Báo cáo khoa học: The transcription factor ZBP-89 suppresses p16 expression through a histone modification mechanism to affect cell senescence doc

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The transcription factor ZBP-89 suppresses p16 expressionthrough a histone modification mechanism to affectcell senescenceYunpeng Feng*, Xiuli Wang*, Liang Xu, Hong Pan, Shan Zhu, Qian Liang, Baiqu Huangand Jun LuInstitute of Genetics and Cytology, Northeast Normal University, and the Key Laboratory of Molecular Epigenetics of Ministry of Education(MOE), Northeast Normal University, Changchun, ChinaIntroductionZBP-89 is a ubiquitously expressed four-zinc fingertranscription factor that binds to the GC-rich DNAelements, functioning either as a repressor or as anactivator of the known target genes. For instance,when acting as an activator, ZBP-89 recruits thecoactivator p300 to the p21 promoter, resulting inKeywordshistone deacetylase 3 (HDAC3); histonedeacetylase 4 (HDAC4); p16; senescence;ZBP-89CorrespondenceJ. Lu, Institute of Genetics and Cytology,and the Key Laboratory of MolecularEpigenetics of MOE, Northeast NormalUniversity, 5268 Renmin Street, Changchun130024, ChinaFax: +86 431 85099768Tel: +86 431 85099798E-mail: luj809@nenu.edu.cn*These authors contributed equally to thiswork(Received 1 April 2009, revised 29 May2009, accepted 3 June 2009)doi:10.1111/j.1742-4658.2009.07128.xThe transcription factor ZBP-89 has been implicated in the induction ofgrowth arrest and apoptosis. In this article, we demonstrate that ZBP-89was able to restrain senescence in NCI-H460 human lung cancer cells,through epigenetically regulating p16INK4aexpression. Specifically, ourresults indicate that knockdown of ZBP-89 by RNA interference stimulatedcellular senescence in NCI-H460 cells, as judged by the senescence-associated b-galactosidase activity assay and senescence-associated hetero-chromatin foci assay, and this process could be reversed by RNAinterference-mediated p16INK4asilencing. We also show that histone deacet-ylase (HDAC) 3 and HDAC4 inhibited p16INK4apromoter activity in adose-dependent manner. Furthermore, chromatin immunoprecipitationassays verified that HDAC3 was recruited to the p16INK4apromoter byZBP-89 through an epigenetic mechanism involving histone acetylationmodification. Moreover, immunofluorescence and coimmunoprecipitationassays revealed that ZBP-89 and HDAC3 formed a complex. These datasuggest that ZBP-89 and HDAC3, but not HDAC4, can work coordinatelyto restrain cell senescence by downregulating p16INK4aexpression throughan epigenetic modification of histones.Structured digital abstractlMINT-7144512: HDAC4 (uniprotkb:P56524) physically interacts (MI:0914) with ZBP-89(uniprotkb:Q9UQR1)byanti tag coimmunoprecipitation (MI:0007)lMINT-7144482, MINT-7144499: ZBP-89 (uniprotkb:Q9UQR1) physically interacts (MI:0914)with HDAC3 (uniprotkb:O15379)byanti tag coimmunoprecipitation (MI:0007)lMINT-7144469: ZBP-89 (uniprotkb:Q9UQR1) and HDAC3 (uniprotkb:O15379) colocalize(MI:0403)byfluorescence microscopy (MI:0416)AbbreviationsCDK, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; Co-IP, coimmunoprecipitation; DAPI, 4¢,6-diamidino-2-phenylindole;GFP, green fluorescent protein; HAT, histone acetyltransferase; HDAC, histone deacetylase; RNAi, RNA interference; SAHF, senescence-associated heterochromatin foci; SA-b-gal, senescence-associated b-galactosidase; siRNA, small interfering RNA; TRITC,tetramethylrhodamine isothiocyanate.FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS 4197upregulation of the gene [1]. Bai and Merchant alsoreported that elevated expression of ZBP-89 inducedgrowth arrest and apoptosis through promoting p21expression upon treatment with the histone deacetylase(HDAC) inhibitor butyrate, or through stabilizing p53protein, indicating that ZBP-89 plays a role in cellcycle progression [2]. Recently, Wu et al. [3] reportedthat ZBP-89 functioned as a repressor by recruitingHDAC1 to the vimentin promoter. ZBP-89 shares withSp1 and other Sp-like factors the ability to recognizeGC-rich sequences in target genes. To depict this over-lapping DNA recognition, a competitive model of inhi-bition has been proposed, in which ZBP-89 repressesgene transcription by displacing