Oncogene (1998) 16, 1931 ± 1938  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Retinoblastoma protein associates with SP1 and activates the hamster dihydrofolate reductase promoter

Ve ronique Noe 1, Cristina Alemany1, Lawrence A Chasin2 and Carlos J Ciudad1

1Unit of Biochemistry, School of Pharmacy, University of Barcelona, 08028 Barcelona, Spain and 2Department of Biological Sciences, Columbia University, New York, USA

The dihydrofolate reductase (dhfr) promoter is power- site (minimum promoter). Deletion of this GC box fully activated by the transcription factor Sp1. It has practically abolishes transcription of the dhfr gene been suggested that Sp1 is a potential target for (Ciudad et al., 1992) despite the presence of the E2F transcriptional regulation by the cell cycle regulator binding element located just downstream of the major retinoblastoma protein (Rb), and so we have explored transcriptional start site. this possibility using the hamster dhfr gene as a model. On the one hand, Sp1 interacts with other proteins By the use of DNA probes from the hamster dhfr gene such as p107 (Datta et al., 1995), p53 (Borellini and promoter, containing the most proximal GC box Glazer, 1993) and E2F (Karlseder et al., 1996; Lin et (minimal promoter), and nuclear extracts from cultured al., 1996); the interaction with p107 represses Sp1- hamster cells (CHO K1), we show that polyclonal and dependent transcription whereas the interaction with monoclonal antibodies against Rb supershift the binding E2F leads to synergistic activation of dhfr transcription. of Sp1. Nuclear extract immunoprecipitation with anti- On the other hand, the tumor suppressor Rb is Rb followed by Western analysis using anti-Sp1 also involved in the control of transcription of several shows that Rb is complexed to Sp1. Complementary genes. It can repress positive transcription factors such Immunoprecipitation/WB analysis shows both forms of as E2F, either by sequestering this factor (Helin et al., Rb protein in the anti-Sp1 immunoprecipitates. More- 1992; Kaelin et al., 1992) or by turning E2F into an over, nuclear extract immunodepletion of Rb abolishes active repressor (Weintraub et al., 1992, Weintraub et Sp1 gel-shift. The interaction between Rb and Sp1 is al., 1995); but Rb can also stimulate the IGF-II (Kim et maintained in all the phases of the cell cycle. Transient al., 1992a), c-fos,c-myc,TGF-b1 (Kim et al., 1991; overexpression of Rb in dhfr negative cells co-transfected Udvadia et al., 1993), ATF-2, TGF-b2 (Kim et al., with a dhfr minigene driven by its minimal promoter 1992b), c-jun (Chen et al., 1994), cyclin D1 (MuÈ ller et increases DHFR activity and potentiates transcription al., 1994), IL-6 (Santhanam et al., 1991), neu (Yu et al., when overexpressing Sp1. Both e€ects are severely 1992), TK and DHFR (Udvadia et al., 1993) promoters reduced when the co-transfections are performed with a through stimulation of Sp1-mediated transcription. homologous dhfr minigene containing a single point However, although there appears to be a functional mutation in the GC box. Thus, the activation by Rb of interaction between Rb and Sp1, their physical the dhfr gene may be exerted through Sp1. Stable interaction has not been demonstrated. Here, we show transfectants of pCMVRb in K1 cells show an increase that Rb associates with Sp1 in solution, and that both in both mRNA and DHFR activity. It is concluded that proteins are present together, throughout the cell cycle, Sp1 is physically associated with Rb, and that this in the transcriptional complex that binds to the hamster association increases Sp1-mediated transcription of the dhfr promoter. Transient overexpression of Rb leads to hamster dhfr gene. an increase in DHFR expression in a homologous system, which is mediated through the Sp1 binding site. Keywords: DHFR; Sp1; Rb The e€ect of Rb on DHFR expression is also observed in permanently transfected cells.

