The transcription factor ZBP-89 suppresses p16 expression through a histone modification mechanism to affect cell senescence Yunpeng Feng*, Xiuli Wang*, Liang Xu, Hong Pan, Shan Zhu, Qian Liang, Baiqu Huang and Jun Lu Institute of Genetics and Cytology, Northeast Normal University, and the Key Laboratory of Molecular Epigenetics of Ministry of Education (MOE), Northeast Normal University, Changchun, China Introduction ZBP-89 is a ubiquitously expressed four-zinc finger transcription factor that binds to the GC-rich DNA elements, functioning either as a repressor or as an activator of the known target genes. For instance, when acting as an activator, ZBP-89 recruits the coactivator p300 to the p21 promoter, resulting in Keywords histone deacetylase 3 (HDAC3); histone deacetylase 4 (HDAC4); p16; senescence; ZBP-89 Correspondence J. Lu, Institute of Genetics and Cytology, and the Key Laboratory of Molecular Epigenetics of MOE, Northeast Normal University, 5268 Renmin Street, Changchun 130024, China Fax: +86 431 85099768 Tel: +86 431 85099798 E-mail: luj809@nenu.edu.cn *These authors contributed equally to this work (Received 1 April 2009, revised 29 May 2009, accepted 3 June 2009) doi:10.1111/j.1742-4658.2009.07128.x The transcription factor ZBP-89 has been implicated in the induction of growth arrest and apoptosis. In this article, we demonstrate that ZBP-89 was able to restrain senescence in NCI-H460 human lung cancer cells, through epigenetically regulating p16 INK4a expression. Specifically, our results indicate that knockdown of ZBP-89 by RNA interference stimulated cellular 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 RNA interference-mediated p16 INK4a silencing. We also show that histone deacet- ylase (HDAC) 3 and HDAC4 inhibited p16 INK4a promoter activity in a dose-dependent manner. Furthermore, chromatin immunoprecipitation assays verified that HDAC3 was recruited to the p16 INK4a promoter by ZBP-89 through an epigenetic mechanism involving histone acetylation modification. Moreover, immunofluorescence and coimmunoprecipitation assays revealed that ZBP-89 and HDAC3 formed a complex. These data suggest that ZBP-89 and HDAC3, but not HDAC4, can work coordinately to restrain cell senescence by downregulating p16 INK4a expression through an epigenetic modification of histones. Structured digital abstract l MINT-7144512: HDAC4 (uniprotkb:P56524) physically interacts (MI:0914) with ZBP-89 (uniprotkb: Q9UQR1)byanti tag coimmunoprecipitation (MI:0007) l MINT-7144482, MINT-7144499: ZBP-89 (uniprotkb:Q9UQR1) physically interacts (MI:0914) with HDAC3 (uniprotkb: O15379)byanti tag coimmunoprecipitation (MI:0007) l MINT-7144469: ZBP-89 (uniprotkb:Q9UQR1) and HDAC3 (uniprotkb:O15379) colocalize ( MI:0403)byfluorescence microscopy (MI:0416) Abbreviations CDK, 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 4197 upregulation of the gene [1]. Bai and Merchant also reported that elevated expression of ZBP-89 induced growth arrest and apoptosis through promoting p21 expression upon treatment with the histone deacetylase (HDAC) inhibitor butyrate, or through stabilizing p53 protein, indicating that ZBP-89 plays a role in cell cycle progression [2]. Recently, Wu et al. [3] reported that ZBP-89 functioned as a repressor by recruiting HDAC1 to the vimentin promoter. ZBP-89 shares with Sp1 and other Sp-like factors the ability to recognize GC-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 represses gene transcription by displacing proteins such as Sp1 and Sp3 [4,5]. An analysis of the proximal promoter of the ornithine decarboxylase gene revealed that Sp1 and ZBP-89 bound to the GC elements in a mutually exclusive manner [6]. In other cases, ZBP-89 appears to inhibit gene activity by binding to DNA indepen- dently of Sp1 [7]. Reversible acetylation of internal lysine residues of the N-terminal domains of nucleosomal histones and the resultant changes in the chromatin structure are important epigenetic mechanisms in the regulation of gene transcription. The interplay between histone acet- yltransferases (HATs) and HDACs is critical to the dynamics of chromatin structure and function, thus regulating