Enhanced Deacetylation of p53 by the Anti-apoptotic ProteinHSCO in Association with Histone Deacetylase 1*

Received for publication, October 17, 2006, and in revised form, March 12, 2007 Published, JBC Papers in Press, March 12, 2007, DOI 10.1074/jbc.M609751200

Hisako Higashitsuji, Hiroaki Higashitsuji1, Tomoko Masuda, Yu Liu, Katsuhiko Itoh, and Jun Fujita2

From the Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawaharacho,Kyoto 606-8507, Japan

HSCO (hepatoma subtracted-cDNA library clone one, alsocalled ETHE1) was originally identified by its frequent overex-pression in hepatocellular carcinomas. HSCO inhibits functionofNF-�Bbybinding toRelAandaccelerating its export from thenucleus.We showhere thatHSCOexhibits anti-apoptotic activ-ity in cells exposed to DNA-damaging agents by suppressingtranscriptional activity of p53. Induction of pro-apoptoticgenes,Noxa, Perp, PIG3, and Baxwere suppressed in cells over-expressingHSCO.By increasingubiquitylation anddegradationof p53, HSCO reduces p53 protein levels. HSCO specificallyassociates with histone deacetylase 1 (HDAC1) independentlyof Mdm2 and facilitates deacetylation of p53 at Lys-373/382 byHDAC1. The metallo-�-lactamase family consensus sequencein HSCO is important for its effect on p53 deacetylation. Co-immunoprecipitation and immunofluorescence studies sug-gested that HSCO, HDAC1, and p53 form a complex in thenucleus. Thus,HSCO is a cofactor that increases the deacetylaseactivity of HDAC1 toward p53, leading to suppression of apo-ptosis. Treatment of hepatocellular carcinomas that retainwild-type p53 and overexpress HSCO with anti-HSCO agents mightre-establish the p53 response and revert chemoresistance.

The p53 tumor suppressor is mutated in �50% of many dif-ferent cancers (1) and is probably rendered inactive by a rangeof indirectmechanisms such asMdm2 amplification and loss ofp14ARF in the remaining 50% (2). In unstressed cells, p53 isnormallymaintained at low levels by continuous ubiquitylationcatalyzed by E33 ubiquitin ligases such as Mdm2, COP1, Pirh2,TOPORS, and ARF-BP1/Mule, and subsequent degradation bythe 26 S proteasome (3–5). In response to genotoxic stress, p53is rapidly stabilized and activated. The activated p53 mainly

functions as a sequence-specific DNA-binding transcriptionfactor to activate or repress a large number of target genes,which mediate cell-cycle arrest, apoptosis, senescence, differ-entiation, DNA repair, and inhibition of angiogenesis andmetastasis (6). The activity of p53 is largely controlled by thecellular p53 level, its DNA-binding activity, subcellular local-ization, and recruitment of transcriptional co-activators or co-repressors. Although the precise mechanisms of p53 activationare not fully elucidated, accumulating evidence indicates thatpost-translational modifications of p53, including phosphoryl-ation of Ser and Thr residues and ubiquitylation, acetylation,and sumoylation of Lys residues, play important roles in regu-lating its stability and transcriptional activity (5, 7). Further-more, these modifications are interrelated. For example,phosphorylation of Ser-15 or Ser-33/37 increases the affinityof p53 for p300 and promotes acetylation of p53 at Lys-373/382 (7, 8). Because the Lys residues acetylated in p53 overlapwith those that are ubiquitylated, p53 acetylation has beenconsidered to be important for p53 degradation as well astranscriptional activation (2, 5).Histone deacetylase (HDAC) activity has been linked to diet,

premalignant cell changes, aging, and development of diseases,including cancer. Eighteen potential HDACs have been identi-fied in humans (9, 10), which are classified into three groupsbased on homology to yeast proteins. The enzymatic activitiesof HDACs in Class I (Rpd3-like) such as HDAC-1, -2, and -3,andClass II (Hda1-like) are zinc-dependent and sensitive to theinhibitor trichostatin A (TSA). Class I HDACs are ubiquitouslyexpressed small nuclear proteins, whereas Class II HDACs arelarger proteins that shuttle between the cytoplasm and thenucleus. The NAD-dependent enzymatic activities of Class III(Sir2-like) HDACs are inhibited by nicotinamide but not byTSA (11). Histone acetylation can be reversed by HDACs.HDAC recruitment to promoter regions by p53 through inter-action with mSin3A, which directly binds to HDACs, down-regulates gene expression by core histone deacetylation (6).Thus,HDACs act as p53 co-repressors.HDACs can deacetylatenon-histone proteins as well (10). HDAC1 interacts with p53possibly through mSin3A or PID/MTA2, deacetylates p53 invitro and in vivo, and down-regulates p53 transcriptional activ-ity (12–14). Deacetylation of p53 is required for its effectivedegradation mediated by the ubiquitin ligase Mdm2, and Mdm2canpromotep53deacetylationby recruitingacomplexcontainingHDAC1 (15). In addition to HDAC1, a Class III HDAC, SIRT1,interacts with p53 and deacetylates it at Lys-382 (2).Hepatocellular carcinoma (HCC) is currently the fifth most

common solid tumorworldwide and the fourth leading cause of

* This work was supported by Grants-in-Aid from the Ministry of Science,Culture, Sports and Education of Japan. The costs of publication of thisarticle were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence may be addressed. Tel.: 81-75-751-3753; Fax:81-75-751-3750; E-mail: hhigashi@virus.kyoto-u.ac.jp.

2 To whom correspondence may be addressed. Tel.: 81-75-751-3751; Fax:81-75-751-4977; E-mail: jfujita@virus.kyoto-u.ac.jp.

3 The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; HSCO,hepatoma subtracted-cDNA library clone one; HCC, hepatocellular carci-noma; HDAC, histone deacetylase; TSA, trichostatin A; HEK, human embry-onic kidney; DKO-MEF, doubly knocked-out mouse embryonic fibroblast;shRNA, short hairpin RNA; HA, hemagglutinin; CMV, cytomegalovirus; GFP,green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-trans-ferase; TRITC, tetramethylrhodamine isothiocyanate; ActD, Actinomycin D;WB, Western blot; IP, immunoprecipitation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 18, pp. 13716 –13725, May 4, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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cancer-related death (16). Although screening of high risk pop-ulations by ultrasonography and measurement of the serum�-fetoprotein level has facilitated the early detection of HCC, amajority of patients present with advanced disease. Even forthose patients who undergo surgical resection, the recurrencerates are as high as 50% at 2 years, and nonsurgical treatmentsare ineffective or minimally effective at best (16, 17). It is there-fore important to identifymolecules that can be used to developnovel diagnostic, preventive, or therapeutic strategies.By constructing subtracted cDNA libraries, we have previ-