Introduction Results It has been suggested that Sp1-mediated transcription Interaction of Rb and Sp1 in binding to the hamster dhfr is stimulated by Rb (Kim et al., 1992a; Udvadia et al., minimal promoter 1993; Chen et al., 1994). Here we examine a possible association of Rb with Sp1 in the control of the First, to test whether there was an interaction between expression of the dhfr gene, which is needed for the Sp1 and Rb, we performed supershift experiments synthesis of purines, thymidylate and glycine required using a probe from the hamster dhfr promoter, nuclear for the replication of DNA in S phase of the cell cycle. extracts from CHO K1 cells, and antibodies against The dhfr gene promoter contains cis ± acting elements Rb. As DNA probes we used the two fragments 410f for the transcription factor Sp1, and only the most and sp1f described in the Materials and methods proximal GC box, at least in hamster cells, is needed to section. Gel shift with 410f probe gave rise to three correctly initiate transcription at the major proximal retarded bands. The upper and lower bands corre- sponded to Sp3 and the central one to Sp1, as Correspondence: CJ Ciudad identi®ed by speci®c antibodies against Sp1 (PEP 2) Received 23 January 1997; revised 11 November 1997; accepted 11 and Sp3 (D20) (Figure 1b). The binding of these two November 1997 Sp proteins was abolished (Figure 1c) when using the Association of Rb with Sp1 VNoeÂet al 1932 gc probe containing a single point mutation (G to C) in Western blotting. Nuclear extracts from K1 cells were the ®fth position of the Sp hexanucleotide core binding incubated with anti-Rb N9 antibody, or non immune sequence in the proximal GC box. Polyclonal antibody serum in the primary immunoprecipitation; then the N9 (against aminoacids 330-610) (Shirodkar et al., washed immunoprecipitates were subjected to Western 1992), and monoclonal antibody XZ77 (against blot and detection of Sp1 using the speci®c antibody aminoacids 444-535 and 620-665) (Hu et al., 1991), PEP 2. It can be observed in Figure 2a that Sp1 co- produced a supershift of the three 410f shifted bands immunoprecipitates with Rb (lanes N9), while no (Figure 1a). N9 antibody also supershifted the binding signal was detected when the immunoprecipitation of Sp1 observed with probe sp1f (Figure 1d). A non- was performed with nonimmune serum (lane NIS). immune serum (NIS) did not produce the supershift PEP 2 antibody speci®c for Sp1 recognized hamster with either probe (Figure 1a and d). As a negative Sp1 protein (Figure 2b). N9 antibody against Rb does control, the antibodies were incubated in the same not cross-react with Sp1 protein (Figure 2c). We also experimental conditions with a radiolabeled Oct-1 performed the complementary immunoprecipitation probe and nuclear extracts from K1 cells. In no case using anti-Sp1 antibody in the primary immunopreci- was a supershift observed with this probe (Figure 1e). pitation, followed by detection of Rb in the Western We also tested the lack of reactivity of antibody N9 blot, using C-15 antibody. Figure 2d shows that the with a non-related binding, that produced by the speci®c anti-Sp1 antibody PEP2 co-immunoprecipitates interaction of PPAR/RXR with the PPRE from the Rb in the hypo- and hyperphosphorylated forms. mitochondrial HMG-CoA synthase promoter. The These results demonstrate that the two proteins, Sp1 addition of N9 antibody did not alter the mobility of and Rb, form part of the same complex and that the the shifted band (Figure 1f). Sp1-Rb complex is formed in the absence of DNA.

Co-immunoprecipitation of Rb and Sp1 E€ect of immunodepletion of Rb or Sp1 on nuclear protein binding to the hamster dhfr minimal promoter. To con®rm the presence of Sp1 and Rb in the same complex, we analysed the immunoprecipitate obtained As a further test of the presence of Sp1-Rb complexes, with anti-Rb antibody for the presence of Sp1 using we measured the binding of Sp1 to the dhfr promoter