gene expression in eukaryotes [8]. Several HATs have been identified that act as transcriptional coactivators. In contrast, HDACs form part of tran- scriptional corepressor complexes [9]. The INK4A locus encodes a cyclin-dependent kinase (CDK) inhibitor, p16 INK4a (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 phosphorylation and blocking cell cycle progression [10,11]. Expression of p16 is regulated primarily at the transcriptional level. The p16 promoter lacks a distinct TATA box, and is GC-rich. The GC-rich regions represent the putative binding sites for the ubiquitously expressed Sp1 transcription factor [12]. As ZBP-89 also binds to the GC-rich DNA elements, it raises the question of whether ZBP-89 participates in p16 transcriptional reg- ulation. In this article, we present experimental data showing that knockdown of ZBP-89 in human lung cancer cells by a specific small interfering RNA (siR- NA) vector (ZBP-89i) increased expression of p16 and induced cell senescence. Moreover, overexpression of HDAC3 and HDAC4 resulted in repression of p16 expression, and HDAC3 was recruited to the p16 pro- moter through ZBP-89. On the basis of these data, we discuss the possible mechanisms of the functional interactions among ZBP-89, HDAC3 and HDAC4 in p16 transcriptional inhibition and their effects on cell senescence. Results Knockdown of endogenous ZBP-89 promoted NCI-H460 cell senescence Previously, a study showed that ZBP-89 was able to induce cell growth arrest and apoptosis [2]. However, whether ZBP-89 affects cancer cell senescence has not been investigated. To test this effect, we constructed an siRNA vector specific to ZBP-89 (ZBP-89i) to knock down ZBP-89 expression in the human lung cancer cell line NCI-H460. Western blots verified the exogenous expression of the ZBP-89 vector (Fig. 1A), and the efficiency of inhibition of ZBP-89 expression by ZBP- 89i (Fig. 1B). The transfected NCI-H460 cells were then 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 in human cells [13]. As shown in Fig. 1C, a 1.5-fold increase in SA-b-gal activity was seen after 7 days of ZBP-89i transfection, whereas overexpression of ZBP- 89 led to a reverse effect. In addition, cells transfected with ZBP-89i exhibited phenotypic changes that are typical of cells undergoing replicative senescence. These changes include increased SA-b-gal staining, flattened cell morphology, and enlarged cell size (Fig. 1D). Meanwhile, the senescence-associated heterochromatin foci (SAHF) assays were performed using antibodies against 3MeK9H3 and HP1 proteins, and the reactions of these antibodies were visualized by confocal micros- copy. As shown in Fig. 1E, both marker proteins were localized to the specific heterochromatic foci in cells transfected with ZBP-89i. Also, 3MeK9H3 and HP1 proteins were found to be colocalized in discrete foci in the senescent cells, as observed by confocal micros- copy. Together, these data implied that ZBP-89 played a role in restraint of human lung cancer NCI-H460 cell senescence. ZBP-89 interacted with the p16 promoter to repress its transcription It has been well documented that p16 plays a critical role in inducing cell senescence; we were therefore curi- ous to know whether ZBP-89 induced senescence through p16 regulation in NCI-H460 cells. We used a p16 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 FEBS fection with ZBP-89i alone, cotransfection of cells with ZBP-89i and p16i vectors failed to induce NCI-H460 cell senescence (Fig. 2A). Western blotting demon- strated that p16 protein expression was decreased on ZBP-89 ectopic expression, whereas it was enhanced by knockdown of the endogenous ZBP-89 in NCI- H460 cells (Fig. 2B). Furthermore, overexpression of ZBP-89 greatly inhibited p16 promoter activity (Fig. 2C). Also, it can be seen from Fig. 2D that the p16 mRNA level was decreased on ectopic expression of ZBP-89, but increased by knockdown of endoge- nous ZBP-89. To determine whether ZBP-89 was truly present at the p16 promoter to regulate the gene as a transcription factor, we designed a series of primers coordinate to the three regions in the p16 promoter for chromatin