proteins such as Sp1and Sp3 [4,5]. An analysis of the proximal promoter ofthe ornithine decarboxylase gene revealed that Sp1 andZBP-89 bound to the GC elements in a mutuallyexclusive manner [6]. In other cases, ZBP-89 appearsto inhibit gene activity by binding to DNA indepen-dently of Sp1 [7].Reversible acetylation of internal lysine residues ofthe N-terminal domains of nucleosomal histones andthe resultant changes in the chromatin structure areimportant epigenetic mechanisms in the regulation ofgene transcription. The interplay between histone acet-yltransferases (HATs) and HDACs is critical to thedynamics of chromatin structure and function, thusregulating gene expression in eukaryotes [8]. SeveralHATs have been identified that act as transcriptionalcoactivators. In contrast, HDACs form part of tran-scriptional corepressor complexes [9].The INK4A locus encodes a cyclin-dependent kinase(CDK) inhibitor, p16INK4a(hereafter p16), which func-tions as a negative regulator of cyclin–CDK com-plexes. It binds preferentially to CDK4 and CDK6,and prevents their association with D-type cyclins,thus inhibiting retinoblastoma protein phosphorylationand blocking cell cycle progression [10,11]. Expressionof p16 is regulated primarily at the transcriptionallevel. The p16 promoter lacks a distinct TATA box,and is GC-rich. The GC-rich regions represent theputative binding sites for the ubiquitously expressedSp1 transcription factor [12]. As ZBP-89 also binds tothe GC-rich DNA elements, it raises the question ofwhether ZBP-89 participates in p16 transcriptional reg-ulation. In this article, we present experimental datashowing that knockdown of ZBP-89 in human lungcancer cells by a specific small interfering RNA (siR-NA) vector (ZBP-89i) increased expression of p16 andinduced cell senescence. Moreover, overexpression ofHDAC3 and HDAC4 resulted in repression of p16expression, and HDAC3 was recruited to the p16 pro-moter through ZBP-89. On the basis of these data, wediscuss the possible mechanisms of the functionalinteractions among ZBP-89, HDAC3 and HDAC4 inp16 transcriptional inhibition and their effects on cellsenescence.ResultsKnockdown of endogenous ZBP-89 promotedNCI-H460 cell senescencePreviously, a study showed that ZBP-89 was able toinduce cell growth arrest and apoptosis [2]. However,whether ZBP-89 affects cancer cell senescence has notbeen investigated. To test this effect, we constructed ansiRNA vector specific to ZBP-89 (ZBP-89i) to knockdown ZBP-89 expression in the human lung cancer cellline NCI-H460. Western blots verified the exogenousexpression of the ZBP-89 vector (Fig. 1A), and theefficiency of inhibition of ZBP-89 expression by ZBP-89i (Fig. 1B). The transfected NCI-H460 cells werethen lysed and assayed for the activity of senescence-associated b-galactosidase (SA-b-gal; pH 6.0), a bio-marker that is tightly associated with senescence inhuman cells [13]. As shown in Fig. 1C, a 1.5-foldincrease in SA-b-gal activity was seen after 7 days ofZBP-89i transfection, whereas overexpression of ZBP-89 led to a reverse effect. In addition, cells transfectedwith ZBP-89i exhibited phenotypic changes that aretypical of cells undergoing replicative senescence. Thesechanges include increased SA-b-gal staining, flattenedcell morphology, and enlarged cell size (Fig. 1D).Meanwhile, the senescence-associated heterochromatinfoci (SAHF) assays were performed using antibodiesagainst 3MeK9H3 and HP1 proteins, and the reactionsof these antibodies were visualized by confocal micros-copy. As shown in Fig. 1E, both marker proteins werelocalized to the specific heterochromatic foci in cellstransfected with ZBP-89i. Also, 3MeK9H3 and HP1proteins were found to be colocalized in discrete fociin the senescent cells, as observed by confocal micros-copy. Together, these data implied that ZBP-89 playeda role in restraint of human lung cancer NCI-H460 cellsenescence.ZBP-89 interacted with the p16 promoter torepress its transcriptionIt has been well documented that p16 plays a criticalrole in inducing cell senescence; we were therefore curi-ous to know whether ZBP-89 induced senescencethrough p16 regulation in NCI-H460 cells. We used ap16 siRNA vector (p16i) to knock down p16 expres-sion [14]. The results showed that, as