ously identified 19 genes overexpressed inHCCs, including twonovel genes (18, 19). One of these genes was named HSCO(hepatoma subtracted-cDNA library clone one) (20). HSCOmRNA was overexpressed in 20 of 30 HCCs analyzed. Overex-pression of HSCO inhibits caspase 9 activation and apoptosisinduced by DNA-damaging agents such as adriamycin and eto-poside, whereas it augments apoptosis induced by tumornecrosis factor-�. HSCO is a nuclear-cytoplasmic shuttlingprotein that binds to RelA and sequesters it in the cytoplasm byaccelerating its export from the nucleus, resulting in inhibitionof NF-�B activity. This activity of HSCO underlies the abroga-tion of p53-induced apoptosis in Saos-2 cells. In addition to ourdiscoveries, Oue et al. (21) used serial analysis of gene expres-sion and found that HSCO (also called YF13H12) was overex-pressed in 52% of 46 gastric carcinomas. Tiranti et al. (22), viapositional cloning, identified HSCO as the gene responsible forethylmalonic encephalopathy. Ethylmalonic encephalopathy ischaracterized by neurodevelopmental delay and regression,prominent pyramidal and extrapyramidal signs, recurrent pete-chiae, orthostatic acrocyanosis, and chronic diarrhea, leadingto death in the first decade of life. They proposed the name ofthe gene be changed fromHSCO to ETHE1 and suggested thatits product is a mitochondrial protein. Here, we show thatHSCO can control the transcriptional activity of p53 byenhancing its deacetylation in association with HDAC1.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—Adenovirus-transformedhuman embryonic kidney (HEK) 293 cells (p53wild type), SV40large T antigen-expressing 293T cells (p53 wild type), humanosteosarcoma U-2 OS cells (p53 wild type), human non-smallcell lung carcinoma A549 cells (p53 wild type), and p53�/�/mdm2�/� doubly knocked-out mouse embryonic fibroblasts(DKO-MEFs, kindly provided by Dr. D. P. Lane) were main-tained in Dulbecco’s modified Eagle’s medium supplementedwith 10% fetal bovine serum. Human lung adenocarcinomaH1299 cells (p53-null) were maintained in RPMI 1640mediumsupplemented with 10% fetal bovine serum. Mouse NIH/3T3cells were maintained in Dulbecco’s modified Eagle’s mediumsupplemented with 10% calf serum. All cells were grown at37 °C in a humidified atmosphere of 5% CO2 in air. DNA trans-fection was performed by using the calcium phosphate methodor FuGENE6 reagent (Roche Applied Science).Induction of Apoptosis and Assays for Caspase Activity—Ex-

ponentially proliferating cells (2 � 104) were plated into a35-mm plate and treated with actinomycin D (7.5 nM), Adria-mycin (1 �g/ml), etoposide (50 �g/ml), or tumor necrosis fac-tor-� and cycloheximide (50 ng/ml and 100 �g/ml, respec-

tively), or transfected with p53 cDNA in pcDNA3.1(�)-Neo orvector alone. Cell numbers were determined in triplicates atdifferent time points. Viability of cells was determined by stain-ing with trypan blue, and cell numbers were counted under amicroscope. Caspase 3 activity and caspase 9 activity weredetermined by using the Caspase-3 Colorimetric Assay Kit andCaspase-9 Colorimetric Assay Kit (Medical & Biological Labo-ratories), respectively.Reporter Gene Assays—Luciferase reporter plasmids, p53-

Luc or pAP-1-Luc, containing p53- or AP-1-binding sites,respectively (Stratagene), and pRL-TK (Promega, Madison,WI) were co-transfected with wild-type or mutant (H79N orR159H) HSCO cDNA fused to HA or FLAG tag in expressionvector pCMV4–3HA or pcDNA3.1(�)-Neo as described (20).24 or 48 h later, cells were assayed for luciferase activity ortreated with actinomycin D and assayed 24 h later. Luciferaseactivity was measured by the Dual Luciferase Reporter AssaySystem (Promega) according to themanufacturer’s protocol. Insome experiments, plasmids expressing p53 were co-trans-fected aswell. pFC-MEKKplasmid (Stratagene) served as a pos-itive control for the AP-1 reporter assay.Analyses of Gene Expression and Protein-Protein Interactions—

RNA extraction, Northern blot analysis, immunoprecipitation,and Western blot analysis were performed as described (20).For immunoprecipitation, anti-p53 antibody (DO-1 andFL-393, Santa Cruz Biotechnology), anti-HA antibody (12CA5,Roche Applied Science), anti-FLAG antibody (M2, Sigma),anti-GFP antibody (Nacalai), agarose-immobilized anti-p53antibody (DO-1, Santa Cruz Biotechnology), and agarose-im-mobilized anti-FLAG antibody (M2, Sigma) were used. About5–10% (10–25 �g of protein) of total cellular lysates used forimmunoprecipitation was also analyzed by Western blotting.Protein bands were visualized by using the Enhanced Chemilu-minescence kit (AmershamBiosciences).Western blot analysiswas performed using the antibody against p53 (DO-1, SantaCruz Biotechnology, DO-7, BDPharmingen), HA, FLAG,�-ac-tin (C4, Chemicon), ubiquitin (FL-76, Santa Cruz Biotechnol-ogy), Mdm2 (SMP14, Santa Cruz Biotechnology), HDAC1(Santa Cruz Biotechnology), HDAC2 (Santa Cruz Biotechnol-ogy), HDAC3 (Santa Cruz Biotechnology), SIRT1 (Santa CruzBiotechnology), acetylated Lys (Cell Signaling Technology),acetylated histone H4 (Upstate Biotechnology), acetylated p53(Lys-320, Upstate), and acetylated p53 (Lys-373/382, Upstate).In some experiments, biotinylated anti-GFP antibody (B-2,Santa Cruz Biotechnology) and anti-FLAG antibody (BioM2,Sigma) were used. Histones were isolated from cells by HClextraction and acetone precipitation (23).For GST pulldown assays, full-length p53, HSCO, RelA, and

HDAC1 cDNAs were cloned into the expression vectorpGEX-4T or pGEX-6P-1 (Amersham Biosciences) andexpressed as proteins fused toGST inEscherichia coli (DH5� orBL21 strain). The fusion proteins and GST were immobilizedon glutathione-Sepharose and incubated with recombinantHSCO or HDAC1 protein from which GST had been removedafter cleavage with PreScission protease (Amersham Bio-sciences), or immunoprecipitates prepared from cell lysates.After incubation at 4 °C for 60 min, bound proteins were ana-lyzed by SDS-PAGE and Western blotting.