immunoprecipitation (ChIP) assays (Fig. 2E). P1 locates far upstream of the p16 promoter ()1800 bp) as a negative control, whereas P2 and P3 locate downstream of the p16 promoter at )700 and )400 bp, which represent the important regulatory regions of the p16 gene. The ChIP data shown in Fig. 2F reveal that ZBP-89 was enriched at the P2 and P3 regions of the p16 promoter upon ZBP-89 overex- pression. These results suggest that ZBP-89 was able to inhibit p16 expression at the promoter activity, mRNA and protein levels. HDAC3 and HDAC4 downregulated p16 by inducing histone hypoacetylation We previously reported that the HAT p300 stimulated p16 expression [14], and this prompted us to speculate whether HDAC(s) also plays a role in p16 regulation as the opposing enzyme(s) to the HATs. To test this assumption, we transfected 293T cells with the p16 promoter reporter together with the expression vectors of HDAC1–HDAC6; of the six HDACs tested, HDAC3 and HDAC4 had much more prominent effects on p16 repression (Fig. 3A). Also, p16 promoter activity was inhibited by HDAC3 and HDAC4 overexpression in a dose-dependent manner (Fig. 3B,C). The endogenous p16 mRNA level was also decreased upon HDAC3 and HDAC4 overexpres- sion, as revealed by real-time PCR (Fig. 3D). Addi- tionally, ChIP assays with antiacetylated histone H3 and histone H4 antibodies showed that the acetylation level of histone H3 was significantly changed by exoge- nous expression of HDAC4, whereas the acetylation of Fig. 1. Knockdown of endogenous ZBP-89 promoted human lung cancer NCI-H460 cell senescence. Western blot analysis of the ZBP-89 protein in NCI-H460 cells transfect- ed with ZBP-89–Flag, or pcDNA3.1 as a control (A), or ZBP-89i, or ZBP-89–Flag plus ZBP-89i vectors, and an irrelevant siRNA vector as a control (B). (C) ZBP-89i increased the SA-b-gal activity. NCI-H460 cells transfected with ZBP-89 or ZBP-89i vectors were lysed and tested for SA-b-gal activity, using o-nitrophenyl- D-galactopyrano- side as substrate at pH 6.0. The controls were the pcDNA3.1 empty vector and an irrelevant siRNA vector. ** P < 0.01, * P < 0.05 (n = 3). (D) Representative photo- micrographs of the SA-b-gal staining at day 7 post-ZBP-89i transfection. The irrele- vant siRNA vector was used as the control. (E) NCI-H460 cells were transfected with ZBP-89i for 7 days. Cells were stained with DAPI, and heterochromatic foci were visual- ized by fluorescence microscopy. 3MeK9H3 was immunostained in red, and HP1 in green. The nuclei were counterstained with DAPI (blue). It can be seen that HP1 and 3MeK9H3 were colocalized in senescent cells in discrete SAHF (white and yellow spots), as shown by confocal microscopy. Y. Feng et al. ZBP-89 affects senescence through p16 FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS 4199 histone H4 was markedly affected by overexpression of HDAC3 (Fig. 3E,F). These experiments demon- strate that repression of p16 expression by HDAC3 and HDAC4 coincided with histone hypoacetylation. HDAC3 interacted with ZBP-89 We next sought to investigate whether physical interac- tions among HDAC3 ⁄ HDAC4 and ZBP-89 occur. In 293T cells cotransfected with HDAC3 ⁄ 4–green fluores- cent protein (GFP) and ZBP-89, HDAC3 and ZBP-89 were colocalized in the nuclei, but HDAC4 and ZBP-89 were not colocalized remarkably, as revealed by confocal laser scanning microscopy (Fig. 4A). Moreover, coim- munoprecipitation (Co-IP) assays revealed that com- plexes containing HDAC3–GFP ⁄ HDAC4–GFP and ZBP-89–Flag were precipitated by antibodies against GFP and Flag, and they were detected in immunoblots by antibodies against Flag and GFP (Fig. 4B), suggest- ing that HDAC3 and ZBP-89 were present in the same complexes, but not HDAC4. These data provide evi- dence that the transcription factor ZBP-89 and the core- pressor HDAC3 interacted and worked coordinately to contribute to the repression of p16 expression. HDAC3 was recruited to the p16 promoter by ZBP-89 To determine whether HDAC3 and HDAC4 were recruited to the p16 promoter by ZBP-89, we examined the binding of HDAC3 and HDAC4 in different regions of the p16 promoter upon knockdown of the endogenous ZBP-89, and the