochondrial as well as mitochondrial protein (Fig. mitochondrial presequence processing motifs are site-directed mutagenesis analysis. mitochondrial import of HDAC7 is a for N-terminal processing of HDAC7, we that of structurally basic amino acid residues in the N-terminal presequence α-helix mitochondrial import and processing of of C-terminal HDAC7 containing the in C4-2 cells in expression of HDAC7 (Fig. a we similarly the C4-2 cells both the of (Fig. as well as the (Fig. of HDAC7 were we demonstrate that HDAC7 mitochondrial import is a structurally targeting presequence and that localization of HDAC7 to mitochondria results in of the targeting HDAC7 Is a Mitochondrial secondary targeting peptides the mitochondrial targeting presequence a Kyte-Doolittle hydrophobicity plotting and II software analysis von Heijne G. 10: Google Scholar) both HDAC7 as a protein with of hydrophobicity commonly with membrane proteins not Mitochondrial subfractionation of untreated C4-2 mitochondria that HDAC7 with known soluble mitochondrial proteins AIF and Smac/DIABLO (Fig. Tom20 and the oxidative phosphorylation complex V as for the outer membrane and inner of mitochondria, HDAC7 is a soluble mitochondrial permeabilization of the mitochondrial outer membrane in release of Mitochondrial outer were with either the saponin (16Schulz I. Methods Enzymol. 1990; 192: 280-300Crossref PubMed Scopus (124) Google Scholar) or of the pro-apoptotic protein G. M. R. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to mitochondria has been to mitochondrial outer membrane permeabilization in the release of pro-apoptotic proteins. membrane permeabilization in a of mitochondrial HDAC7 staining and relocalization of HDAC7 to nuclear that with concentration (Fig. GFP-Bax mitochondrial release of AIF, cytochrome and HDAC7 in addition to nuclear (Fig. we demonstrate that HDAC7 is a soluble mitochondrial protein the of is an mitochondrial outer HDAC7 mitochondrial release the of several proteins implicated in programmed cell death. Cytoplasmic of HDAC7 of HDAC7 have demonstrated regulated nucleocytoplasmic in the differentiation of (11Kao H.Y. Verdel A. Tsai C.C. Simon C. Juguilon H. Khochbin S. J. Biol. Chem. 2001; 276: 47496-47507Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Cytoplasmic relocalization of HDAC7 has been implicated in cell death (8Verdin E. Dequiedt F. Kasler H. Novartis Found. Symp. 2004; 259 (163-169): 115-131Crossref PubMed Google Scholar). of and that HDAC7 can to including the mitochondrial IMS, we characterized the of HDAC7 pro-apoptotic for apoptosis as by GFP-Bax a of known apoptosis including serum and to mass apoptosis in cells as by and cleavage as well as phosphorylation not demonstrate that a of the apoptotic in C4-2 cells was the and protein inhibitor with 100 and both and cleavage as well as phosphorylation of (Fig. treated C4-2 cells with 100 for h and cells and nuclear demonstrate a complete of HDAC7 from mitochondrial to cytoplasmic (Fig. we demonstrate live cell imaging of C4-2 cells GFP-HDAC7 a complete of GFP-HDAC7 in the after 9 h (Fig. This was most in the of cells GFP-HDAC7 initially was localized to the Cytoplasmic HDAC7 sequestration for the of the not Here we the localization of a Class II HDAC to the mitochondrial of human prostate epithelial cells. Similar to other nuclear encoded mitochondrial we demonstrate that mitochondrial import of HDAC7 results in N-terminal and residence in the inner membrane Similar to other pro-apoptotic mitochondrial HDAC7 is released from mitochondria the of programmed cell death is in the cytoplasm. Here we describe the novel of a human Class II HDAC localized to the mitochondrial inner membrane space of human prostate cancer cells. we similar localization of HDAC7 in other human cell lines including AT5BIVA and MR5CV1 fibroblasts as well as PC-3 and not prostate cancer we that such a phenomenon is not the to