compared trans-ZBP-89 affects senescence through p16 Y. Feng et al.4198 FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBSfection with ZBP-89i alone, cotransfection of cells withZBP-89i and p16i vectors failed to induce NCI-H460cell senescence (Fig. 2A). Western blotting demon-strated that p16 protein expression was decreased onZBP-89 ectopic expression, whereas it was enhancedby knockdown of the endogenous ZBP-89 in NCI-H460 cells (Fig. 2B). Furthermore, overexpression ofZBP-89 greatly inhibited p16 promoter activity(Fig. 2C). Also, it can be seen from Fig. 2D that thep16 mRNA level was decreased on ectopic expressionof ZBP-89, but increased by knockdown of endoge-nous ZBP-89. To determine whether ZBP-89 was trulypresent at the p16 promoter to regulate the gene as atranscription factor, we designed a series of primerscoordinate to the three regions in the p16 promoterfor chromatin immunoprecipitation (ChIP) assays(Fig. 2E). P1 locates far upstream of the p16 promoter()1800 bp) as a negative control, whereas P2 and P3locate downstream of the p16 promoter at )700 and)400 bp, which represent the important regulatoryregions of the p16 gene. The ChIP data shown inFig. 2F reveal that ZBP-89 was enriched at the P2 andP3 regions of the p16 promoter upon ZBP-89 overex-pression. These results suggest that ZBP-89 was ableto inhibit p16 expression at the promoter activity,mRNA and protein levels.HDAC3 and HDAC4 downregulated p16 byinducing histone hypoacetylationWe previously reported that the HAT p300 stimulatedp16 expression [14], and this prompted us to speculatewhether HDAC(s) also plays a role in p16 regulationas the opposing enzyme(s) to the HATs. To test thisassumption, we transfected 293T cells with the p16promoter reporter together with the expression vectorsof HDAC1–HDAC6; of the six HDACs tested,HDAC3 and HDAC4 had much more prominenteffects on p16 repression (Fig. 3A). Also, p16promoter activity was inhibited by HDAC3 andHDAC4 overexpression in a dose-dependent manner(Fig. 3B,C). The endogenous p16 mRNA level wasalso decreased upon HDAC3 and HDAC4 overexpres-sion, as revealed by real-time PCR (Fig. 3D). Addi-tionally, ChIP assays with antiacetylated histone H3and histone H4 antibodies showed that the acetylationlevel of histone H3 was significantly changed by exoge-nous expression of HDAC4, whereas the acetylation ofFig. 1. Knockdown of endogenous ZBP-89promoted human lung cancer NCI-H460 cellsenescence. Western blot analysis of theZBP-89 protein in NCI-H460 cells transfect-ed with ZBP-89–Flag, or pcDNA3.1 as acontrol (A), or ZBP-89i, or ZBP-89–Flag plusZBP-89i vectors, and an irrelevant siRNAvector as a control (B). (C) ZBP-89iincreased the SA-b-gal activity. NCI-H460cells transfected with ZBP-89 or ZBP-89ivectors were lysed and tested for SA-b-galactivity, using o-nitrophenyl-D-galactopyrano-side as substrate at pH 6.0. The controlswere the pcDNA3.1 empty vector andan irrelevant siRNA vector.**P < 0.01,*P < 0.05 (n = 3). (D) Representative photo-micrographs of the SA-b-gal staining atday 7 post-ZBP-89i transfection. The irrele-vant siRNA vector was used as the control.(E) NCI-H460 cells were transfected withZBP-89i for 7 days. Cells were stained withDAPI, and heterochromatic foci were visual-ized by fluorescence microscopy. 3MeK9H3was immunostained in red, and HP1 ingreen. The nuclei were counterstained withDAPI (blue). It can be seen that HP1 and3MeK9H3 were colocalized in senescentcells in discrete SAHF (white and yellowspots), as shown by confocal microscopy.Y. Feng et al. ZBP-89 affects senescence through p16FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS 4199histone H4 was markedly affected by overexpressionof HDAC3 (Fig. 3E,F). These experiments demon-strate that repression of p16 expression by HDAC3and HDAC4 coincided with histone hypoacetylation.HDAC3 interacted with ZBP-89We next sought to investigate whether physical interac-tions among HDAC3 ⁄ HDAC4 and ZBP-89 occur. In293T cells cotransfected with HDAC3 ⁄ 4–green fluores-cent protein (GFP) and ZBP-89, HDAC3 and ZBP-89were colocalized in the nuclei, but HDAC4 and ZBP-89were not colocalized remarkably, as revealed by confocallaser scanning microscopy (Fig. 4A). Moreover, coim-munoprecipitation (Co-IP) assays revealed that com-plexes containing HDAC3–GFP ⁄ HDAC4–GFP andZBP-89–Flag were precipitated by antibodies againstGFP and Flag, and they were detected in immunoblotsby antibodies against Flag and GFP (Fig. 4B), suggest-ing that HDAC3 and ZBP-89 were present in the samecomplexes, but not HDAC4. These data provide evi-dence that the transcription factor ZBP-89 and the core-pressor HDAC3 interacted and worked coordinately tocontribute to the repression of p16 expression.HDAC3 was recruited to the p16 promoter byZBP-89To determine whether HDAC3 and HDAC4 wererecruited to the p16 promoter by ZBP-89, we examinedthe binding of HDAC3 and HDAC4 in differentregions of the p16 promoter upon knockdown of theendogenous ZBP-89, and the results showed that thebinding of HDAC3 was indeed significantly reduced byknockdown of the endogenous ZBP-89, whereas that ofHDAC4 was not affected (Fig. 5A). We then analyzedthe relationship between histone H3 ⁄ H4 acetylationand ZBP-89 expression. The ChIP data indicated thatthe histone H4 acetylation level was decreased by over-expression of ZBP-89, whereas that of histone H3 wasFig. 2. ZBP-89 restrained cancer cell senescence by repressing p16 expression. (A) ZBP-89 restrained senescence of NCI-H460 cellsthrough p16 repression. Representative photomicrographs of the SA-b-gal staining at day 7 after ZBP-89i transfection, or ZBP-89i plus p16itransfection. An irrelevant siRNA vector was used as the control. (B) ZBP-89 repressed p16 protein expression. Western blot analysis of thep16 protein in NCI-H460 cells transfected with ZBP-89 or ZBP-89i vector. (C) ZBP-89 inhibited p16 promoter activity. 293T cells were trans-fected with ZBP-89 vector, and the p16 promoter activity was examined by luciferase reporter assay. The control was the pcDNA3.1 emptyvector.**P < 0.01 (n = 3). (D) ZBP-89 repressed endogenous p16 mRNA. 293T cells were transfected with ZBP-89 or ZBP-89i vector. TotalRNA was isolated and reverse transcribed, and p16 mRNA was measured by PCR. b-Actin was used as an internal control. (E) Diagram ofthe 5¢-flanking region of p16 gene. Lines denote the three regions of the p16 promoter (P1, P2, and P3) amplified by specific primers in ChIPanalysis. (F) Binding of ZBP-89 on the p16 promoter. ChIP assays with antibody against Flag in 293T cells transfected with the ZBP-89–Flagexpression vector. No Ab: samples with no antibody. Input: DNA prior to immunoprecipitation.ZBP-89 affects senescence through p16 Y. Feng et al.4200 FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBSnot affected (Fig. 5B). Moreover, the acetylation levelsof both histone H3 and histone H4 were increased byZBP-89i transfection (Fig. 5C). Furthermore, we mea-sured the binding of endogenous HDAC3 andHDAC4, as well as ZBP-89, at the p16 promoter inNCI-H460 cells. The results showed that the binding ofZBP-89 and HDAC3 was reduced by knockdown ofthe endogenous ZBP-89 (Fig. 5D). Thus, these dataclearly indicate that HDAC3, but not HDAC4, wasrecruited to the p16 promoter via ZBP-89.DiscussionZBP-89 is a four zinc finger transcription factor thateither represses or activates several target genes [15],although it is more commonly known as a transcrip-tional repressor [16]. This transcription factor containsa transcription activation domain at its C-terminusand a repression domain at its N-terminus [17]. It hasbeen shown that ZBP-89 binds to the GC-rich pro-moter elements of genes that are involved in cellgrowth regulation, e.g. genes coding for gastrin, orni-thine decarboxylase, and the CDK inhibitor p21[1,4,6,18]. It was reported that elevated expression ofZBP-89 induced growth arrest and apoptosis throughpromoting p21 expression upon treatment with theHDAC inhibitor butyrate, or through p53 protein sta-bilization [2]. We report here that ZBP-89 was capableof restraining human lung cancer NCI-H460 cell senes-cence, and this process could be reversed by inhibitionof p16 expression through RNA interference (RNAi)(Fig. 2A). Our data also show that ZBP-89 was ableto decrease both p16 promoter activity (Fig. 2C) andthe endogenous p16 mRNA level (Fig. 2D), as well asto decrease the p16 protein level (Fig. 2B). Theseexperimental results supported our assumption that thecell senescence induced