results showed that the binding of HDAC3 was indeed significantly reduced by knockdown of the endogenous ZBP-89, whereas that of HDAC4 was not affected (Fig. 5A). We then analyzed the relationship between histone H3 ⁄ H4 acetylation and ZBP-89 expression. The ChIP data indicated that the histone H4 acetylation level was decreased by over- expression of ZBP-89, whereas that of histone H3 was Fig. 2. ZBP-89 restrained cancer cell senescence by repressing p16 expression. (A) ZBP-89 restrained senescence of NCI-H460 cells through p16 repression. Representative photomicrographs of the SA-b-gal staining at day 7 after ZBP-89i transfection, or ZBP-89i plus p16i transfection. An irrelevant siRNA vector was used as the control. (B) ZBP-89 repressed p16 protein expression. Western blot analysis of the p16 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 empty vector. ** P < 0.01 (n = 3). (D) ZBP-89 repressed endogenous p16 mRNA. 293T cells were transfected with ZBP-89 or ZBP-89i vector. Total RNA was isolated and reverse transcribed, and p16 mRNA was measured by PCR. b-Actin was used as an internal control. (E) Diagram of the 5¢-flanking region of p16 gene. Lines denote the three regions of the p16 promoter (P1, P2, and P3) amplified by specific primers in ChIP analysis. (F) Binding of ZBP-89 on the p16 promoter. ChIP assays with antibody against Flag in 293T cells transfected with the ZBP-89–Flag expression 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 FEBS not affected (Fig. 5B). Moreover, the acetylation levels of both histone H3 and histone H4 were increased by ZBP-89i transfection (Fig. 5C). Furthermore, we mea- sured the binding of endogenous HDAC3 and HDAC4, as well as ZBP-89, at the p16 promoter in NCI-H460 cells. The results showed that the binding of ZBP-89 and HDAC3 was reduced by knockdown of the endogenous ZBP-89 (Fig. 5D). Thus, these data clearly indicate that HDAC3, but not HDAC4, was recruited to the p16 promoter via ZBP-89. Discussion ZBP-89 is a four zinc finger transcription factor that either represses or activates several target genes [15], although it is more commonly known as a transcrip- tional repressor [16]. This transcription factor contains a transcription activation domain at its C-terminus and a repression domain at its N-terminus [17]. It has been shown that ZBP-89 binds to the GC-rich pro- moter elements of genes that are involved in cell growth 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 of ZBP-89 induced growth arrest and apoptosis through promoting p21 expression upon treatment with the HDAC inhibitor butyrate, or through p53 protein sta- bilization [2]. We report here that ZBP-89 was capable of restraining human lung cancer NCI-H460 cell senes- cence, and this process could be reversed by inhibition of p16 expression through RNA interference (RNAi) (Fig. 2A). Our data also show that ZBP-89 was able to decrease both p16 promoter activity (Fig. 2C) and the endogenous p16 mRNA level (Fig. 2D), as well as to decrease the p16 protein level (Fig. 2B). These experimental results supported our assumption that the cell senescence induced by ZBP-89 might be p16- dependent. A number of previous reports suggested that p16 was required for cellular senescence in normal human fibroblasts [19]. A more recent study by Herbig et al. [20] indicated that p16 and p21 acted through independent pathways to influence cellular senescence. Taking together all of these data, we speculate that ZBP-89 is a multiple-function factor that participates in a variety of cell processes by regulating different genes. These functions include the induction of apop- tosis through p21 and p53 [2], and the restraint of cell senescence through p16, as shown in this study. Fig. 3. HDAC3 and HDAC4 downregulated p16 by inducing histone hypoacetylation. (A) HDAC3 and HDAC4 downregulated p16 promoter activity. 