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Electrophoretic Mobility Shift Assays—A 27-bp double-stranded oligonucleotide probe selected from the humangadd45� promoter (5�-TACAGAACATGTCTAAGCAT-GCTGGGG-3�) was labeled with [�-32P]ATP and purified withMicroSpin TM G25 columns (Amersham Biosciences). As acontrol, Sp1 binding-site probe (5�-GGATAGGGGCGGGGC-GAGG-3�) was used. Nuclear extracts (10 �g) from HEK293cells were incubated with 5 ng of labeled probe in a 20-�l reac-tion buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5 mM

dithiothreitol, 50 mM NaCl, and 5% glycerol) containing 10 �gof bovine serum albumin and 1 �g of poly(dI-dC):poly(dI-dC)for 20 min at room temperature. For competition tests, 50-foldexcess (250 ng) of unlabeled wild-type or mutant (5�-TACA-GAATCGCTCTAAGCATGCTGGGG-3�) gadd45� probeswere added to each reaction. The reactionmixture was electro-phoresed in 4% polyacrylamide gel. The gels were dried andexposed to film at �80 °C with an intensifying screen.Analysis of p53 Stability and Ubiquitylation in Vivo—For in

vivo p53 degradation assays, U-2 OS cells stably expressingHSCO-FLAG or FLAG alone (three clones each) and H1299cells co-transfected with plasmids expressing p53 alone or incombination with HSCO-FLAG were used and analyzed asdescribed (24). To analyze the effects of HSCO down-regula-tion,U-2OS cells transfectedwithHSCO-specific short hairpinRNA (shRNA) were used.For in vivo ubiquitylation assays, H1299 cells or DKO-MEFs

in 60-mm dish were co-transfected with plasmids expressingp53 (1.0 �g), HSCO-FLAG (1.0 �g), HA-Mdm2 (1.0 �g), andHis-tagged ubiquitin in various combinations. Prior to collec-tion after 48 h, cells were treated with proteasome inhibitors,MG115 (10 �M) and MG132 (10 �M), for 6 h. Cells were thenlysed in buffer (6.0 M guanidium-HCl, 0.1 M Na2HPO4/NaH2PO4, 10mMTris-HCl, pH 8.0, 5mM imidazole, and 10mM

2-mercaptoethanol), and sonicated. The lysates were incubatedwith nickel-nitrilotriacetic acid-agarose beads. After extensivewashing, bound proteins were eluted and analyzed byWesternblotting.In Vivo p53 Acetylation and Deacetylation Assays—For in

vivo p53 acetylation assays, 293T cells were transfected withplasmids expressing wild-type or mutant (H79N or R159H)HSCO, Mdm2, and p300. The cells were cultured in the pres-ence or absence of 5 �M TSA and lysed in buffer (20 mM Tris-HCl, pH 7.6, 170 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1�M dithiothreitol) supplemented with proteinase inhibitorsand TSA. To detect acetylated p53, cell lysates (250–500 �gof proteins) were incubated with 1 �g of agarose-immobi-lized antibody specific to human p53 (DO-1) for 4 h at 4 °C.The p53 content in each precipitated sample was equalizedand subjected to Western blotting to assess the amount ofacetylated p53.For in vivo p53 deacetylation assays, p53-null H1299 cells

were co-transfected with plasmids expressing p53, wild-type ormutant (H141A) HDAC1, HSCO, and p300. In some experi-ments, H1299 and U-2 OS cells were co-transfected with plas-mids expressing p53, p300, FLAG-tagged HSCO, and HDAC1-specific shRNA. The cells were lysed and analyzed by Westernblotting as described above.

In Vitro p53 Acetylation Assays—The cDNA for humanHSCO was cloned into pGEX6P-1 (Amersham Biosciences)and expressed as GST fusion protein in E. coli strain BL21. TheGST tag was cleaved using PreScission protease (AmershamBiosciences). p300HATdomain fused toGST (1.0�g, Upstate)was preincubated with the purified HSCO orMdm2 for 10minat room temperature. Substrate (1.0�g ofGST-p53)was added,and the mixture was incubated with 50 nCi of [14C]acetyl-CoAin 20 �l of reaction buffer (50 mM Tris-HCl, pH 8.0, 10% glyc-erol, 1.0 mM dithiothreitol, 0.1 mM EDTA, 1.0mM phenylmeth-ylsulfonyl fluoride) for another 60min at 37 °C. Acetylationwasanalyzed by SDS-PAGE followed by autoradiography.In Vitro p53 Deacetylation Assays—The expression vector

for wild-type or mutant (H141A) HDAC1 tagged with FLAGwas co-transfected with HSCO cDNA in expression vectorpcDNA3.1(�)-Neo or vector alone onto 293T cells. The cellswere lysed in low stringency buffer (50 mM Tris-HCl, pH 7.5,120 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40) in the pres-ence of protease inhibitors. After pre-clearing with protein Abeads, the extracts were immunoprecipitated with anti-FLAGantibody in the presence of protein A beads for 2 h at 4 °C.Then, the beads were washed twice with low stringency buffer,twice with low stringency buffer containing 0.5 M NaCl, andtwicewith deacetylation buffer (10mMTris-HCl, pH8.0, 10mM

NaCl, 10% glycerol). The immune complexes were then incu-batedwith acetylated p53 in 20�l of deacetylation buffer for 2 hat 37 °C and analyzed by Western blotting. Acetylated p53 wasprepared via an in vitro p53 acetylation reaction (25).Inhibition of Endogenous Gene Expression by shRNA—For

production of small interfering RNA in the cells, we used thepSuper vector expressing shRNAas described (24). To suppressexpression of endogenous HSCO, the pSuper plasmid wasdigested with BglII and HindIII, and the annealed oligonucleo-tides corresponding to human HSCO (wt1: 5�-CTCTATGCT-GTGAATACCC-3�, wt2: 5�-CAGGCTGACTTACACATTG-3�, and mutant: 5�-CAGGTCGACTATCCAATGT-3�) werecloned into it. To suppress expression of endogenous HDAC1and p53, oligonucleotides corresponding to human HDAC1(5�-CTATGGTCTCTACCGAAAA-3�) and p53 (5�-GACTC-CAGTGGTAATCTAC-3�), respectively, were cloned into thepSuper plasmid. Transfection of pSuper plasmids was per-formed using FuGENE6 Reagent (Roche Applied Science).Immunofluorescence Staining—Immunofluorescence stain-

ing was performed as described (20). FLAG-tagged HSCO wasdetectedwith anti-FLAGantibody (BioM2, Sigma) and strepta-vidin-allophycocyanin (BD Pharmingen). HA-tagged p53 wasdetected with anti-HA antibody (Sigma) and TRITC-conju-gated anti-rabbit IgG (DAKO). EGFP-tagged HDAC1 wasdetected by GFP fluorescence. They were observed using aconfocal laser microscope (Olympus). In some experiments,cells stably expressing HSCON- or C-terminally tagged withFLAG were treated with MitoTracker (Molecular Probes).After fixation, FLAG was detected with anti-FLAG antibody(M2, Sigma), fluorescein isothiocyanate-conjugated anti-mouse IgG (DAKO), and MitoTracker with its fluorescenceusing a confocal microscope.