a bc –– PEP2 Ab D20 Ab – NIS N9 XZ77 NIS NE –+ ++ NE – +

Sp1 Sp3

GS SS

410f probe gc probe K1 NE 410f probe K1 NE

d e f Ab ––N9 NIS Ab – NIS N9 XZ77 Ab – N9 NE –+++ SS GS SS GS GS GS

Sp1f probe Oct-1 probe K1 NE PPRE probe K1 NE purified PPAR/RXR

Figure 1 Interaction of Rb with Sp1 in the binding to the dhfr promoter. (a) Gel retardation analysis using 410f probe and Rb- antibodies or NIS. One ml of a non-immune serum (NIS), 4 ml of a polyclonal anti-Rb antibody (N9) or 10 ml of a monoclonal antibody (XZ77) against Rb was added to the binding mixture containing 2 mg of nuclear extract from K1 exponentially growing cells before (XZ77) or after (NIS and N9) the addition of radiolabeled 410f probe. The reaction was incubated for a further 15 min before electrophoresis. (b) Identi®cation of Sp proteins binding to the 410f probe. One ml of speci®c antibodies (Ab) against Spl (PEP 2) or Sp3 (D20) were added to the binding reaction. (c) Gel shift performed with probe gc containing a single point mutation in the Sp binding site of the most proximal GC box in the minimal promoter of the hamster dhfr gene. (d) Same as A, but using labeled sp1f probe and N9 antibody or NIS. (e) Gel retardation using the Oct-1 probe and 1 ml of NIS, 4 mlofN9or10ml of XZ77 antibodies with K1 cell nuclear extract. (f) Gel retardation using the PPRE probe and 4 ml of N9 with puri®ed proteins PPAR/RXR. All the mobility retardations, gel shift (GS) and supershift (SS), are indicated by arrows. Each probe was used at 20 000 c.p.m./assay. Poly [d(I-C)] (2 mg) was used as non speci®c competitor Association of Rb with Sp1 VNoeÂet al 1933 aba PEP2 PEP2 Ext NIS PEP2 N9 N9 410f GS Sp1 0 NIS 10 12 15 20 30 100 100 100 10 100 RB-Ab (ng) in IP (ng of Ab used in the IP) K1NE rSp1 b cd N9 N9 Ext NIS PEP2 sp1f GS Rb-p Rb Rb 100 100 rSp1 K1NE 0 NIS 10 12 15 20 30 100 (ng of Ab used in the IP) RB-Ab (ng) in IP Figure 2 Sp1 coimmunoprecipitates with Rb. (a) Coimmunopre- cipitation using Rb-antibody. Immunoprecipitates were obtained c N9 from 30 mg of K1 cell nuclear extract by incubation with the NIS indicated amounts of NIS, PEP 2 or N9 antibodies. After washing, the bound proteins were resolubilized and Sp1 protein oct-1 GS was then detected by Western analysis with PEP 2 antibody. The ®rst lane (Ext) shows the signal corresponding to Sp1 obtained directly from 20 mg of K1 nuclear extract. (b) Detection of 100 10 100 hamster Sp1 by Western blot using 5 mg of K1 nuclear extract Ab (ng) in IP (lane KlNE) or 50 ng of recombinant Sp1 protein (lane rSp1) and PEP2 antibody against Sp1. (c) Detection of hamster Rb using Figure 3 E€ect of immunoprecipitating Rb or Sp1 on nuclear 15 mg of K1 nuclear extract and N9 antibody against Rb. The protein binding to dhfr promoter sequences. Thirty micrograms of absence of cross-reactivity of N9 antibody was tested with nuclear extract from K1 exponentially growing cells were recombinant Sp1 protein (lane rSp1). (d) Coimmunoprecipitation immunoprecipitated with the indicated amounts of anti-Rb using Sp1-antibody. Twenty mg of K1 cell nuclear extract was antibody N9. After removal of the immune complexes with the immunoprecipitated with anti-Sp1 antibody PEP2 or NIS. The aid of insoluble Protein A, the supernatants were used in gel-shift washed immunoprecipitates were subjected to Western analysis analyses with probes 410f (a), sp1f (b) or Oct-1 (c). NIS, a gel using anti-Rb antibody C-15. The ®rst lane (Ext) shows the signal shift performed after immunoprecipitation under the same corresponding to Rb obtained directly from 10 mg of K1 nuclear conditions with 100 ng of nonimmune serum extract