293T cells were transfected with p16 luciferase plasmid together with HDAC constructs expressing HDAC1–HDAC6. Results are shown as fold repression relative to that of the cells transfected with empty plasmid. (B, C) One microgram of the p16 reporter vector, plus different 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) Quantitative estimation of p16 mRNA level. Cells were transfected with HDAC3 or HDAC4 vector. Total RNA was isolated and reverse transcribed, and p16 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 and HDAC4 participated in p16 regulation by inducing histone hypoacetylation. Cells were transfected with HDAC3 or HDAC4. The presence of acety-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 to immunoprecipitation. **P < 0.01, *P < 0.05 (n = 3). Y. Feng et al. ZBP-89 affects senescence through p16 FEBS Journal 276 (2009) 4197–4206 ª 2009 The Authors Journal compilation ª 2009 FEBS 4201 It has been suggested that, as a transcription factor, ZBP-89 can function through multiple mechanisms. These mechanisms include the competition of ZBP-89 with transcription activators such as Sp1 for over- lapping binding sites, thereby decreasing promoter activity and transcription intensity [4]. Others include the ability of ZBP-89 to recruit the coactivator p300 to the promoter of the target gene, resulting in upregula- tion of gene expression [1]. A third model suggests that ZBP-89 recruits a corepressor to a promoter, and that this corepressor either negatively regulates other fac- tors that are present, or alters the local chromatin structure, 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 been extensively investigated prior to this study. Here, we discovered that the siRNA-mediated knockdown of endogenous ZBP-89 expression markedly reduced the enrichment of HDAC3 on the p16 promoter (Fig. 5A,D). Our experimental evidence also supports important roles of the HDAC activity of HDAC3 in repression of p16 expression (Fig. 3). It is likely that the inhibition of on p16 gene expression by ZBP-89 fits the model described by Wu et al. [3], which involves the recruitment of corepressor and chromatin modifiers to the gene promoter. Our Co-IP evidence for the coexistence of HDAC3 and ZBP-89 in the same com- plex (Fig. 4B) further supports this notion, and the data in Fig. 5A,D show that knockdown of ZBP-89 failed to decrease the binding of HDAC4 to the p16 promoter. We suspect that the interaction between Fig. 4. Interactions among ZBP-89 and HDAC3 ⁄ 4. (A) Colocalization of ZBP-89 and HDAC3 ⁄ 4. 293T cells were plated onto glass slides and transfected with HDAC3–GFP or HDAC4–GFP plus ZBP-89– Flag. Cells were fixed in formaldehyde and stained with antibody against FLAG and then a TRITC secondary antibody, and visualized under a fluorescence microscope. ZBP-89 was immunostained in red and HDAC3 ⁄ 4 in green. The nuclei were counterstained with Hoechst 33342 (blue). Cells were examined under a Confocal Laser Scanning Microscope (Olympus, FV-1000, Japan). (B) Co-IP assays for the association of HDAC3 ⁄ 4 with ZBP-89. The cell nuclear extracts were prepared and precipitated with antibodies against Flag and GFP, and detected by using immunoblotting with respec- tive antibodies. Lanes 1, 3, 5 and 7: cells were transfected with HDAC3–GFP and ZBP-89–Flag vectors. Lanes 2, 4, 6 and 8: cells were 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-Flag serum. Lanes 7 and 8: IP with anti-Flag serum, IB with anti-GFP serum. 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 cells were transfected with ZBP-89i vector together with HDAC3–Flag or HDAC4–Flag vector. Samples were immunoprecipitated with anti- body against Flag. DNA was then amplified using PCR. (B, C) ChIP assays for detection of the presence of acetylated histone H3 and histone H4 on the p16 promoter. 293T cells were transfected with ZBP-89 (B) or ZBP-89i vector (C). Cells were then harvested, and DNA was sheared and immunoprecipitated with antibodies against acetylated histone H3 and acetylated histone H4. Input: DNA prior to immunoprecipitation. (D) NCI-H460 cells were transfected with ZBP-89i vector. Samples were immunoprecipitated with antibodies against HDAC3, HDAC4, or ZBP-89. DNA was then amplified using PCR. 