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RESULTS

HSCO Increases Resistance to p53-dependent Apoptosis—Inhuman U-2 OS cells, HSCO confers resistance to apoptosisinduced by DNA-damaging agents but not tumor necrosis fac-tor-� (20). DNA damage induces p53-dependent apoptosis,whereas tumor necrosis factor-� triggers apoptosis p53-inde-pendently (26). As shown in Fig. 1A, expression of HSCOincreased the survival of U-2 OS cells exposed to actinomycinD. The reduced activation of caspases 3 and 9 indicated that theincreased survival was due to suppression of apoptosis (Fig. 1B).The anti-apoptotic effect of HSCOwas also observed in actino-mycin D-treated A549 cells, a human lung carcinoma cell lineexpressing wild-type p53, but not in actinomycin D-treated

HLE cells, a human HCC cell line expressing mutant p53 (datanot shown). In p53-null humanH1299 cells, apoptosis and acti-vation of caspase 9 induced by introduction of p53 were alsoinhibited by overexpression of HSCO (Fig. 1A and data notshown).When HSCO-specific shRNAwas expressed in U-2 OS cells,

the cytotoxic effect of actinomycin D and activation of caspase9 were enhanced (Fig. 1, C andD). The effect of HSCO-specificshRNA on caspase-9 activity was reduced by concomitant sup-pression of p53 expression. Expression of shRNAs specific toHSCO, its mutant, or p53 did not affect the caspase-9 activitywithout actinomycin D treatment (data not shown). Takentogether, these results suggest that HSCO suppresses the pro-apoptotic signaling pathway mediated by p53.HSCO Suppresses Transcriptional Activity of p53—After

genotoxic stress, p53 transactivates many genes involved inapoptotic pathways (3). To analyze the effects of HSCO on thetranscriptional activity of p53, we transfected U-2OS cells witha luciferase p53-cis reporter plasmid. Actinomycin D inducedluciferase activity, which was suppressed dose dependently byHSCO (Fig. 2A). Interestingly, the mutant (H79N) HSCO hav-ing a missense mutation within the consensus sequence con-served throughout the metallo-�-lactamase family (20) did not

FIGURE 1. p53-sensitive anti-apoptotic activity of HSCO. A, increased sur-vival. Three clones each of U-2 OS (upper panel) and H1299 (lower panel) cellsstably expressing HSCO-FLAG (E) or FLAG alone (▫) were exposed to actino-mycin D (upper panel) or transfected with p53-expressing plasmids (lowerpanel). Cell numbers were counted at the indicated times after treatment.Data represent mean � S.D. of triplicates. B, decreased activation of caspases3 and 9. Caspase activities were determined in U-2 OS cells expressing HSCO-FLAG or FLAG alone 24 h after treatment with actinomycin D (ActD) as indi-cated. Data represent mean � S.D. of triplicates. C, effects of HSCO down-regulation on apoptosis. U-2 OS cells were transfected with pSuper-HSCOwt1(black bars), pSuper-HSCOmut (striped bars), or pSuper vector alone (whitebars), which produce shRNAs for wild-type HSCO, its mutant, or none, respec-tively. Cells were treated with ActD, and the cell numbers were counted at theindicated times. Data represent mean � S.D. of triplicates. D, effects of HSCOdown-regulation on caspase activity. U-2 OS cells were transfected withpSuper-p53, which produces shRNA for p53, pSuper-HSCOwt1, pSuper-HS-COwt2, or pSuper-HSCOmut and treated with ActD as indicated. pSuper-HS-COwt2 also encodes shRNAs for wild-type HSCO, but the targeted region is dif-ferent from pSuper-HSCOwt1. Caspase 9 activity was determined as in B. Datarepresent mean � S.D. of triplicates (upper panel). Cell lysates were also analyzedby Western blotting using the indicated antibodies (lower panels).

FIGURE 2. Effects of HSCO on transcriptional activity of p53. A, reducedtranscriptional activity of p53. U-2 OS cells were co-transfected with a p53-responsive luciferase reporter, pRL-TK, and plasmids expressing wild-type(�wt) HSCO, mutant (H79N) HSCO, or vector alone. 24 h after exposure toactinomycin D (ActD), luciferase activity was assayed (upper panel). The resultswere normalized to Renilla luciferase activity and represent the mean � S.D.of triplicates. Cell lysates were also analyzed by Western blotting using theindicated antibodies (lower panels). B, p53-null H1299 cells were co-transfectedand analyzed as in A except that p53-expressing plasmids were co-transfectedinstead of ActD treatment. C, U-2 OS cells were co-transfected and analyzed as inA except that an AP-1-responsive luciferase reporter was used instead of a p53-responsive reporter. pFC-MEKK served as a positive control. D, mdm2�/�/p53�/�

DKO-MEFs were co-transfected and analyzed as in B. E, mdm2�/�/p53�/� DKO-MEFs were co-transfected and analyzed as in C. F, reduced induction of p53-inducible genes. U-2 OS cells were transfected with plasmids expressing HSCO-FLAG or FLAG alone (Mock). After incubation with (�) or without (�) ActD, geneexpression was analyzed by Northern blotting using the indicated cDNA probes.

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suppress the induction of luciferase. In the p53-null H1299 andSaos-2 cells, expression of HSCO suppressed the p53-cisreporter activity induced by exogenous p53 (Fig. 2B, and datanot shown). The inhibition was specific to the p53 transactiva-tion, since HSCO did not affect the luciferase AP-1-cis reporteractivity (Fig. 2C). Essentially similar results were obtained usingmdm2�/�/p53�/� DKO-MEFs (Figs. 2D and 2E), indicatingthat the observed effect is independent of Mdm2. In U-2 OScells exposed to actinomycin D, induction of p53-induciblegenes involved in apoptosis, such asNoxa, Perp, PIG3, and Baxwas suppressed by HSCO (Fig. 2F).HSCO Reduces the Amount of p53 Bound to the p53-re-

sponsive Element—To clarify the mechanisms by whichHSCO decreases the transcriptional activity of p53, we ana-lyzed the effect of HSCO on binding of p53 to the p53-re-sponsive element in vitro. As shown in Fig. 3A, incubation ofthe labeled p53-binding consensus sequences with nuclearextracts from actinomycin D-treated U-2 OS cells produceda slow migrating band in the electrophoretic mobility shift

assay. Co-expression of HSCOdecreased the band intensity. Theband was supershifted in the pres-ence of anti-p53 antibody (data notshown). In p53-null H1299 cells, theband appeared only when exoge-nous p53 was expressed, and theband intensity was decreased byco-expression of HSCO (Fig. 3A).The band disappeared in the pres-ence of excess unlabeled wild-type, but not mutant, p53-bindingconsensus sequences. These resultsindicate that the DNA-bindingactivity and/or the amount ofnuclear p53 are decreased by over-expression of HSCO.HSCO Accelerates Degradation of

p53 by the Ubiquitin-ProteasomeSystem—We therefore assessed theeffects of HSCO on the p53 proteinlevel. When exogenous p53 wasexpressed alone or in combinationwith HSCO in p53-null H1299cells, the p53 protein level waslower in the presence of HSCO,whereas the p53 mRNA levels wereequivalent (Fig. 3B). In U-2 OS cellsstably overexpressing HSCO, thelevel of endogenous p53 protein wasdecreased, and its half-life wasshortened (Fig. 3C). Reciprocally,when endogenous HSCO proteinlevel was down-regulated, theendogenous p53 protein level wasincreased (Fig. 3D), and its half-lifewas lengthened (Fig. 3E).The degradation of p53 is mainly

mediated by the ubiquitin-protea-some system (5). Expression of an E3 for p53 (Mdm2, COP1, orPirh2) decreased the exogenous p53 protein level in H1299cells, and co-expression of HSCO further decreased it (data notshown). In HeLa cells, in which p53 is predominantly degradedthrough the human papillomavirus 18 E6 pathway (27), overex-pression of HSCO decreased the p53 protein level (data notshown). These results suggest that the activity of HSCO is inde-pendent of the kind of E3 for p53.We next examined the effects of HSCO on ubiquitylation of

p53. As shown in Fig. 3F, expression of HSCO increased theubiquitylation of exogenous p53 by Mdm2 in DKO-MEFs.These results suggest that HSCO accelerates degradation ofp53 by increasing its ubiquitylation and that the decreased p53-cis reporter activity in the presence of HSCO is due, at leastpartly, to this effect.Interaction of HSCO with p53—When exogenous p53 and