after immunoprecipitation with Rb antibody. Nuclear –+–+–+–+–+–+ extracts from exponentially growing cells were exposed RB Ab to various concentrations of N9 antibody, followed by incubation with insoluble protein A. After centrifuga- SS tion, the supernatants were used for gel shift analysis GS of either 410f or sp1f radiolabeled probes. Immuno- precipitation with Rb antibody eliminated the Sp1 shift (Figure 3a and b). As a control we performed gel shift GO G1 G1/S S G2/M Exp analysis with Oct-1 probe and supernatants from the 0 9 11 15 19 immunoprecipitation with antibodies against Rb. The binding to Oct-1 probe was unchanged in these Hours after serum stimulation conditions, con®rming the speci®city of the immuno- Figure 4 Interaction of Rb with Sp1 throughout the cell cycle. depletion (Figure 3c). Two mg of nuclear extract from synchronized populations of K1 cells in each of the phases of the cell cycle were subjected to supershift analysis using radiolabeled 410f probe and Rb- Interaction of Rb and Sp1 throughout the cell cycle antibody. Four ml of anti-Rb antibody (N9) were added to the gel shift binding mixture and the reaction was incubated for a At this point, we were interested in knowing whether further 15 min period before electrophoresis. All the mobility the observed interaction between Rb and Sp1 was retardations, gel shift (GS) and supershift (SS), are indicated by arrows. Each probe was used at 20 000 c.p.m./assay. Poly [d(I-C)] restricted to a particular phase of the cell cycle. To (2 mg) was used as non speci®c competitor. Exp, exponentially check this idea, and considering that Rb switches growing cells. between hypo- and hyperphosphorylated forms during the cell cycle, we performed supershift analyses using nuclear extracts prepared from populations of Co-transfection of dhfr minigenes plus expression vectors synchronized CHO K1 cells in each phase of the for Sp1 and Rb cell cycle. These nuclear extracts were incubated with radiolabeled 410f as the probe in the absence or the We then examined the e€ect of co-transfecting Sp1 presence of anti-Rb antibody N9. We have checked and/or Rb, driven by eukaryotic expression vectors, on that this antibody is able to recognize the di€erent DHFR expression using an homologous system. We states of phosphorylation of Rb. As shown in Figure transfected plasmid p410 (hamster dhfr minigene driven 4, the anti-Rb antibody is able to produce a by its minimal promoter) into hamster DG44 cells supershift of Sp1 in all the phases of the cell cycle. (deletion mutant of the dhfr locus ) and the resulting Therefore, Rb and Sp1 interact throughout the cell DHFR activity was determined by incorporation of cycle. tritiated deoxyuridine into DNA. This assay provides a Association of Rb with Sp1 VNoeÂet al 1934 rapid and convenient means of testing DHFR colonies obtained from transfection with the empty expression when DHFR activity is the limiting factor vector are also shown (K1 CMV). in the synthesis of DNA (Ciudad et al., 1988). In the experiments reported here, DHFR activity was limiting since 1 nM and 5 nM methotrexate decreased by 30% Discussion and 100%, respectively, the DHFR activity provided by the expression of plasmid p410 in DHFR-de®cient The dhfr minimal promoter contains regulatory recipient cells. Then, we proceeded to co-transfect, elements for the binding of the transcription factors along with p410, increasing amounts of either Sp1 and E2F. The role of E2F in cell growth regulated pCMVSpl or pCMVRb. Overexpression of either transcription of the dhfr promoter has been emphasized protein resulted in an increased DHFR transient by the groups of Azizkhan (Blake and Azizkhan, 1989; activity (Figure 5a and b). The combined overexpres- Wade et al., 1992) and Farnham (Means et al., 1992; sion of Sp1 and Rb together with p410 had a Slansky et al., 1993; Li et al., 1994). On the other hand, synergistic e€ect on DHFR transient activity (Figure Sp1 powerfully activates DHFR transcription (Farn- 5c). However, overexpression of Sp1 plus Rb using ham and Shimke, 1986; Swick et al., 1989; Blake et al., pGC as DHFR minigene failed to increase DHFR 1990; Ciudad et al., 1992), and given this essential role transient activity (Figure 5c) indicating that the e€ect of Sp1 in activating transcription, we