by ZBP-89 might be p16-dependent. A number of previous reports suggestedthat p16 was required for cellular senescence in normalhuman fibroblasts [19]. A more recent study by Herbiget al. [20] indicated that p16 and p21 acted throughindependent pathways to influence cellular senescence.Taking together all of these data, we speculate thatZBP-89 is a multiple-function factor that participatesin a variety of cell processes by regulating differentgenes. These functions include the induction of apop-tosis through p21 and p53 [2], and the restraint of cellsenescence through p16, as shown in this study.Fig. 3. HDAC3 and HDAC4 downregulated p16 by inducing histone hypoacetylation. (A) HDAC3 and HDAC4 downregulated p16 promoteractivity. 293T cells were transfected with p16 luciferase plasmid together with HDAC constructs expressing HDAC1–HDAC6. Results areshown as fold repression relative to that of the cells transfected with empty plasmid. (B, C) One microgram of the p16 reporter vector, plusdifferent amounts of HDAC3 (B) or HDAC4 (C), were cotransfected into 293T cells. Luciferase activity was determined 24 h after transfec-tion and normalized to the Renilla activity. The pcDNA3.1 vector was used as the control. **P < 0.01, *P < 0.05 (n = 3). (D) Quantitativeestimation of p16 mRNA level. Cells were transfected with HDAC3 or HDAC4 vector. Total RNA was isolated and reverse transcribed, andp16 mRNA was measured by real-time PCR. b-Actin was used as an internal control. **P < 0.01, *P < 0.05 (n = 3). (E, F) HDAC3 andHDAC4 participated in p16 regulation by inducing histone hypoacetylation. Cells were transfected with HDAC3 or HDAC4. The presence ofacety-H3 (E) or acety-H4 (F) in each region was measured by real-time PCR. The input was used as an internal control. Input: DNA prior toimmunoprecipitation. **P < 0.01, *P < 0.05 (n = 3).Y. Feng et al. ZBP-89 affects senescence through p16FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS 4201It has been suggested that, as a transcription factor,ZBP-89 can function through multiple mechanisms.These mechanisms include the competition of ZBP-89with transcription activators such as Sp1 for over-lapping binding sites, thereby decreasing promoteractivity and transcription intensity [4]. Others includethe ability of ZBP-89 to recruit the coactivator p300 tothe promoter of the target gene, resulting in upregula-tion of gene expression [1]. A third model suggests thatZBP-89 recruits a corepressor to a promoter, and thatthis corepressor either negatively regulates other fac-tors that are present, or alters the local chromatinstructure, through factors such as HDAC1 [3]. How-ever, the precise link between ZBP-89 and the chroma-tin-modifying factors, e.g. the HDACs, has not beenextensively investigated prior to this study. Here, wediscovered that the siRNA-mediated knockdown ofendogenous ZBP-89 expression markedly reduced theenrichment of HDAC3 on the p16 promoter(Fig. 5A,D). Our experimental evidence also supportsimportant roles of the HDAC activity of HDAC3 inrepression of p16 expression (Fig. 3). It is likely thatthe inhibition of on p16 gene expression by ZBP-89 fitsthe model described by Wu et al. [3], which involvesthe recruitment of corepressor and chromatin modifiersto the gene promoter. Our Co-IP evidence for thecoexistence of HDAC3 and ZBP-89 in the same com-plex (Fig. 4B) further supports this notion, and thedata in Fig. 5A,D show that knockdown of ZBP-89failed to decrease the binding of HDAC4 to the p16promoter. We suspect that the interaction betweenFig. 4. Interactions among ZBP-89 and HDAC3 ⁄ 4. (A) Colocalizationof ZBP-89 and HDAC3 ⁄ 4. 