HSCO were co-expressed in H1299 cells, HSCO co-immuno-precipitated with p53 (Fig. 4A). Mutant HSCO (H79N) alsoco-immunoprecipitated with p53 under similar conditions. As

FIGURE 3. Effects of HSCO on p53 protein levels. A, inhibition of nuclear p53 DNA-binding activity by HSCOoverexpression. Equal amounts of nuclear extracts were subjected to electrophoretic mobility shift assays withradiolabeled p53-binding site oligonucleotide DNAs. U-2 OS cells and H1299 cells were transfected with plas-mids expressing FLAG-tagged HSCO or p53 and treated with actinomycin D (ActD, �) or vehicle alone (�) asindicated. Nuclear lysates were prepared from them and analyzed by electrophoretic mobility shift assays. Forcompetition analysis, a 50-fold excess of unlabeled wild-type (wt) or mutant (mut) p53-site oligonucleotideDNAs were added to the reaction mixture. Comparability of the nuclear extracts was verified by electrophoreticmobility shift assays with a radiolabeled Sp1 probe. B, effects of HSCO overexpression on p53 levels. H1299 cellstransiently transfected with plasmids expressing p53, HSCO-FLAG, and GFP as indicated were analyzed byWestern blotting (WB, upper panels) and Northern blotting (NB, lower panels) using indicated antibodies andcDNA probes, respectively. C, enhanced p53 degradation by HSCO overexpression. Three stable clones each ofU-2 OS cells expressing HSCO-FLAG (▫) or FLAG alone (control, E) were treated with cycloheximide, andharvested at indicated times. Lysates were analyzed by WB, and a representative result is shown (upper panels).The intensity of the bands was quantified, and the result represents the mean � S.D. of triplicates (lower panel).D, effects of HSCO down-regulation on p53 levels. U-2 OS cells transiently transfected with plasmids expressingshRNA specific to HSCO (�) or vector alone (�) were analyzed as in B. E, decreased p53 degradation by HSCOdown-regulation. Three stable clones each of U-2 OS cells expressing shRNA specific to HSCO (E) or vectoralone (control, ▫) were analyzed as in C. F, increased ubiquitylation of p53 by HSCO overexpression. DKO-MEFswere co-transfected with plasmids expressing p53, Mdm2, HSCO-FLAG, and His-ubiquitin and cultured in thepresence of MG132 as indicated. Cell lysates and those affinity-purified using nickel-nitrilotriacetic acid-agar-ose beads were analyzed by WB using the indicated antibodies.

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shown in Fig. 4B, exposure of U-2 OS cells to actinomycin Dincreased the level of endogenous p53 protein but not mRNA.Interestingly, the HSCO protein level was also increased 2–4 hafter the stress. In U-2 OS cells treated with actinomycin D for4 h, endogenous HSCO was co-immunoprecipitated with p53(Fig. 4C).In vitro GST-pulldown assays did not show binding of

recombinant HSCO and recombinant p53 (data not shown),suggesting that the interaction between p53 and HSCO is indi-rect or modification of HSCO and/or p53 is necessary for thebinding. We, therefore, used GST-HSCO and the lysates fromU-2 OS cells treated with actinomycin D in the pulldown assay.The amount of lysates was adjusted so that each input con-tained an equal amount of p53 protein. As shown in Fig. 4D,only a small amount of p53 was captured by the immobilizedHSCO from lysates prepared from untreated cells. Treatmentof cells with actinomycin D markedly increased the amount ofp53 bound toHSCO.A similar effect on the p53-HSCObindingwas observed when cells were treated with Adriamycin or eto-poside. These results suggest that one or more moleculesinduced by DNA damage and/or modification of p53 are nec-essary for the binding of HSCO to p53.HSCODecreases Acetylation of p53—The stability and activ-

ity of p53 are affected by post-translational modifications (2).Phosphorylation of multiple Ser residues of p53 has been pro-posed to interfere with the ability of Mdm2 to negatively regu-

late p53. HSCO did not affect the phosphorylation of Ser resi-dues induced by actinomycin D (Fig. 5A).p53 is specifically acetylated at multiple Lys residues of the

C-terminal regulatory domain by p300/CBP and p300/CBP-associated factor, and the acetylation levels of p53 correlatewellwith its activation and stabilization induced by stress (6). Asshown in Fig. 5B, HSCO as well as Mdm2 reduced the p300-induced p53 acetylation in 293T cells. The reduction at Lys-373/382wasmore prominent than that at Lys-320. The effect ofHSCO was reversed by an HDAC inhibitor TSA, but not nico-tinamide. The missense mutations within the metallo-�-lacta-mase family consensus sequence (H79N or R159H) abolishedthe effect of HSCO on p53 acetylation (Fig. 5C and data notshown). Overexpression of HSCO did not change the globalhistone H4 acetylation status (data not shown). Whenexpression of HSCO was suppressed in the presence of pro-teasome inhibitors in U-2 OS cells, the level of acetylated p53was increased (Fig. 5D), demonstrating that HSCO nega-tively regulates p53 acetylation in vivo.We further analyzed the effect ofHSCOonp53 acetylation in

vitro. In contrast toMdm2,HSCOdid not affect the acetylationof p53 by p300 (Fig. 5E). HSCO did not show deacetylase activ-ity by itself (data not shown), suggesting that HSCO mightinduce p53 deacetylation by recruiting a TSA-sensitivedeacetylase. In vitro deacetylase assay using acetylated p53 andthe immunoprecipitates prepared from cells expressing FLAG-tagged HDACs with anti-FLAG antibody demonstrated thatHSCO enhances deacetylase activity of HDAC1, but notHDAC2 nor HDAC3 (Fig. 5F). No effect on p53 deacetylationwas observedwhenHSCOwas incubatedwith immunoprecipi-tates containing mutant (H141A) HDAC1 with no enzymaticactivity.To determine whether HSCO facilitates deacetylation of p53

by HDAC1 in vivo, HSCO and/or HDAC1 were co-expressedwith p53 and p300 in H1299 cells. As shown in Fig. 5G, p53acetylation was decreased by expression of HDAC1 alone.HSCO alone also decreased it, probably by enhancing theendogenous HDAC1 activity. Co-expression of HSCO andHDAC1 further decreased the p53 acetylation. Upon co-ex-pressionwithmutant (H141A)HDAC1, the effect ofHSCOwaslost and p53 acetylation was restored, indicating that H141AHDAC1 acts as a dominant negative mutant.When expression of HDAC1 was down-regulated in H1299

cells, the effect of HSCO on acetylation of exogenous p53 wasabrogated (Fig. 5H, left). A similar effect of HDAC1 down-reg-ulation was additionally observed on endogenous p53 (Fig. 5H,right), suggesting that the effect of HSCO on p53 acetylation isHDAC1-dependent.Interaction of HSCOwith HDAC1—Whenwe co-transfected