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 FEBS ZBP-89 and HDAC4 might be indirect. There have been indications that HDAC3 can interact with HDAC4 [26]. These data have led us to speculate that ZBP-89 may interact with HDAC4 through HDAC3, thus forming a complex that works coordinately to contribute to the repression of p16 expression. HDACs are expressed in a variety of tissue types. In mammalian cells, HDACs normally function as repres- sors of gene expression by forming large protein com- plexes [21]. Also, HDACs could directly interact with transcription factors to repress gene expression. For instance, 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 to vimentin’s proximal promoter by ZBP-89 [3]. In this study, HDAC3, but not HDCA4, was found to be the specific deacetylase recruited to the p16 promoter, further verifying the gene specificity of HDACs. To summarize, we demonstrate in this article, for the first time, that the multifunctional transcription factor ZBP-89 was able to restrain human lung cancer NCI-H460 cell senescence through inhibition of p16 expression, and this process involved the recruitment of HDAC3 to the p16 promoter by ZBP-89. Moreover, we provide experimental evidence that ZBP-89 and HDAC3 coexisted in the same complex and worked coordinately to contribute to the repression of p16 expression, which, in turn, induced cell senescence. Noticeably, current data indicate that, as a bifunc- tional transcription factor, ZBP-89 can interact with p300 ⁄ 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. Further experiments will be required to fully elucidate the details of the regulatory mechanisms of ZBP-89. Experimental procedures Cell culture, transfection, and luciferase reporter assay The human lung cancer cell line NCI-H460 and the human embryonic kidney cell line 293T were maintained in IMDM supplemented with 10% fetal bovine serum, 100 mg ÆmL )1 penicillin and 100 mgÆmL )1 streptomycin in a humidified atmosphere containing 5% CO 2 at 37 °C. The 293T cells were transfected using a standard calcium phosphate method. Cells were then incubated for 5 h before the culture medium was changed. After another 24 or 48 h, cells were harvested for luciferase activity, RT-PCR, western blot or ChIP assays. The luciferase activities were measured on a Turner 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. The NCI-H460 cells were transfected using Fu GENE HD trans- fection reagent (Roche, Basel, Switzerland). Plasmid constructs The p16 promoter reporter ()869 to +1 bp from the ATG translation initiation site) ligated to the luciferase reporter gene (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, San Francisco, CA, USA). Flag-ZBP-89-myc was provided by J. L. Merchant (Department of Internal Medicine and Physiology, University of Michigan, USA). The plasmids expressing human HDAC4 (fused to the GFP-epitope) were generously provided by R. Bassel-Duby (Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA). RNA extraction and real-time quantitative PCR Total cellular RNA was extracted from the 293T cells according 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-time quantitative PCR analyses for mRNA levels were performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with an SYBR Green kit (Toyobo, Osaka, Japan). The primer pairs for p16 were as follows: sense, 5¢-TTCCTGGACACGCTGGT-3¢; and antisense, 5¢-CAATCGGGGATGTCTGAG-3¢. The b-actin primer pairs were as follows: sense, 5¢-TCGTGCGT GACATTAAGGAG-3¢; and antisense, 5¢-ATGCCAGGGT ACATGGTGGT-3¢. The 25 lL reaction mixture contained 1nm each primer. Data were analyzed by using the 2 )DDCt method [26]. ChIP The protocol for ChIP has been described previously [27]. Briefly, the chromatin solution was precleared with 50 lLof protein A–agarose beads (Upstate Biotechnology, Santa Cruz, CA, USA). The soluble fraction was collected, and 5 lg of antibodies against acetyl-histone H3 (Upstate Bio- technology), acetyl-histone H4 (Upstate Biotechnology), HDAC3 (Santa Cruz; sc-11417), HDAC4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-11418) or Flag Y. Feng et al. ZBP-89 affects senescence through p16 FEBS 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 or real-time quantitative PCR. The sequences of the primers used were as follows: P1 sense, 5¢-AGTTTCGCTCTTGTCT CCCAG-3¢; P1 antisense, 5¢-ATGGCGAAACCCTGTCTC