have explored the exerted by Rb is mediated through Sp1. possibility of the interaction of Sp1 with the cell cycle regulator Rb. The main conclusion of this study is that the Levels of Rb protein, dhfr mRNA and DHFR activity in retinoblastoma gene product Rb forms a complex CHO cells stably transfected with Rb with Sp1, as indicated by the supershift, co-immuno- Finally, we examined whether the e€ect of Rb on precipitation, and immunodepletion experiments. DHFR activity observed in co-transfection experiments Using CHO nuclear extracts and radiolabeled 410f as is reproduced in cells stably transfected with Rb. First, a probe, both anti-Rb antibodies (polyclonal N9 or a pool of permanent transformants (K1-RbO3) over- monoclonal XZ77) supershift this probe. Polyclonal expressing Rb was developed by transfecting CHO K1 antibody N9 against Rb also supershifts the sp1f cells with pCMVRb (0.3 mg) and further selection with probe. Sp1 protein is present in the immunoprecipitate G418. This pool showed a threefold level of over- obtained with N9 anti-Rb antibody, and both hypo- expression of Rb as determined by Western analysis and hyperphosphorylated forms of Rb protein are (Figure 6a). Then, DHFR mRNA and activity were present in the immunoprecipitate obtained with PEP2 determined. The levels of DHFR mRNA showed a anti-Sp1 antibody. Rb-immunodepletion of the nuclear threefold increase (Figure 6b) as determined by RT- extracts abolishes the formation of Sp1-gel shifts. The PCR, and DHFR activity doubled (Figure 6c) as association between Sp1 and Rb is speci®c since non- measured by the incorporation of tritiated deoxyur- immune serum does not produce either supershift, co- idine. The results attained with a similar pool of immunoprecipitation, or immunodepletion of Sp1. As a further control, the Rb-antibodies used do not alter the shift mobility produced by Sp1-unrelated probes such as Oct-1 or PPRE. Moreover, the physical interaction between Rb and Sp1 is maintained throughout the cell cycle, regardless the phosphoryla- tion state of Rb. Our results extend those obtained by Datta et al., 1995, since they only found an association between Sp1 and p107. However, they did not rule out the possibility of interaction of Sp1 with Rb, p130 or p300. The antibodies or the protein fractions they used detected only interactions with p107. A second conclusion is that the association of Rb with Sp1 leads to activation of dhfr gene transcription as seen in transient and stable transfections. In co- transfection experiments using wild type or a mutated version of dhfr minigenes containing the minimal promoter for this gene, we demonstrate that over- expression of either Rb or Sp1 activates the dhfr Figure 5 DHFR transient activity in co-transfections of p410 promoter through the Sp1 binding site. Overexpressing with pCMVSpl and/or pCMVRb. (a) Dose response of co- transfection of p410 with pCMVSp1. DG44 cells (250 000/35 mm both Rb and Sp1 has a synergistic e€ect on dish) were co-transfected with 1 mg of p410 and the indicated transcription. These results agree with those obtained amounts of pCMVSp1. After 24 h expression, 2mCi of 6-[3H]- by Udvadia et al., 1993. These authors used a deoxyuridine was added for an additional 24 h. The incorporation heterologous system cotransfecting, into Drosophila of radioactivity to DNA was measured in a scintillation counter. The control value corresponds to DG44 cells tranfected with p410 cells, a hamster dhfr promoter driving the CAT gene. only. (b) Dose response of co-transfection of p410 with pCMVRb. In contrast, we used an homologous system transfect- The experiment is the same as in a, but using the indicated ing (into dhfr de®cient hamster cells) a hamster dhfr amounts of pCMVRb in the co-transfection. (c) Co-transfections minigene, containing intron-1 which is needed for of p410 or pGC with pCMVSp1 and/or pCMVRb. DG44 cells correct expression, and determining DHFR dependent were co-transfected with 1 mg of p410 or 1 mg pGC, plus 2 mgof pCMVSp1 and/or 1 mg of pCMVRb. Results are presented as the activity. Also, stable overexpression of Rb results in an mean+s.e. (4 exp) increase in both DHFR mRNA and activity. Association of Rb with Sp1 VNoeÂet al 1935 ab aprt Rb dhfr K1 K1 K1 K1 K1 K1 CMV Rb03 CMV Rb03