293T cells were plated onto glass slidesand transfected with HDAC3–GFP or HDAC4–GFP plus ZBP-89–Flag. Cells were fixed in formaldehyde and stained with antibodyagainst FLAG and then a TRITC secondary antibody, and visualizedunder a fluorescence microscope. ZBP-89 was immunostained inred and HDAC3 ⁄ 4 in green. The nuclei were counterstained withHoechst 33342 (blue). Cells were examined under a Confocal LaserScanning Microscope (Olympus, FV-1000, Japan). (B) Co-IP assaysfor the association of HDAC3 ⁄ 4 with ZBP-89. The cell nuclearextracts were prepared and precipitated with antibodies againstFlag and GFP, and detected by using immunoblotting with respec-tive antibodies. Lanes 1, 3, 5 and 7: cells were transfected withHDAC3–GFP and ZBP-89–Flag vectors. Lanes 2, 4, 6 and 8: cellswere transfected with HDAC4–GFP and ZBP-89–Flag vectors.Lanes 1 and 2: input and with anti-GFP serum. Lanes 3 and 4:immunoprecipitation (IP) with anti-Flag serum, immuno-blotting (IB)with anti-GFP serum. Lanes 5 and 6: input and with anti-Flagserum. Lanes 7 and 8: IP with anti-Flag serum, IB with anti-GFPserum. Input: protein prior to immunoprecipitation.Fig. 5. HDAC3 was recruited to the p16 promoter by ZBP-89. (A)HDAC3 was recruited to the p16 promoter by ZBP-89. 293T cellswere transfected with ZBP-89i vector together with HDAC3–Flag orHDAC4–Flag vector. Samples were immunoprecipitated with anti-body against Flag. DNA was then amplified using PCR. (B, C) ChIPassays for detection of the presence of acetylated histone H3 andhistone H4 on the p16 promoter. 293T cells were transfected withZBP-89 (B) or ZBP-89i vector (C). Cells were then harvested, andDNA was sheared and immunoprecipitated with antibodies againstacetylated histone H3 and acetylated histone H4. Input: DNA priorto immunoprecipitation. (D) NCI-H460 cells were transfected withZBP-89i vector. Samples were immunoprecipitated with antibodiesagainst HDAC3, HDAC4, or ZBP-89. DNA was then amplified usingPCR. No Ab: samples with no antibody.ZBP-89 affects senescence through p16 Y. Feng et al.4202 FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBSZBP-89 and HDAC4 might be indirect. There havebeen indications that HDAC3 can interact withHDAC4 [26]. These data have led us to speculate thatZBP-89 may interact with HDAC4 through HDAC3,thus forming a complex that works coordinately tocontribute to the repression of p16 expression.HDACs are expressed in a variety of tissue types. Inmammalian cells, HDACs normally function as repres-sors of gene expression by forming large protein com-plexes [21]. Also, HDACs could directly interact withtranscription factors to repress gene expression. Forinstance, HDAC1 can directly interact with the tran-scription factor MyoD to silence p21 gene expression[22]. HDAC2 and HDAC4 interact with the tran-scription factor YY1 to repress gene expression [23,24],and HDAC3 interacts with c-Jun to mediate AP-1-dependent gene repression [25]. HDAC1 is recruited tovimentin’s proximal promoter by ZBP-89 [3]. In thisstudy, HDAC3, but not HDCA4, was found to bethe specific deacetylase recruited to the p16 promoter,further verifying the gene specificity of HDACs.To summarize, we demonstrate in this article, forthe first time, that the multifunctional transcriptionfactor ZBP-89 was able to restrain human lung cancerNCI-H460 cell senescence through inhibition of p16expression, and this process involved the recruitmentof HDAC3 to the p16 promoter by ZBP-89. Moreover,we provide experimental evidence that ZBP-89 andHDAC3 coexisted in the same complex and workedcoordinately to contribute to the repression of p16expression, which, in turn, induced cell senescence.Noticeably, current data indicate that, as a bifunc-tional transcription factor, ZBP-89 can interact withp300 ⁄ CBP on the p21 promoter to enhance gene activ-ity [1], or with HDAC3 on the p16 promoter to sup-press p16 expression, as shown in this study. Furtherexperiments will be required to fully elucidate thedetails of the regulatory mechanisms of ZBP-89.Experimental proceduresCell culture, transfection, and luciferase reporterassayThe human lung cancer cell line NCI-H460 and the humanembryonic kidney cell line 293T were maintained in IMDMsupplemented with 10% fetal bovine serum, 100 mg ÆmL)1penicillin and 100 mgÆmL)1streptomycin in a humidifiedatmosphere containing 5% CO2at 37 °C. The 293T cellswere transfected using a standard calcium phosphatemethod. Cells were then incubated for 5 h before the culturemedium was changed. After another 24 or 48 h, cells wereharvested for luciferase activity, RT-PCR, western blot orChIP assays. The luciferase activities were measured on aTurner Designs TD-20 ⁄ 20 Luminometer in the Dual-Lucif-erase Assay System (Promega, Madison, WI, USA) mode,which uses a second luciferase gene from Renilla reniformis,providing constitutive activity as an internal