TAC-3¢; P2 sense, 5¢-AGACAGCCGTTTTACACGCAG-3¢; P2 antisense, 5¢-CACCGAGAAATCGAAATCACC-3¢;P3 sense, 5 ¢-TAGGAAGGTTGTATCGCGGAGG-3¢; and P3 antisense, 5¢-CAAGGAAGGAGGACTGGGCTC-3¢ [28]. The locations of P1, P2 and P3 at the p16 promoter are illustrated in Fig. 2E. All of the PCR experiments were repeated at least three times, and one of the representative results is shown. Western blot and Co-IP assays NCI-H460 and 293T cells were harvested after treatments, and 1 · 10 6 cells were digested and lysed in the lysis buffer for 30 min at 4 °C. Total cell extracts were separated by 12% SDS ⁄ PAGE, and then transferred to a poly(vinylidene difluoride) membrane. The membrane was incubated with antibodies against p16 (Santa Cruz; sc-468), Flag, ZBP-89 (Santa Cruz; sc-19408), or b-actin (Sigma; A1978), and visualized by using the chemiluminescent substrate method with the SuperSignal West Pico kit provided by Pierce Co., (Rockford, IL, USA). b-Actin was used as an internal control 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. The supernatant was incubated with the antibodies against Flag and GFP (Upstate; 06-896) with gentle shacking for 1 h at 4 °C, and this was followed by the addition of 40 lL of pro- tein A–agarose for another 3 h. The beads were resuspended in 100 lLof2· loading buffer and boiled for 10 min. The proteins were separated on a 12% SDS ⁄ PAGE gel and then transferred to a poly(vinylidene difluoride) membrane for immunoblot detection with antibodies against Flag or GFP. RNAi The ZBP-89-targeting and p16-targeting siRNAs were synthesized according to published data. The target RNAi sequence for ZBP-89 was 5¢-GAGCAGAAGCAGGTG CAGA-3¢ [29]. The p16-targeting siRNA sequence was 5¢-GAGGAGGTGCGGGCGCTGC-3¢ [18]. An oligo- nucleotide that represents the small hairpin RNA targeting the ZBP-89 sequence was designed and cloned into the pSli- encer2.0-U6 vector (Ambion, Austin, TX, USA) between the BamHI and HindIII restriction sites, according to the manufacturer’s instructions. Cells were seeded in six-well plates, cultured for 18 h, and then transfected with 5 lgof ZBP-89 siRNA, p16 siRNA, or control vectors. Cells were incubated for another 48 h, and collected for immunoblot- ting analysis. Immunofluorescence staining and SAHF assay The treated 293T cells were washed twice in NaCl ⁄ P i , fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 at room temperature, and then quenched in ice-cold NaCl ⁄ P i . After blocking with 5% BSA, collected cells were incubated with rabbit anti-Flag serum for 1 h and stained with tetramethylrhodamine isothio- cyanate (TRITC)-conjugated goat anti-(rabbit serum) as secondary antibody (Zhongshan, Beijing, China) for 45 min at 4 °C. Cells were examined under an Olympus FV1000 (Olympus, Tokyo, Japan) confocal microscope. For SAHF assay, cells were incubated with rabbit anti-HP1 serum for 1 h and stained with fluorescein isothiocyanate-conjugated goat anti-(rabbit serum) as secondary antibody, incubated with rat anti-3MeK9H3 serum and stained with TRITC- conjugated goat anti-(rat serum) as secondary antibody, and finally stained with 4¢,6-diamidino-2-phenylindole (DAPI). Cells were visualized under an Olympus FV1000 (Olympus, Japan) confocal microscope. Senescence-associated galactosidase activity assay Cells were lysed in reporter lysis buffer. Cell lysates containing equal amounts of protein were diluted in equal volumes of 2· assay buffer containing 1.33 mgÆmL )1 o-nitrophenyl-d-galactopyranoside, 2 mm MgCl 2 and 100 lL of 2-mercaptoethanol in 200 mm phosphate buffer (pH 6.0), and incubated at 37 °C for 4 h. The absorbance at 420 nm was measured after the addition of an equal volume of 1 m Na 2 CO 3 . 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Cytokine 31, 46–51. 28 Kondo Y, Shen L & Issa JP (2003) Critical role of histone methylation in tumor suppressor gene silencing in 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. Cell 117, 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¢-ATGGCGAAACCCTGTCTC TAC-3¢; P2 sense, 5¢-AGACAGCCGTTTTACACGCAG-3¢; P2 antisense, 5¢-CACCGAGAAATCGAAATCACC-3¢;P3 sense, 5 ¢-TAGGAAGGTTGTATCGCGGAGG-3¢; and P3 antisense,