the Similar to other mitochondrial nuclear encoded HDAC7 a targeting presequence that is necessarily by mitochondrial by an incompletely understood HDAC7 is in a mitochondrial previously to contain several pro-apoptotic proteins and in response to apoptotic similar to of other pro-apoptotic factors such as cytochrome c and apoptosis is an well conserved mechanism, not be to HDAC7 in the mitochondria of other human cell HDAC7 in apoptosis (5Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). HDAC7 nuclear during T cell expression of the to apoptosis. a pro-apoptotic of cytoplasmic at HDAC7, was demonstrated that a HDAC7 to the to the T cell receptor-mediated apoptosis (5Dequiedt F. Kasler H. Fischle W. Kiermer V. Weinstein M. Herndier B.G. Verdin E. Immunity. 2003; 18: 687-698Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). HDAC7 has been to the expression of the the domain in the N-terminal of HDAC7 that the mitochondrial targeting Mitochondrial targeting presequences of a positively charged of often by not the of to that the presequence not the as HDAC7 nuclear Western of GFP-HDAC7 C4-2 cells for an not is a protein suggesting that the additional amino from the mitochondrial targeting presequence of These are with The of the N-terminal of and rat HDAC7 is unknown with to mitochondrial localization and we observed N-terminally GFP-HDAC7 in the mitochondria of we that N-terminal peptide additions to the targeting presequence may not mitochondrial is that whereas we often observed HDAC7 in both the and of both live and HDAC7 is localized to mitochondria is often robust and the HDAC7 mitochondrial import are is of either enhanced HDAC7 mitochondrial import or 14-3-3 proteins cytoplasmic sequestration of HDAC7, and mitochondria are in the is to that mitochondrial HDAC7 is for as both the and nuclear the is at a more N-terminal the that mitochondrial processing be an This the that mitochondrial HDAC7 have enhanced nuclear import to were initially to HDAC7 in mitochondria as have cytoplasmic and nuclear localization of whereas we observed localization of HDAC7 in both the and of cells (Fig. we mitochondrial HDAC7 in cells was robust and mitochondrial more often we observed both mitochondrial localization of GFP-HDAC7 in a of general cytoplasmic staining (Fig. of HDAC7 we that mitochondrial HDAC7 localization at be by a more general cytoplasmic HDAC7 as the was N-terminal in and known mitochondrial proteins N-terminal targeting peptide removed endoproteolytic we that the cytoplasmic and nuclear GFP-HDAC7 that we observed at in the mitochondrial inner membrane of HDAC7 is to mitochondria as the targeting presequence be mitochondria as an intracellular in to HDAC7 that be for and may a novel of of the mitochondrial HDAC7 is in biological events to HDAC7, we that the of proteins be from previously for HDAC7 (10Dressel U. Bailey P.J. Wang S.C. Downes M. Evans R.M. Muscat G.E. J. Biol. Chem. 2001; 276: 17007-17013Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). HDAC7 is a regulated and can in either or mitochondrial of cells or the we that the of HDAC7 intracellular localization is we have demonstrated the novel that HDAC7 can to mitochondria in addition to cytoplasmic and nuclear HDAC7 mitochondrial release of N-terminally HDAC7 and subsequent cytoplasmic sequestration be in programmed cell death apoptotic we observed and cleavage as well as phosphorylation in response to we to phosphorylation has been as a of to apoptosis K. K. M. P. A. J. Allis C.D. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). may at the and at we observed HDAC7 to the to apoptosis not be as HDAC7 to be the known HDAC localized to mitochondria, we that HDAC7 has a and complex in apoptosis. are to cytoplasmic substrates and the of mitochondrial HDAC7 in the of programmed cell death. Dr. Tomas for the GFP-Bax expression and Dr. Eric Verdin for the HDAC7-FLAG expression