The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor
2005; Springer Nature; Volume: 24; Issue: 3 Linguagem: Inglês
10.1038/sj.emboj.7600550
ISSN1460-2075
AutoresGaynor J. Bates, Samantha M. Nicol, Brian J. Wilson, Anne-Marie F Jacobs, Jean‐Christophe Bourdon, Julie Wardrop, David J. Gregory, David P. Lane, Neil D. Perkins, Frances V. Fuller-Pace,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle20 January 2005free access The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor Gaynor J Bates Gaynor J Bates Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Samantha M Nicol Samantha M Nicol Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Brian J Wilson Brian J Wilson Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Anne-Marie F Jacobs Anne-Marie F Jacobs Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Jean-Christophe Bourdon Jean-Christophe Bourdon Department of Surgery & Molecular Oncology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Julie Wardrop Julie Wardrop Department of Surgery & Molecular Oncology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author David J Gregory David J Gregory Division of Gene Expression and Regulation, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author David P Lane David P Lane Department of Surgery & Molecular Oncology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Neil D Perkins Neil D Perkins Division of Gene Expression and Regulation, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Frances V Fuller-Pace Corresponding Author Frances V Fuller-Pace Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Gaynor J Bates Gaynor J Bates Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Samantha M Nicol Samantha M Nicol Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Brian J Wilson Brian J Wilson Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Anne-Marie F Jacobs Anne-Marie F Jacobs Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Jean-Christophe Bourdon Jean-Christophe Bourdon Department of Surgery & Molecular Oncology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Julie Wardrop Julie Wardrop Department of Surgery & Molecular Oncology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author David J Gregory David J Gregory Division of Gene Expression and Regulation, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author David P Lane David P Lane Department of Surgery & Molecular Oncology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Neil D Perkins Neil D Perkins Division of Gene Expression and Regulation, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Frances V Fuller-Pace Corresponding Author Frances V Fuller-Pace Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK Search for more papers by this author Author Information Gaynor J Bates1,‡, Samantha M Nicol1,‡, Brian J Wilson1, Anne-Marie F Jacobs1, Jean-Christophe Bourdon2, Julie Wardrop2, David J Gregory3, David P Lane2, Neil D Perkins3 and Frances V Fuller-Pace 1 1Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK 2Department of Surgery & Molecular Oncology, University of Dundee, Ninewells Hospital & Medical School, Dundee, UK 3Division of Gene Expression and Regulation, School of Life Sciences, University of Dundee, Dundee, UK ‡These authors contributed equally to this work *Corresponding author. Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee DD1 9SY, UK. Tel.: +44 1382 496370; Fax: +44 1382 633952; E-mail: [email protected] The EMBO Journal (2005)24:543-553https://doi.org/10.1038/sj.emboj.7600550 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. Here, we show that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53-dependent promoters and that endogenous p68 and p53 co-immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53-dependent apoptosis, but does not influence p53 stabilisation or expression of non-p53-responsive genes. We also show, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53-dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock-down does not significantly affect NF-κB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity. Introduction The DEAD box family of RNA helicases includes a large number of conserved proteins, which are found in all organisms from bacteria to humans and have been shown to be involved in virtually all cellular processes that require manipulation of RNA structure, including transcription, pre-mRNA processing, RNA degradation, RNA export, ribosome assembly and translation. Although the characteristic biochemical properties for this family are RNA-dependent ATPase and RNA helicase activities, relatively few