control. TheNCI-H460 cells were transfected using Fu GENE HD trans-fection reagent (Roche, Basel, Switzerland).Plasmid constructsThe p16 promoter reporter ()869 to +1 bp from the ATGtranslation initiation site) ligated to the luciferase reportergene (pGL2 basic; Promega) was provided by E. Hara(Imperial Cancer Research Fund Laboratories, London,UK). Plasmids expressing human HDAC3 and HDAC4(fused to the FLAG-epitope) were gifts from W. C. Greene(Gladstone Institute of Virology and Immunology, SanFrancisco, CA, USA). Flag-ZBP-89-myc was provided byJ. L. Merchant (Department of Internal Medicine andPhysiology, University of Michigan, USA). The plasmidsexpressing human HDAC4 (fused to the GFP-epitope) weregenerously provided by R. Bassel-Duby (Department ofMolecular Biology, University of Texas SouthwesternMedical Center, Dallas, TX, USA).RNA extraction and real-time quantitative PCRTotal cellular RNA was extracted from the 293T cellsaccording to the Promega Total RNA Isolation System man-ual. RNA was resuspended in RNase-free water and quanti-tated by spectrophotometry before being reverse transcribed.PCR products were resolved in 2% agarose gel. Real-timequantitative PCR analyses for mRNA levels were performedusing an ABI Prism 7000 Sequence Detection System(Applied Biosystems, Foster City, CA, USA) with an SYBRGreen kit (Toyobo, Osaka, Japan). The primer pairs for p16were as follows: sense, 5¢-TTCCTGGACACGCTGGT-3¢;and antisense, 5¢-CAATCGGGGATGTCTGAG-3¢. Theb-actin primer pairs were as follows: sense, 5¢-TCGTGCGTGACATTAAGGAG-3¢; and antisense, 5¢-ATGCCAGGGTACATGGTGGT-3¢. The 25 lL reaction mixture contained1nm each primer. Data were analyzed by using the 2)DDCtmethod [26].ChIPThe protocol for ChIP has been described previously [27].Briefly, the chromatin solution was precleared with 50 lLofprotein A–agarose beads (Upstate Biotechnology, SantaCruz, CA, USA). The soluble fraction was collected, and5 lg of antibodies against acetyl-histone H3 (Upstate Bio-technology), acetyl-histone H4 (Upstate Biotechnology),HDAC3 (Santa Cruz; sc-11417), HDAC4 (Santa CruzBiotechnology, Santa Cruz, CA, USA; sc-11418) or FlagY. Feng et al. ZBP-89 affects senescence through p16FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS 4203(Sigma, St Louis, MO, USA; F3165) were added. The immu-noprecipitated chromatin DNAs were analyzed by PCR orreal-time quantitative PCR. The sequences of the primersused were as follows: P1 sense, 5¢-AGTTTCGCTCTTGTCTCCCAG-3¢; P1 antisense, 5¢-ATGGCGAAACCCTGTCTCTAC-3¢; P2 sense, 5¢-AGACAGCCGTTTTACACGCAG-3¢;P2 antisense, 5¢-CACCGAGAAATCGAAATCACC-3¢;P3sense, 5 ¢-TAGGAAGGTTGTATCGCGGAGG-3¢; and P3antisense, 5¢-CAAGGAAGGAGGACTGGGCTC-3¢ [28].The locations of P1, P2 and P3 at the p16 promoter areillustrated in Fig. 2E. All of the PCR experiments wererepeated at least three times, and one of the representativeresults is shown.Western blot and Co-IP assaysNCI-H460 and 293T cells were harvested after treatments,and 1 · 106cells were digested and lysed in the lysis bufferfor 30 min at 4 °C. Total cell extracts were separated by12% SDS ⁄ PAGE, and then transferred to a poly(vinylidenedifluoride) membrane. The membrane was incubated withantibodies against p16 (Santa Cruz; sc-468), Flag, ZBP-89(Santa Cruz; sc-19408), or b-actin (Sigma; A1978), andvisualized by using the chemiluminescent substrate methodwith the SuperSignal West Pico kit provided by Pierce Co.,(Rockford, IL, USA). b-Actin was used as an internalcontrol for normalizing the loading materials.Coprecipitation was performed in 293T cells, using a pro-tocol described elsewhere [24]. Total cell extracts were precle-ared with 40 lL of protein A–agarose at 4 °C for 1 h. Thesupernatant was incubated with the antibodies against Flagand GFP (Upstate; 06-896) with gentle shacking for 1 h at4 °C, and this was followed by the addition of 40 lL of pro-tein A–agarose for another 3 h. The beads were resuspendedin 100 lLof2· loading buffer and boiled for 10 min. Theproteins were separated on a 12% SDS ⁄ PAGE gel and thentransferred to a poly(vinylidene difluoride) membrane forimmunoblot detection with antibodies against Flag or GFP.RNAiThe ZBP-89-targeting and p16-targeting siRNAs weresynthesized according to published data. The target