c

Figure 6 Analysis of stable transfectants of pCMVRb. K1 cells were transfected by the calcium phosphate method with 0.3 mgof pCMVRb (K1-RbO3) or the empty vector (KlCMV) and selected with Geneticin for 3 weeks. The surviving colonies were pooled and used in the following determinations: (a) Detection of Rb protein levels. Nuclear extracts (10 mg) from K1 cells, and pools from permanent transfectants KlCMV and K1-RbO3 were used for Rb detection as described in Materials and methods. The top panel shows a representative autoradiography of the Western blot and the bottom panel displays the results of the densitometry. (b) DHFR mRNA levels. Total RNA from K1, KlCMV and K1-RbO3 transfectants was used in the RT-PCR reaction using the combination of primers for dhfr and aprt described in Materials and methods. The signals corresponding to the ampli®ed products are shown in the top panel of the Figure, and the dhfr mRNA level is expressed as the ratio of intensities between the dhfr and aprt signals in the bottom panel. (c) DHFR activity. DHFR activity was measured in K1, K1CMV and K1-RbO3 transfectants by the incorporation of [3H]-deoxyuridine to DNA, as described in Materials and methods. Results are presented as the mean+s.e. (3 exp)

It has been reported that recombinant Rb protein when fused to the DNA binding domain of GAL4 enhances Sp1-binding activity (Chen et al., 1994) and (Adnane et al., 1995). According with this result pRb that the IGF-II (Kim et al., 1992a), c-fos,c-myc can decrease transcription of the TGF-b1 promoter in (Udvadia et al., 1993) and c-jun (Chen et al., 1994) NIH3T3 and AKR-2B mouse cells (Kim et al., 1991); promoters can be regulated by Rb through stimulation also it can turn E2F into an active repressor of Sp1-mediated transcription. Interestingly, cotrans- (Weintraub et al., 1995). However, pRb has also been fection of Rb with Sp1 in Drosophila Schneider SL2 described to stimulate transcription from the IGF-II cells resulted in a marked increase in dhfr transcription, (Kim et al., 1992a), c-fos,c-myc, ATF-2, TGF-b2 (Kim irrespective of the presence of E2F sites within the dhfr et al., 1992b), c-jun (Chen et al., 1994), cyclin D1 promoter (Udvadia et al., 1993). Another tumor (MuÈ ller et al., 1994), IL-6 (Santhanam et al., 1991), neu suppressor, p53, has also been shown to increase in (Yu et al., 1992), TK and DHFR (Udvadia et al., concentration in response to the growth factor GM- 1993) promoters. In addition pRb can increase CSF and to form heterocomplexes with Sp1 that transcription of TGF-bl (Kim et al., 1991; Udvadia stimulate the DNA-binding activity of the latter in et al., 1993) in CCL-64 mink lung epithelial cells and proliferating TF-1 cells (Borellini and Glazer, 1993). It A-549 human lung adenocarcinoma. Therefore, pRb has been proposed (Chen et al., 1994; Shew et al., can both stimulate and repress transcription on 1990) that the mechanism of the increase in the Sp1- di€erent sets of genes, and this may depend on the mediated transcription by Rb is through the capture by recipient cell used in the co-transfection. Recently, Rb of an inhibitor of Sp1. This proposal was based on Strauss and co-workers have reported, using the the lack of evidence of an association between Rb and DDRT-PCR technique, that 16 genes are stimulated Sp1 and on the identi®cation of two proteins of by introducing a wild type Rb cDNA into Rb-de®cient 20 KDa (Chen et al., 1994; Shew et al., 1990) and human mammary carcinoma cells, and two in a mouse 74 KDa (Murata et al., 1994) with Sp1-inhibitor system aside from the 15 that are repressed (Rhode et characteristics. Our evidence of a direct association al., 1996). This behaviour may be inherent to the between Rb and Sp1, as shown by the supershift and characteristic of Rb as a key regulator of the cell cycle co-immunoprecipitation experiments, does not preclude in the G1 phase. such an inhibitor-mediated mechanism. To date, the role of Rb in regulating the dhfr gene Two reports describe a novel interaction between has been assumed to be the sequestering of E2F in Sp1 and members of the E2F1 family, including the arrested cells, thus preventing activation of E2F- superactivation of Sp1-mediated transcription by E2F responsive promoters. In this model, stimulation of (Karlseder et al., 1996; Lin et al., 1996). Therefore, Sp1 cell proliferation results in phosphorylation of Rb is bound by both E2F and Rb proteins, each of which (Buchkovich et al., 1989; DeCaprio et al., 1989) and can increase Sp1-mediated transcription. This is in E2F (Fagan et al., 1994) with the concomitant release keeping with the picture evolving that regulation of of free E2F, which then directly stimulates the transcription results from interactions that produce transcription of genes containing E2F-binding sites. di€erent combinations of transcription factors. On the other hand, we now show a physical interaction It has to be stressed that indeed there is good between Rb and Sp1 which is maintained throughout evidence that Rb can cause a repression of transcrip- the cell cycle, regardless the phosphorylation state of tion when it is tethered to a promoter, for instance Rb. Therefore, Rb can have di€erent behaviours in its Association of Rb with Sp1 VNoeÂet al 1936 interaction with either E2F or Sp1. This does not Nuclear extracts exclude the possibility that the phosphorylation state of Nuclear extracts were prepared as described (Ciudad et al., Rb may be important for Sp1-mediated transcription. 1992) from exponentially growing CHO K1 cells, or cells At present, what seems very clear is that the level of throughout the cell cycle. Protein concentrations were expression of Rb a€ects positively Sp1 transcriptional determined using the Bio-Rad protein assay based on the activity. Given the reported activation of Sp1 by E2F, method of Bradford M, 1976, using BSA as standard

and our present observation that Rb associates and (Sigma), and extracts were frozen in liquid N2 and stored at activates Sp1, Rb protein may act at a higher level of 7808C. regulation controlling in a dual fashion genes contain- ing cis-acting elements for E2F and Sp1, such as dhfr. Gel shift and supershift analysis Gel shift experiments were carried out in 2O-ml reaction mixtures containing 2 mg of poly [d(I-C)] (poly [d(I-C)]-ds,

Pharmacia), 5% glycerol, 1 mM MgC12,60mMKCl, 2 mg Materials and methods of nuclear extract protein, 0.5-1 ng (20 000 c.p.m.) of DNA probe and 25 mM Tris-HCI, pH 8. The mixture was incubated on ice for 15 min in the absence of the Cell culture labeled probe and then for a further 30 min in its Conditions for the monolayer culture of CHO cells were as presence. Immediately afterwards, the samples were described previously (Urlaub et al., 1985). CHO K1 and subjected to polyacrylamide gel electrophoresis (5% CHO DG44 cells were grown in Ham's F12 medium polyacrylamide: bis 30 : 1, 5% glycerol in 0.56TBE supplemented with 7% fetal calf serum (FCS, above two (16TBE is 90 mM Tris borate, pH 8, 2 mM EDTA) for from GIBCO) and maintained at 378C in a humidi®ed 5% 3 ± 4 h at a maximum of 20 mA. The gel was dried and the