members appear to be true processive helicases and it is clear that many are likely to be involved in unwinding of short base-paired regions of RNA or indeed in the disruption or rearrangement of RNA–protein interactions (Tanner and Linder, 2001). p68 is a prototypic member of the DEAD box family (Ford et al, 1988) and is an established ATPase and RNA helicase (Hirling et al, 1989; Iggo and Lane, 1989). Previous reports have shown that p68 expression is growth and developmentally regulated, and that p68 is overexpressed and abnormally polyubiquitylated in colorectal tumours (Stevenson et al, 1998; Causevic et al, 2001). Recently, p68 has been shown to be essential for pre-mRNA splicing in vitro (Liu, 2002) and to play a role in the regulation of c-H-ras alternative splicing (Guil et al, 2003). Dbp2p, the yeast homologue of p68 (Iggo et al, 1991), was found to be important for both rRNA processing and nonsense-mediated mRNA decay (Bond et al, 2001), while, in an earlier study, overexpressed human p68 was found to stabilise T7 mRNAs in bacteria (Iost and Dreyfus, 1994). Although the roles of p68 in pre-mRNA/rRNA processing and mRNA decay/stability are consistent with its function as an RNA helicase, p68 has also been reported to act as a transcriptional coactivator for oestrogen receptor alpha (ERα), a function that appears to be independent of helicase activity (Endoh et al, 1999; Watanabe et al, 2001). Moreover, p68 has recently been shown to be recruited to the promoter of the ERα target gene pS2 (Metivier et al, 2003), consistent with it playing a role in ERα-dependent transcriptional initiation. p68 has also been reported to interact with the transcriptional coactivators CBP/p300 as well as RNA polymerase II and to stimulate transcriptional activation mediated by CBP/p300 although, in this case, p68 ATPase/RNA helicase activity appeared to be required (Rossow and Janknecht, 2003). These findings therefore suggest that, in addition to its role in RNA processing, p68 may also have an important function as a transcriptional regulator. Given the implied role of p68 in growth regulation and tumour progression (Stevenson et al, 1998; Causevic et al, 2001), we investigated the ability of p68 to coactivate other transcription factors that are important in tumour development. One such protein is the critical tumour suppressor p53, a latent and labile transcription factor that is induced and activated in response to several stresses, including DNA damage (Vogelstein et al, 2000; Balint and Vousden, 2001). Activated p53 induces transcription of a host of downstream target genes, which are mainly involved in growth arrest, apoptosis and DNA repair. In addition, p53 also induces expression of its negative regulatory partner Mdm2 (Vogelstein et al, 2000; Balint and Vousden, 2001). In this report, we show that p68 is a potent transcriptional coactivator of p53, as shown by its ability to synergise with p53 to activate transcription from p53-responsive promoters. Additionally, endogenous p53 and p68 co-immunoprecipitate from nuclear protein extracts, suggesting that these proteins interact in the cell. Furthermore, by RNAi-mediated suppression of p68 expression in cells that express wild-type (WT) p53, we show that p68 is specifically required for the induction of expression of the cellular p53 target genes p21WAF-1, mdm2, Fas/APO1 and PIG3 in response to treatment with the DNA-damaging agent etoposide, while it has no effect on non-p53-responsive genes. This activity is specific to p68 since RNAi suppression of the highly related RNA helicase p72 (Lamm et al, 1996) has no effect on the induction of p53 transcriptional activity by DNA damage. We also show that p68 knock-down results in a reduction in apoptosis in response to p53 induction. Finally, we show by chromatin immunoprecipitation (ChIP) that p68 is recruited to the p21 promoter. These findings are therefore consistent with p68 being an important regulator of the p53 response and suggest a novel mechanism for regulating p53 transcriptional activity. Results p68 acts as a coactivator of p53 transcriptional activity To determine initially whether p68 has the potential to modulate the transcriptional activity of p53, we transfected H1299 (p53-null) cells with p68 and p53 cytomegalovirus (CMV) expression plasmids together with the p53-responsive reporter plasmid PG13-luciferase and measured luciferase activity. p68 potently synergised with p53 to activate transcription from the PG13 promoter (Figure 1A), supporting the hypothesis that p68 might regulate p53 transactivation function, with the most dramatic