U-2OS cellswith plasmids expressing FLAG-taggedHSCOandEGFP-tagged HDAC1, HDAC1 co-immunoprecipitated withHSCO from the cell lysates (Fig. 6A). This interaction was spe-cific, because HDAC2, HDAC3, and SIRT1 did not co-immu-noprecipitate with HSCO under similar conditions (data notshown). The HSCO point mutant (H79N) co-immunoprecipi-tated with HDAC1 (Fig. 6B), although it is defective in enhanc-ing deacetylation of p53 (Fig. 5C). The interaction ofHSCOandHDAC1 was also observed in p53-null H1299 cells (Fig. 6C),

FIGURE 4. Interaction of HSCO with p53. A, co-immunoprecipitation (IP) ofexogenous proteins. H1299 cells were co-transfected with plasmids express-ing wild-type or mutant (H79N) HSCO tagged with FLAG and EGFP-p53 asindicated. Cell lysates (10% input) and immunoprecipitates prepared by IPwith indicated antibodies were analyzed by Western blotting (WB). Arrowsindicate mobilities of specific bands. B, effects of actinomycin D (ActD) ongene expression. U-2 OS cells were treated with ActD, and expression of HSCOand p53 were analyzed at indicated times by Northern blotting (NB) and WBusing cDNA probes and antibodies as indicated. C, Co-IP of endogenousHSCO. U2-OS cells were treated with ActD, and 4 h later cell lysates wereanalyzed by IP and WB as indicated. D, GST-pulldown assays. GST-HSCOfusion protein or GST was incubated with lysates of U2-OS cells treated withActD for the indicated times (left panels) or with DNA-damaging agents for 4 has indicated (right panels). Bound proteins and 10% inputs were analyzed byWB using anti-p53 antibody.

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indicating that the binding was not mediated by p53. By using aseries of HSCO deletion mutants in co-immunoprecipitationand Western blot analysis, full-length HSCO was found to benecessary for the binding (data not shown).As shown in Fig. 6D,endogenous HSCO was co-immunoprecipitated with endoge-nous HDAC1 from A549 cell lysates. When A549 cells weretreated with actinomycin D, the endogenous protein levels ofHSCOandp53 increased andmore endogenousHSCOandp53co-immunoprecipitated with HDAC1.

Although no direct bindingbetween recombinant HSCO andrecombinant HDAC1 was observed(Fig. 6E), a GST-pulldown assayusing lysates prepared from U-2 OScells expressing FLAG-HDAC1 andtreated with actinomycin D demon-strated an interaction betweenGST-HSCO and HDAC1 (Fig. 6F).The binding was also observedusing lysates from p53-null H1299cells expressing FLAG-HDAC1(Fig. 6G). These results suggest thatHDAC1 indirectly interacts withHSCO and/or a modification ofHDAC1 is necessary for this p53-in-dependent interaction. As shown inFig. 6H, HSCO co-immunoprecipi-tatedwithHDAC1 from lysates pre-pared from DKO-MEFs, indicatingthat neither Mdm2 nor p53 is nec-essary for the binding of HSCO toHDAC1.HSCO co-immunoprecipitated

with p53 (Fig. 4) and with HDAC1independently of p53 (Fig. 6). We,therefore, asked whether HDAC1affects the interaction betweenp53 and HSCO. From U-2 OS celllysates, HSCO was co-immuno-precipitated with p53, the amountof which was increased by co-ex-pression of HDAC1 (Fig. 7A). Theamount of p53 co-immunopre-cipitated with HSCO was alsoincreased in the presence ofHDAC1. When the expression ofendogenous HDAC1 was down-regulated, the binding of HSCO andp53 was decreased (Fig. 7B).Results with confocal micros-

copy were consistent with a notionthat HSCO, p53, and HDAC1 forma complex in the nucleus (Fig. 7C).HSCO shuttles between nucleusand cytoplasm (20). Recently,HSCO was suggested as a mito-chondrial protein responsible forethylmalonic encephalopathy (22).

In U-2 OS cells HSCO C-terminally tagged with FLAG wasobserved mainly in the nucleus, cytoplasm, or both (22.3 � 3.6,10.7 � 2.6, and 67.0 � 2.9%, respectively, of transfected cells,n � 3), and some of the cytoplasmic HSCOwas localized in themitochondria (Fig. 7D). The subcellular localization of N-ter-minally tagged HSCO was not different from that of C-termi-nally tagged HSCO (data not shown). In summary, we havedemonstrated that HSCO inhibits p53-dependent apoptosis bysuppressing transcriptional activity of p53.HSCO forms a com-

FIGURE 5. Effects of HSCO on p53 acetylation. A, no effect of HSCO on DNA damage-induced phospho-rylation of p53. U-2 OS cells stably overexpressing HSCO-FLAG or FLAG alone were treated with actino-mycin D (ActD). Cell lysates were prepared at 0 and 4 h after the treatment and analyzed by Westernblotting (WB) using indicated antibodies. B, suppression of p300-induced acetylation of p53 by HSCOoverexpression in vivo. 293T cells were co-transfected with plasmids expressing HSCO, Mdm2, and p300 asindicated and cultured in the presence (�) or absence (�) of TSA or nicotinamide. Cell lysates wereimmunoprecipitated with agarose-immobilized anti-p53 antibody and analyzed by WB after equalizationof p53 content in each precipitate. C, effects of HSCO mutation. 293T cells were co-transfected withplasmids expressing wild-type (wt) or mutant (H79N) HSCO, Mdm2, and p300 as indicated and analyzed asin B. D, effects of HSCO down-regulation. U-2 OS cells were transfected with pSuper vectors expressingshRNAs for wild-type (wt) HSCO or its mutant (mut), and cultured in the presence (�) or absence (�) ofproteasome inhibitors. Cell lysates or immunoprecipitates prepared with agarose-immobilized anti-p53antibody were analyzed by WB using indicated antibodies. E, effects of HSCO on p53 acetylation in vitro.p300 HAT domain fused to GST was preincubated with HSCO or Mdm2, and then GST-p53 was added assubstrate in the presence of [14C]acetyl-CoA. Acetylation of p53 was visualized by SDS-PAGE and autora-diography. F, effects of HSCO on p53 deacetylation in vitro. 293T cells were co-transfected with plasmidsexpressing HSCO, FLAG-tagged wild-type HDAC1, mutant (H141A) HDAC1, HDAC2, or HDAC3 as indi-cated. Immunoprecipitates were prepared from cell lysates with anti-FLAG antibody, incubated withacetylated p53, and analyzed by WB using indicated antibodies. G, effects of HSCO and HDAC1 overex-pression on p53 acetylation in vivo. H1299 cells were co-transfected with plasmids expressing HSCO,FLAG-tagged wild-type or mutant (H141A) HDAC1, p300, and p53 as indicated and analyzed as inB. H, effects of HDAC1 down-regulation on HSCO activity. H1299 cells (left panels) and U-2 OS cells (rightpanels) were co-transfected with plasmids expressing HSCO-FLAG, p300, p53, and shRNA specific toHDAC1 as indicated, treated with ActD (�) or vehicle alone (�). Cell lysates or immunoprecipitates pre-pared with agarose-immobilized anti-p53 antibody were analyzed by WB using indicated antibodies afterequalization of p53 content.