RNAisequence for ZBP-89 was 5¢-GAGCAGAAGCAGGTGCAGA-3¢ [29]. The p16-targeting siRNA sequence was5¢-GAGGAGGTGCGGGCGCTGC-3¢ [18]. An oligo-nucleotide that represents the small hairpin RNA targetingthe ZBP-89 sequence was designed and cloned into the pSli-encer2.0-U6 vector (Ambion, Austin, TX, USA) betweenthe BamHI and HindIII restriction sites, according to themanufacturer’s instructions. Cells were seeded in six-wellplates, cultured for 18 h, and then transfected with 5 lgofZBP-89 siRNA, p16 siRNA, or control vectors. Cells wereincubated for another 48 h, and collected for immunoblot-ting analysis.Immunofluorescence staining and SAHF assayThe treated 293T cells were washed twice in NaCl ⁄ Pi, fixedin 4% paraformaldehyde for 15 min, permeabilized with0.2% Triton X-100 at room temperature, and thenquenched in ice-cold NaCl ⁄ Pi. After blocking with 5% BSA,collected cells were incubated with rabbit anti-Flag serumfor 1 h and stained with tetramethylrhodamine isothio-cyanate (TRITC)-conjugated goat anti-(rabbit serum) assecondary antibody (Zhongshan, Beijing, China) for 45 minat 4 °C. Cells were examined under an Olympus FV1000(Olympus, Tokyo, Japan) confocal microscope. For SAHFassay, cells were incubated with rabbit anti-HP1 serum for1 h and stained with fluorescein isothiocyanate-conjugatedgoat anti-(rabbit serum) as secondary antibody, incubatedwith rat anti-3MeK9H3 serum and stained with TRITC-conjugated goat anti-(rat serum) as secondary antibody, andfinally stained with 4¢,6-diamidino-2-phenylindole (DAPI).Cells were visualized under an Olympus FV1000 (Olympus,Japan) confocal microscope.Senescence-associated galactosidase activityassayCells were lysed in reporter lysis buffer. Cell lysatescontaining equal amounts of protein were diluted in equalvolumes of 2· assay buffer containing 1.33 mgÆmL)1o-nitrophenyl-d-galactopyranoside, 2 mm MgCl2and 100 lLof 2-mercaptoethanol in 200 mm phosphate buffer (pH6.0), and incubated at 37 °C for 4 h. The absorbance at420 nm was measured after the addition of an equalvolume of 1 m Na2CO3.NCI-H460 cells were transfected with the ZBP-89i vec-tors and p16i vectors, or an irrelevant siRNA vector as thecontrol. At day 7 after transfection, cells were processedusing a Senescence b-Galactosidase Staining Kit (Cell Sig-naling Technology, Danvers, MA, USA). These experi-ments were repeated three times, and one of therepresentative results is shown.AcknowledgementsThis work was supported by grants from The NationalBasic Research Program of China (2005CB522404 and2006CB910506), the Program for Changjiang Scholarsand Innovative Research Team (PCSIRT) in Universi-ties (IRT0519), and the National Natural ScienceFoundation of China (30800557 and 30671184).References1 Bai L & Merchant JL (2000) Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 duringbutyrate activation of p21waf1 transcription in humancells. J Biol Chem 275, 30725–30733.ZBP-89 affects senescence through p16 Y. Feng et al.4204 FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS2 Bai L & Merchant JL (2001) ZBP-89 promotes growtharrest through stabilization of p53. 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ZBP-89 affects senescence through p16FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS 420527 Lu J, Sun H, Wang X, Liu C, Xu X, Li F & Huang B(2005) Interleukin-12 p40 promoter activity is regulatedby the reversible acetylation mediated by HDAC1 andp300. Cytokine 31, 46–51.28 Kondo Y, Shen L & Issa JP (2003) Critical role ofhistone methylation in tumor suppressor gene silencingin colorectal cancer. Mol Cell Biol 23, 206–215.29 Sui G, elAffar B, Shi Y, Brignone C, Wall NR, Yin P,Donohoe M, Luke MP, Calvo D, Grossman SR et al.(2004) Yin Yang 1 is a negative regulator of p53. Cell117, 859–872.ZBP-89 affects senescence through p16 Y. Feng et al.4206 FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS . The transcription factor ZBP-89 suppresses p16 expression through a histone modification mechanism to affect cell senescence Yunpeng Feng*, Xiuli Wang*,. 5¢-ATGGCGAAACCCTGTCTCTAC-3¢; P2 sense, 5¢-AGACAGCCGTTTTACACGCAG-3¢;P2 antisense, 5¢-CACCGAGAAATCGAAATCACC-3¢;P3sense, 5 ¢-TAGGAAGGTTGTATCGCGGAGG-3¢; and P3antisense,
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