effect being observed with 10 ng of the p53 expression plasmid. In addition, titration of the p68 expression plasmid (Figure 1B) confirmed that this was a concentration-dependent effect. Since the highly related RNA helicase p72 was also reported to coactivate ERα (Watanabe et al, 2001), we tested whether p72 could similarly coactivate p53 transcriptional activity by carrying out a similar titration of a p72 expression plasmid. Interestingly, although some stimulation of p53 transcriptional activity by p72 was observed (Figure 1B), this was considerably lower than that seen for p68, suggesting that p72 is not such a potent coactivator of p53. (Both p68 and p72 were expressed at similar levels in these cells; see below and Supplementary data 1.) We then tested whether p68 could stimulate p53 transcriptional activity from other p53-responsive promoters. These included the p21 and Bax promoters as well as the p53-responsive element from the c-Ha-Ras gene (pRasH-Adluc) together with the nonresponsive pAdluc as a control (Deguin-Chambon et al, 2000) (Figure 1C–E). As previously shown with PG13 (Figure 1A and B), p68 synergised strongly with p53 to transactivate the p21 (Figure 1C) and the pRasH-Adluc promoters (Figure 1E), while a weaker effect was seen with the Bax promoter (Figure 1D). Importantly, no cooperative activation was observed with the pAdluc promoter, which lacks p53-binding sites (Figure 1E). These findings thus demonstrate that p68 synergises with p53 to activate transcription from a variety of p53-responsive promoters. A low level of transcriptional activation was observed when p68 alone was transfected (Figure 1C–E), suggesting that p68 has a low level of basal transcriptional activity; however, it should be noted that the amounts of p68 plasmid DNA transfected were higher than those for p53. Since the PG13 reporter plasmid gave the strongest effect in these experiments, we decided to use this to further characterise p68 coactivation activity. To confirm that the observed coactivation of p53 by p68 was not due simply to the transfected p68 affecting p53 levels in the cell, we examined the levels of p53 protein in the presence and absence of transfected p68/p72 by Western blotting (see Supplementary data 1). Although there were some minor variations in the expression of p53 between different transfections, increasing the amounts of transfected p68/p72 had no significant effect on the levels of p53. Figure 1.p68 stimulates p53 transcriptional activity from p53-responsive promoters. Effect of p68 on transactivation of the p53-responsive promoters PG13 (A, B), p21 (C), Bax (D) and pRasH-Adluc (E), fused to the luciferase reporter (pAdluc was used as a non-p53-responsive control (E)). In each case, the relative luciferase activity is shown with the basal activity of the promoter being taken as 1. Panels A and B show titres of the p53 and p68 plasmid DNAs, respectively, and the amounts used per ml of transfection mix are indicated. The amounts of reporter plasmid DNA used per ml of transfection mix were as follows: PG13, 2.5 μg; p21, 3 μg; Bax, 3 μg; pRasH-Adluc/pAdluc, 2.5 μg. Unless otherwise stated, the amounts of p53 plasmid transfected in these experiments had been optimised previously for the different promoters and were as follows: PG13, 10 ng; p21, Bax and pRasH-Adluc/pAdluc, 400 ng. Similarly, unless otherwise stated, 5 μg of p68 plasmid DNA was used. Graphs A and B represent the average results from two independent transfections, while graphs C–E represent average results from three independent experiments. Download figure Download PowerPoint We next tested whether p68 coactivation was dependent on transcriptionally active p53 or whether similar effects could be seen with a transcriptionally inactive (L22Q/W23S) mutant (Venot et al, 1999). As shown in Figure 2A, p68 did not coactivate the L22Q/W23S p53 mutant. (Similar results were obtained with a His 175 p53 mutant; data not shown.) Since previous reports had suggested that coactivation of ERα was not dependent on p68 helicase activity (Endoh et al, 1999), we also tested an ATPase/helicase-inactive mutant of p68 (NEAD p68). Interestingly, NEAD p68, which is expressed at similar levels to WT p68 (data not shown), was equally capable of coactivating p53 (Figure 2B). This suggests that p68 helicase activity is not required for p53 coactivation, although we cannot rule out the possibility that, in this system, the transfected (helicase-inactive) p68 interacts with endogenous WT p68 (Ogilvie et al, 2003) to form a complex that may have sufficient residual helicase activity. Figure 