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plex with HDAC1 and p53, enhances p53 deacetylation, anddecreases p53 stability.

DISCUSSION

HDAC-1, -2, and -3 are all capable of down-regulating p53transcriptional activity, which is dependent on the deacetylaseactivity of HDACs and p53 acetylation mediated by p300/CBP(13). Human SIRT1 also targets p53 for deacetylation (10).Because reduction of p53 acetylation by HSCOwas sensitive toTSA inhibition, but not to nicotinamide, HDAC(s) of Class Iand/or II must be involved. Although HDAC1 and HDAC2 arehighly similar with an overall sequence identity of 82% (9), invitro p53 deacetylation assays demonstrated that HSCO stim-ulates the deacetylase activity of HDAC1, but not HDAC2 orHDAC3. In vivo experiments demonstrated that HSCOreduces p53 acetylation in combination with HDAC1.

HSCO was co-precipitated with HDAC1 in the absence ofp53, although one or more modifications and/or cofactors ofHDAC1 were necessary for the binding. Recombinant p53 didnot interact with HSCO in vitro, but p53 was co-precipitatedwith HSCO in vivo. Co-expression of HDAC1 increased, andsuppression of HDAC1 expression decreased, the p53-HSCOco-precipitation. Thus, HDAC1 probablymediates the interac-tion between p53 and HSCO, and binding with HSCO facili-tates its binding to p53. HDAC1 does not directly interact withp53, and Mdm2, the oncogenic form of PML, and PID/MTA2have been shown to recruit HDAC1 to exert their negative con-trol on p53 function (14, 15, 28). Recently, the nuclear co-re-pressor KAP1 has been shown to inhibit p53 acetylation byinteracting with Mdm2 and stimulating formation of a p53-HDAC1 complex (29). In the p53-HDAC1-HSCO complex,however, Mdm2 is not involved. The molecule mediating thebinding of p53 to HDAC1 remains to be determined.HDAC1 levels are elevated in highly proliferative tissues,

embryonic stem cells, and several transformed cell lines (30).Over-expression of HDAC1 increases resistance of melanomacells to sodium butylate-induced apoptosis by suppressingacetylation of p53 and up-regulation of Bax (31).We found thatHSCO enhances the activity of HDAC1 and decreases acetyla-tion and transcriptional activity of p53. Thus, the suppressiveeffect of HSCO on p53-dependent apoptosis could, at leastpartly, be explained by its effect on HDAC1. Interestingly, byusing gain-of-function p53 acetylationmutants (Gln in place of

FIGURE 6. Interaction of HSCO with HDAC1. A, co-immunoprecipitation (IP)of exogenous proteins. U-2 OS cells were co-transfected with plasmidsexpressing HSCO-FLAG and EGFP-HDAC1 as indicated. Cell lysates (10%input) and immunoprecipitates prepared by IP with indicated antibodieswere analyzed by Western blotting (WB). Arrows indicate mobilities of specificbands. B, co-IP of HDAC1 and mutant HSCO. U-2 OS cells were co-transfectedwith plasmids expressing mutant (H79N) HSCO tagged with FLAG and EGFP-HDAC1as indicated and analyzed as in A except that biotinylated antibodieswere used in WB. C, co-IP in the absence of p53. H1299 cells were co-trans-fected with plasmids expressing HSCO-FLAG and HA-HDAC1 as indicated andanalyzed as in A. D, co-IP of endogenous proteins. A549 cells were treatedwith actinomycin D for 0 or 8 h. Cell lysates (10% input) and immunoprecipi-tates prepared with anti-HDAC1 antibody or control IgG were analyzed by WBusing indicated antibodies. E, GST-pulldown assays. GST-HDAC1 fusion pro-tein, GST-RelA, or GST was incubated with recombinant HSCO, and boundproteins and 10% inputs were analyzed by WB using anti-HSCO antibody(upper panel). GST-HSCO or GST were incubated with recombinant HDAC1,and bound proteins and 10% inputs were analyzed by WB using anti-HDAC1antibody (lower panel). F, GST-pulldown assay using cell lysates. GST-HSCO orGST was incubated with lysates of U2-OS cells transfected with plasmidsexpressing FLAG-HDAC1. Bound proteins and 10% inputs were analyzed byWB using anti-FLAG antibody. G, binding in the absence of p53. GST-HSCO orGST was incubated with lysates of H1299 cells transfected with plasmidsexpressing FLAG-HDAC1 and analyzed as in F. H, co-IP in the absence of p53and Mdm2. DKO-MEFs were co-transfected with plasmids expressing HSCO-FLAG and EGFP-HDAC1 as indicated and analyzed as in A.

FIGURE 7. Complex formation of HSCO with HDAC1 and p53. A, effects ofHDAC1 overexpression on co-immunoprecipitation (IP) of HSCO and p53. U-2OS cells were co-transfected with plasmids expressing HSCO-FLAG, HA-p53,and EGFP-HDAC1 as indicated. Cell lysates (10% input) and immunoprecipi-tates prepared by IP with indicated antibodies were analyzed by Westernblotting (WB) using indicated antibodies. B, effects of HDAC1 down-regula-tion. U-2 OS cells were co-transfected with plasmids expressing HSCO-FLAG,HA-p53, and shRNA for HDAC1 as indicated and analyzed as in A. C, co-local-ization of HSCO, HDAC1, and p53. U-2 OS cells stably expressing HSCO C-ter-minally tagged with FLAG (HSCO-FLAG) were transfected with plasmidsexpressing HA-p53 and EGFP-HDAC1. HSCO-FLAG was detected with anti-FLAG antibody and streptavidin-allophycocyanin and appears blue under theconfocal laser microscope. HA-p53 detected with anti-HA antibody, TRITC-anti-rabbit IgG, and EGFP-HDAC1 detected by GFP fluorescence appear redand green, respectively. D, mitochondrial localization of HSCO. U2-OS cellsstably expressing HSCO-FLAG were incubated with MitoTracker. HSCO-FLAGwas detected with anti-FLAG antibody and fluorescein isothiocyanate-anti-mouse IgG and appears green under the confocal microscope. MitoTrackerappears red.