CO2-containing atmosphere. For synchronization experi- radioactive bands were visualized by autoradiography. In ments, 36105 cells were seeded in 100 mm-diameter dishes supershift experiments, N9 rabbit polyclonal serum raised in F12 medium with 0.5% fetal calf serum for 7 days. against a Trp-Rb fusion protein, XZ77 mouse monoclonal Following serum starvation, cells were refed with medium antibody raised against bacterially expressed Rb, PEP 2 containing 7% FCS for periods of time which corre- rabbit polyclonal antibody against Sp1 non cross-reactive sponded to the di€erent phases of the cell cycle (Noe et al., with Sp2, Sp3 or Sp4, and D20 polyclonal antibody 1997). against Sp3, were added to the above-noted reaction mixture before (XZ77) or after (N9, PEP 2, D20) the addition of the radiolabeled probe. The antibodies were Flow cytometry analysis generously provided by JA DeCaprio (N9), and E Harlow The progression of cells through starvation and the cell and N Dyson (XZ77), or purchased from Santa Cruz cycle after serum stimulation was monitored by ¯ow Biotechnology (PEP 2, D20). cytometry. For this analysis, nuclei were stained with 25 m/ml of propidium iodide (Sigma Quõ mica, Madrid) and Co-immunoprecipitations analysed on a Becton Dickinson ¯ow cytometer. Synchro- nized CHO cells, upon addition of 7% FCS reenter the cell Nuclear extracts from K1 cells were immunoprecipitated cycle, reaching the G1/S border in 11 h, and G2/M after using either N9 anti-Rb antibody, PEP 2 anti-Sp1 antibody 19 h (Noe et al., 1997). Therefore, populations of cells were or nonimmune serum (NIS) with the aid of Protein A- harvested at 0 h (Go), 9 h (G1), 11 h (G1/S), 15 h (S) and Sepharose as above. Then, the pellets were washed twice in 19 h (G2/M), to prepare nuclear extracts. 500 ml of PBS containing 0.1% NP40 plus 0.3 M NaCl, resolubilized in loading bu€er, boiled for 5 min, and subjected to Western analysis as described. DNA probes DNA probe 410f was prepared by PCR ampli®cation from Immunodepletions plasmid DNA p410. This is a BAL-31 deletion mutant minigene that includes just 48 bp upstream of the major Immunoprecipitations were performed on ice for 1 h using transcriptional start site of the hamster dhfr promoter 30 mg of nuclear extracts from exponentially growing CHO (Ciudad et al., 1988). The oligonucleotides used in the PCR K1 cells, and amounts ranging from 10 ± 100 ng, of anti-Rb reaction hybridized with sequences ¯anking the Sp1 antibody N9, in 0.1% NP40 (Sigma Quõ mica). The binding site and the translational start, producing a immunecomplexes were removed by centrifugation with fragment of 150 bp. The forward primer was 5'- 3 ml of insoluble Protein A (Sigma Quõ mica) and the GTTCTAGTCAGCCAGGCAAG-3', and the reverse pri- supernatants were used in gel shift assays with either 410f mer 5'-GTTCAGCGGTCGAACCAT-3'. The ampli®ed and sp1f radiolabeled probes. DNA, 410f, was digested with HpaII (at 774 relative to the translational start), and the 59-bp fragment (splf), Western blot analysis which contains the recognition sequence for Sp1, was gel- puri®ed. Probe gc was generated in the same manner that Di€erent amounts of nuclear extract (stated in the legends) 410f but from plasmid pGC (Ciudad et al., 1992). A ds from CHO K1 cells, 50 ng of recombinant puri®ed Sp1 DNA fragment containing the Oct-1 consensus sequence (Promega), or the washed pellets from the immunoprecipi- (5'-TGTCGAATGCAAATCACTAGAA-3',Promega)was tations, were resolved on SDS-7% polyacrylamide gels used as a control for the shift and supershift experiments. (Laemmli, 1970), and transferred to a PVDF membrane PPAR and RXR (peroxisomal proliferator activated (Immobilon P, Millipore) using a semidry electroblotter. receptor/retinoic acid receptor) were synthesized in vitro The membranes were probed with either anti-Rb antibodies andusedasheterodimerstobindtoHMG-CoAsynthase (N9, 1 : 1500 dilution; or C-15 from Santa Cruz Biotech- PPRE (peroxisomal proliferator response element) in nology, 1 : 100 dilution), or anti-Spl antibody (PEP 2, supershift experiments to test the cross-reactivity of Rb- 1 : 1500 dilution). Signals were detected by secondary antibody N9. The probes were end-labeled with T4 horseradish peroxidase-conjugated antibody (1 : 12000 polynucleotide kinase (BRL) using g-[32P]-ATP (3 000 Ci/ dilution) and enhanced