2.Coactivation requires transcriptionally active p53 but not helicase-active p68. (A) Coactivation of WT and L22Q/W23S (transcriptionally inactive) p53 transcriptional activity by p68. (B) Coactivation of WT p53 transcriptional activity by WT and NEAD (helicase-inactive) p68. Activity is determined by measurement of transactivation of the PG13 promoter fused to the luciferase reporter. The relative luciferase activity is shown, with the basal activity of the promoter being taken as 1. The amounts of p68 plasmid DNA used per ml of transfection mix are indicated and in all cases 10 ng of p53 plasmid DNA and 2.5 μg of PG13 reporter plasmid DNA were used per ml of transfection mix. Graphs represent the average results from two independent transfections. Download figure Download PowerPoint p68 interacts with p53 in vitro and in cultured cells p68 has previously been reported to interact with ERα (Endoh et al, 1999) as well as p300, CBP and RNA polymerase II (Rossow and Janknecht, 2003), suggesting that it may form part of a multiprotein complex to regulate transcription. We therefore investigated whether p68 could interact with p53. We first determined whether p68 and p53 can interact in vitro by performing GST pull-down experiments using purified GST-tagged p68 expressed in mammalian cells and in vitro-translated 35S-labelled p53. As shown in Figure 3A and B, GST-tagged p68, but not a GST vector control, interacted with in vitro-translated p53. Interestingly, p68 also interacted with ΔN-p53 (Figure 3B), which is the product of an alternative internal translation initiation site and lacks much of the amino-terminal transactivation domain of p53 (Courtois et al, 2002; Yin et al, 2002). We also tested whether p72 interacts with p53 in vitro; p72 does interact with p53 but much less efficiently than p68 (Figure 3A and B). To determine whether these proteins interact in the cell, we examined whether endogenous p53 and p68/p72 could co-immunoprecipitate from cell lysates. Nuclear extracts were prepared from U2OS cells, which harbour WT p53, and from SAOS-2 as a p53-null control. p53 was immunoprecipitated using DO-1; immunoprecipitated proteins were resolved by SDS–PAGE and Western blotted for the presence of p68/p72 in the immune complex. Reciprocal immunoprecipitation (IP)/Westerns were also carried out and appropriate irrelevant antibodies were included as additional controls. All nuclear extracts were treated with DNase/RNase prior to IP to exclude the possibility that any observed interaction between p68/p72 and p53 was merely via nucleic acid. As shown in Figure 3C and D, p68 co-immunoprecipitated with p53 from the U2OS extract, indicating that these proteins interact in the cell. In addition, the absence of any p68 in the p53 IP (Figure 3C) from the SAOS-2 extract confirmed that the DO-1 antibody was not immunoprecipitating p68 nonspecifically. As a further control, we performed IP/Westerns for p53/p68 using a different p53 antibody (CM1); this gave similar results (Figure 3E). To determine whether p72 also co-immunoprecipitates with p53, proteins in the p53 IP (Figure 3C) were Western blotted for p72 (Figure 3F). p72 and the alternative upstream translation initiation product p82 (Uhlmann-Schiffler et al, 2002) are both present in the p53 IP. The reciprocal IP/Western gave similar results (Figure 3G). However, in the light of the low p72/p53 in vitro interaction (Figure 3B), the presence of p72/p82 in the p53 IP may be, at least partially, due to the previously reported interaction of p72/82 with p68 (Ogilvie et al, 2003) rather than their specific interaction with p53. Figure 3.p68 and p72 interact with p53 in vitro and in vivo. (A) Expression of GST vector control and GST-tagged p68/p72 used in the GST pull-down experiments as shown by Western blotting of cell lysates with a GST-specific antibody. (B) GST 'pull-down' of in vitro-translated (35S-labelled) p53, showing both input and p53 species interacting with GST-tagged p68/p72. (C) Co-IP of p53 and p68 from nuclear extracts. p53 in U2OS extract was immunoprecipitated with the mouse monoclonal antibody (DO-1) and p68 and p53 in the IP were detected by Western blotting with rabbit polyclonal antibodies 2907 (p68) and CM1 (p53). (D) Reciprocal co-IP of p53 and p68. In this case, p68 was immunoprecipitated using the rabbit polyclonal antibody 2907 and immunoprecipitated p68 and p53 were detected by Western blotting with monoclonal antibodies PAb204 (p68) and DO-1 (p53). (E) Co-IP of p53 and p68 using a different p53-specific immunoprecipitating antibody. p53 in U2OS extract was immunoprecipitated