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Lys) as well as DNA-damaging agents, Knights et al. (32) haveshown that acetylation of Lys-373 enhances the interaction ofp53 with promoters of pro-apoptotic genes, whereas acetyla-tion of Lys-320 promotes cell survival. Consistent with theirfindings, HSCO mainly reduced acetylation of Lys-373/382rather than Lys-320 and suppressed apoptosis after DNA dam-age. Recently, the decision to undergo apoptosis upon DNAdamage has been reported to be mediated through acetylationof p53 at Lys-120 in the DNA-binding domain (33, 34).Whether HSCO affects p53 acetylation at this site is presentlyunknown.Because the C-terminal Lys residues of p53 acetylated in

response to DNA damage are also ubiquitylated by Mdm2, ithas been suggested that p53 acetylation stabilizes p53 (5). Insupport of this notion, increased levels of p53 acetylation bydeacetylase inhibitors inhibit p53 degradation in vivo, and thep53 degradation requires deacetylation (2, 35). However, thephysiological effect of the C-terminal modifications on p53 sta-bilization has been questioned by in vivo knock-in experiments(7, 36, 37). Consistent with the traditional view, we found thatoverexpression of HSCO decreased p53 acetylation andreduced the p53 protein level by enhancing its ubiquitylationand degradation in U-2 OS cells (Fig. 3). Reciprocally, down-regulation of HSCO increased p53 acetylation and suppressedits degradation (Fig. 5). However, exogenous p53 protein levelswere not affected by HSCO in DKO-MEFs, whereas p53 acety-lation was decreased and transcriptional activity of p53 wassuppressed (Fig. 2D).4 This finding suggests that degradation ofp53may not be the dominant mode of action by which HSCOinhibits the function of p53. Reduction of p53 acetylation isprobably more important, which could down-regulate thetranscriptional activity of p53 and reduce apoptosis (2).We found that expression of HSCO enhances p53 deacetyla-

tion, especially at Lys-373/382 and suppresses p53 transcrip-tional activity. Gu and Roeder (38) found that p300/CBP acety-lates the C terminus of p53, specifically, Lys-370/372/373/382,and that acetylated p53 has stronger ability to bind to p53-responsive elements in vitro. Several subsequent studies alsosupported the idea in vitro and in vivo (8, 39). However, the roleof p53 acetylation in stimulating its DNA binding is controver-sial (40), and several studies have shown that p53 acetylation isimportant for the recruitment of coactivators to p53-depend-ent promoters (41, 42). In the present study, although in vitroelectrophoretic mobility shift assay demonstrated a reducedbinding of nuclear p53 to short p53-responsive elements whenHSCO was overexpressed, this could be due to a reducedamount of p53. By repressing p300-mediated p53 acetylation,HSCOprobably inhibits coactivator recruitment and transcrip-tional activation of the p53-inducible pro-apoptotic genes.Recently, p300-mediated acetylation has been shown to pro-

mote the cytoplasmic localization of p53 (43). Apoptotic func-tions of p53 include a nuclear role as a transcription factor anda non-nuclear role in the cytoplasm and mitochondria (44). Inresponse to stress, p53moves to themitochondria and activatespro-apoptotic proteins such as Bid, Bak, and Bax. Thus, by

enhancing deacetylation of p53, HSCO could suppress nuclear-cytoplasmic trafficking of p53 and prevent its interaction withthe apoptosis machinery in mitochondria. Consistent with thispossibility, HSCO expression was inducible after DNA damageand p53 co-localized with HSCO and HDAC1 in the nucleus ofcells overexpressing HSCO. Mutations that replace the aminoacid ofHSCOconservedwithin themetallo-�-lactamase familymembers abrogated its activity to enhance deacetylation andsuppress transcriptional activity of p53. That themutantHSCOretained the ability to bind toHDAC1 and form a complex withp53 suggests a possibility that an unknown enzyme-like activityof HSCO is important for the observed enhancement ofHDAC1 activity. Because mutations in the HSCO gene causeethylmalonic encephalopathy that affects the brain, gastroin-testinal tract, and peripheral vessels (22), it might be worth-while to investigate the effects of mutations on the subcellularlocalization and cytoplasmic function of p53 in these tissues.Death receptor 5 is a pro-apoptotic protein considered to be

a potential target for cancer therapy, and etoposide-induceddeath receptor 5 expression requires cooperation between p53and RelA (45). HSCO binds to and inhibits RelA (20). HSCOalso binds to HDAC1 and inhibits p53 (this study). Thus, theanti-apoptotic effect of HSCO in etoposide-treated cellsexpressing wild-type p53 could, at least partly, be explained bythese effects. HCCs have been regarded as tumors quite resist-ant to chemotherapeutic agents (16). p53 mutation is not fre-quent in HCCs, especially in low grade or low stage tumors,compared with other types of tumors (46). Down-regulation ofHSCO sensitizes human cancer cell lines with wild-type p53 toapoptosis induced by DNA-damaging agents (Ref. 20 and thisstudy). Considering its function and relatively frequent overex-pression in HCCs, we may be able to design effective chemo-therapeutic interventions by inhibiting HSCO and repairingthe apoptotic p53 response in HCCs.

Acknowledgments—We thank Dr. D. P. Lane for DKO-MEFs and Dr.S. Dawson for helpful suggestions.

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HSCO Enhances Deacetylation of p53 by HDAC1

MAY 4, 2007 • VOLUME 282 • NUMBER 18 JOURNAL OF BIOLOGICAL CHEMISTRY 13725

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Jun FujitaHisako Higashitsuji, Hiroaki Higashitsuji, Tomoko Masuda, Yu Liu, Katsuhiko Itoh and

with Histone Deacetylase 1Enhanced Deacetylation of p53 by the Anti-apoptotic Protein HSCO in Association

doi: 10.1074/jbc.M609751200 originally published online March 12, 20072007, 282:13716-13725.J. Biol. Chem. 

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VOLUME 282 (2007) PAGES 13716–13725DOI 10.1074/jbc.AAC120.014613

Correction: Enhanced deacetylation of p53 by the anti-apoptotic protein HSCO in association with histonedeacetylase 1.Hisako Higashitsuji, Hiroaki Higashitsuji, Tomoko Masuda, Yu Liu,Katsuhiko Itoh, and Jun Fujita

There was an error in Fig. 5B. Lanes 7–10 did not show the experi-mental data obtained. This error has now been corrected by removingthese lanes. Accordingly, the legend and the text referring to Fig. 5Bshould be changed from “TSA or nicotinamide” to “TSA” and from“but not nicotinamide” to “but not nicotinamide (data not shown),”respectively. This correction does not affect the results or conclusionsof this work. The authors sincerely apologize for the mistake thatoccurred during figure preparation and for any inconvenience thiserror may have caused readers.

Figure 5B.

9264 J. Biol. Chem. (2020) 295(27) 9264–9264

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