chemiluminiscence, as recom- mmol, Amersham). mended by the manufacturer (Boehringer Mannheim). Association of Rb with Sp1 VNoeÂet al 1937 Transfections, co-transfections and DHFR transient activity for 60 min. Five ml of the cDNA mixture was used directly assay for PCR ampli®cation. PCR reactions were typically carried out as follows. A standard 50 ml mixture contained Stables transfections of CHO K1 cells with eukaryotic 5 ml of the cDNA mixture, 4 mlof106PCR bu€er (Mg2+- expression vector for Rb (pCMVRb, Qin et al., 1992), or free), 1.5 mM MgCl ,0.2mM dNTPs, 2.5 mCi of a-[32P]- the empty vector, were performed by the calcium 2 dATP (3000 Ci/mmol, Amersham), 1 unit of Taq phosphate method (Urlaub et al., 1989). After 24 h polymerase (BRL) and 500 ng of each of four primers. expression, cells were selected with Geneticin (800 mg/ml) The primers used were: 5'-CGCCAAACTTGGGGGAAG- for 3 weeks. The surviving colonies were pooled and used CA-3' and 5'-GAACCAGGTTTTCCGGCCCA-3' for dhfr, for Western, mRNA and DHFR enzymatic analyses. and 5'-ATCCGCAGTTTCCCCCGACTT-3' and 5'-TCA- Co-transfections experiments were carried out in dhfr CACACTCCACCACCTCA-3' for aprt,whichwasusedas de®cient cells (CHO-DG44) by the Polybrene method an internal control. (Chaney et al., 1986) as described in Ciudad et al., 1988. The reaction mixture was separated in two phases by a Plasmid p410 (dhfr minigene driven by its minimal solid paran wax layer (melting T=59 ± 608C, Fluka), which promoter), or plasmid pGC (single point mutant from prevents complete mixing of PCR reactants until the reaction p410 in the Sp1 recognition site, Ciudad et al., 1992), plus has reached a temperature at which nonspeci®c annealing of eukaryotic expression vectors for Rb (pCMVRb), Sp1 primers to non-target DNA is minimized. The lower solution (pCMVSp1), or both, were co-transfected. After 24 h contained the MgCl , the dNTPs, the four primers, the a- expression, the resulting DHFR activity was determined by 2 [32P]-dATP and half the bu€er, and the upper solution incorporation of radiactive deoxyuridine to cellular DNA, as contained the cDNA, the Taq enzyme and the remaining described in Ciudad et al., 1988 with the following bu€er. PCR was performed for 25 cycles, after a 1 min modi®cation: after 24 h labeling with 2 mCi of 6-[3H]- denaturation at 948C; each cycle consisted of denaturation at deoxyuridine, cells were rinsed twice in phosphate-bu€ered 928C for 1 min, primer annealing at 598C for 75 s, and saline and lysed in 100 ml of 0.1% SDS. The lysate was primer extension at 728C for 110 s. Five microliters of each collected on Whatman 31ET papers (2 cm62 cm), which PCR sample was electrophoresed in a 5% polyacrylamide gel. were immediately placed onto ice-cold 66% ethanol The gel was dried and the radioactive bands were visualized containing 250 mM NaCl, in order to precipitate DNA. by autoradiography. After two washes in the same solution, the papers were dried and counted. pCMVSpl was kindly provided by Dr R Tjian, and pCMVRb by Dr W Kaelin. Acknowledgements We thank Mr Robin Rycroft from the Language Advisory mRNA analysis by RT-PCR Service (SAL) for correcting the English manuscript, and Total mRNA was extracted from either CHO K1 cells, K1 Dr Diego Haro for providing the PPRE probe from the rat cells stably transfected with the expression vector for Rb mitochondrial HMG-CoA synthase, and the in vitro (pCMVRb), or the empty vector using the UltraspecTM translated PPAR/RXR heterodimers. This work was RNA reagent (Biotecx) in accordance with the manufac- supported by grants SAF94-177 and SAF96-74 from turer's instructions. cDNA was synthesized in a 20 ml CICYT, and SGR96-84 from ClRlT to CJC and NIH reaction mixture containing 1 mg of RNA (in DEPC grant GM-22529 to LAC. treated water), 125 ng of random hexamers (Promega), CJC was a recipient of a fellowship from CIRIT of 10 mM dithiothreitol, 20 units of RNasin (Promega), Catalonia. VN was a recipient of a predoctoral fellowship 0.5 mM dNTPs (Sigma Quõ mica), 4 mlof56RT bu€er from the Spanish Ministry of Education, and CA is a and 200 units of MLV reverse transcriptase (above two recipient of a predoctoral fellowship from the Catalonian from BRL). The reaction mixture was incubated at 378C Department for Scienti®c Research.

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