with polyclonal antibody (CM1) and p68 and p53 were detected by Western blotting with monoclonal antibodies PAb204 (p68) and DO-1 (p53). (F) Co-IP of p53 and p72. Proteins immunoprecipitated by the p53 antibody (DO-1) (shown in (C)) were also Western blotted for p72 using the rabbit polyclonal antibody K14. (G) Reciprocal co-IP of p53 and p72. p72 was immunoprecipitated with the K14 antibody and p72 and p53 were detected by Western blotting with K14 and DO-1. Note that since only one p72 antibody is available, the same antibody had to be used for IP and Western blotting, giving a strong crossreaction with heavy chain (H). NE: nuclear extract; molecular weight markers (in kDa) are indicated. A nuclear extract from the p53-null cell line SAOS-2 and an irrelevant mouse or rabbit IgG (as appropriate; Cont. IP) were used as controls for IP. Download figure Download PowerPoint p68 is required for the p53 DNA damage response To determine whether p68 was required for p53 function in a physiological context, we 'knocked down' p68 expression by RNAi in MCF-7 cells (which express WT p53) and determined whether loss of p68 affected the p53 response to DNA damage. Using a p68-specific siRNA, we achieved an 80–90% knock-down of p68 protein expression (Figure 4A). Treatment with the DNA-damaging agent etoposide had no effect on p68 expression but, as expected, clearly induced p53 protein expression. Strikingly, however, while suppression of p68 expression had no effect on the induction of p53 protein levels, there was a significant reduction in the ability of p53 to stimulate expression of the p53 target genes p21 and mdm2 following etoposide treatment, as observed by Western blotting for the respective proteins (Figure 4A). These findings therefore indicate that p68 is required for the induction of p53 transcriptional activity in response to DNA damage. (Similar results were obtained with U2OS cells; data not shown.) In addition, the p68 RNAi knock-down specifically suppressed p68 expression, as there was no reduction in expression of the highly related RNA helicase, p72 (Lamm et al, 1996); we instead observed a minor increase in p72 expression (Figure 4A). To rule out the possibility that the effects seen by the p68 siRNA were due to the specific siRNA oligonucleotide chosen, we repeated the p68 knock-down with a second p68 siRNA directed against a different region of the p68 coding region (see Materials and methods). This gave results identical to those obtained with the first siRNA oligonucleotide (data not shown). Figure 4.RNAi depletion of p68, but not p72, inhibits expression of p53 target genes in response to DNA damage. Western blots showing expression of p68, p53, p21 and Mdm2 in MCF-7 cells, which had been transfected with (A) p68-specific or (B) p72-specific siRNA oligonucleotides. In both cases, a control siRNA was used and untransfected (UN) cells served as an additional control. In each case, the effect of treatment with the DNA-damaging agent etoposide (100 μM for 4 h) was examined. Equal amounts of protein (as determined by Bradford reagent (Sigma)) were loaded and detection of actin in the lysates was used as a loading control. Moreover, Western blots showing the levels of p68 and p72/82 in the reciprocal 'knock-downs' confirm the specificity of the siRNAs. (The antibodies used for Western blotting are described in Materials and methods; 2907, K14 and DO-1 were used to detect p68, p72 and p53, respectively.) Download figure Download PowerPoint p72 has been reported to also act as a coactivator of ERα (Watanabe et al, 2001) and to interact with p68 in the cell (Ogilvie et al, 2003). We therefore examined whether a knock-down of p72 similarly affected the p53 DNA damage response. Using a p72-specific siRNA, we achieved efficient suppression of expression of both p72 and the alternative upstream translation initiation product p82, which appears to have similar functions to p72 (Figure 4B) (Uhlmann-Schiffler et al, 2002). Strikingly, however, the p72 RNAi knock-down appeared to have no effect on the ability of p53 to induce p21 and Mdm2 expression upon etoposide treatment (Figure 4B), suggesting that the effect on the p53 DNA damage response is specific to p68. This finding is consistent with the results obtained from the cotransfection experiments, which showed that p72 is not such a potent coactivator of p53 transcription activity (see above and Figure 1B). As expected, the p72 knock-down had no significant effect on p68 expression (Figure 4B). In order to confirm that the lack of induction of p21 and Mdm2 expression was occurring at the mRNA level and to determine whether induction of other cellular p53 target genes in response to etoposide was similarly affected, the p68 RNAi experiment was repeated, RNA was extracted from cells and the levels of mRNA for a range of p53 target genes were determined by quantitative RT–PCR. These included p21 (cell cycle arrest) and mdm2 as well as the apoptosis-promoting genes Fas/APO1 and PIG3. GAPDH was used as a control and in each case the values obtained were normalised against β-actin to avoid discrepancies from differences in overall RNA levels between samples. As shown in Figure 5A and B, suppression of endogenous p68 expression resulted in a lack of induction of p21 and mdm2 mRNA in response to etoposide, consistent with a defect in the ability of p53 to induce transcription of the respective genes. Similar defects were observed in the induction of Fas/APO1 and PIG3 (Figure 5C and D), while there were no significant effects on the levels of GAPDH mRNA either as a result of etoposide treatment or knock-down of p68 (Figure 5E). These findings suggest that depletion of p68 results in a defect in the ability of p53 to induce expression of both cell cycle arrest and apoptosis-promoting genes in response to DNA damage, while it has no significant effect on genes that are not affected by p53, such as GAPDH. Figure 5.RNAi depletion of p68 inhibits expression of cell cycle arrest and apoptosis-promoting p53 target genes. Quantitative RT–PCR of mRNA extracted from MCF-7 cells, which had been transfected with p68-specific siRNA. In both cases, a control siRNA was used and untransfected (UN) cells served as an additional control. Etoposide treatment was performed as in Figure 4. RT–PCR reactions to measure (A) p21, (B) mdm2, (C) Fas/APO1, (D) PIG3 and (E) GAPDH mRNA levels, in each case relative to β-actin, are shown. The average values from three independent RT–PCR reactions are shown. Download figure Download PowerPoint RNAi depletion of p68 has no significant effect on induction of NF-κB transcriptional activity p68 has also been shown to coactivate ERα (Endoh et al, 1999; Watanabe et al, 2001) and to interact with CBP/p300 and RNA polymerase II (Rossow and Janknecht, 2003). Therefore, it was important to determine whether p68 was required for the induction of transcriptional activity of other transcription factors, such as NF-κB. For these experiments, we used a HeLa cell line (HeLa57A), which has an integrated copy of the NF-κB-inducible reporter 3Enhancer-κB-conA-Luc and of the control reporter pRC/RSV-β-galactosidase (Rodriguez et al, 1999). We knocked down p68 expression in these cells by RNAi and examined whether inhibition of p68 expression affected the induction of NF-κB in response to treatment with TNFα, as measured by luciferase activity. Cells transfected with a control siRNA and untreated cells served as controls. We confirmed that p68 was efficiently knocked down and that TNFα treatment had no effect on p68 levels (Supplementary data 2). Interestingly, cells in which p68 expression had been knocked down by RNAi showed no defect in the induction of NF-κB activity in response to TNFα (Figure 6A). In fact, we consistently observed a minor increase in the induction of luciferase activity in these cells compared with controls (Figure 6A). As expected, there was no significant difference in the relative activity of the control pRC/RSV-β-galactosidase reporter in the p68 knock-down and control cells in the presence or absence of TNFα treatment (Figure 6B). These findings thus indicate that p68 is not a general transcriptional coactivator but, instead, acts as a specific coactivator for certain inducible transcription factors, which include ERα (Endoh et al, 1999; Watanabe et al, 2001) and, as shown in the present study, p53. Figure 6.RNAi depletion of p68 does not affect NF-κB activation by TNFα. (A) Induction of NF-κB activity by TNFα in cells transfected with p68 or control siRNA, compared with untransfected cells, as determined by measuring relative luciferase activity. The luciferase activity of untransfected cells, which had not been treated with TNFα, was taken as 1. (B) β-Galactosidase activity in cells. The levels shown represent values relative to that of untransfected and untreated cells, which were taken as 1. Graphs represent the average values from two independent experiments, and reactions were, in each case, performed in duplicate. Download figure Download PowerPoint p68 is recruited to the p21 promoter In order to investigate the mechanism by which p68 stimulates p53
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