Retinoblastoma protein associates with SP1 and activates the ... - Nature

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 VeÂronique NoeÂ1, Cristina Alemany1, Lawrence A Chasin2 and Carlos J Ciudad1 1

Unit 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 powerfully activated by the transcription factor Sp1. It has been suggested that Sp1 is a potential target for transcriptional regulation by the cell cycle regulator retinoblastoma protein (Rb), and so we have explored this possibility using the hamster dhfr gene as a model. By the use of DNA probes from the hamster dhfr gene promoter, containing the most proximal GC box (minimal promoter), and nuclear extracts from cultured hamster cells (CHO K1), we show that polyclonal and monoclonal antibodies against Rb supershift the binding of Sp1. Nuclear extract immunoprecipitation with antiRb followed by Western analysis using anti-Sp1 also shows that Rb is complexed to Sp1. Complementary Immunoprecipitation/WB analysis shows both forms of Rb protein in the anti-Sp1 immunoprecipitates. Moreover, nuclear extract immunodepletion of Rb abolishes Sp1 gel-shift. The interaction between Rb and Sp1 is maintained in all the phases of the cell cycle. Transient overexpression of Rb in dhfr negative cells co-transfected with a dhfr minigene driven by its minimal promoter increases DHFR activity and potentiates transcription when overexpressing Sp1. Both e€ects are severely reduced when the co-transfections are performed with a homologous dhfr minigene containing a single point mutation in the GC box. Thus, the activation by Rb of the dhfr gene may be exerted through Sp1. Stable transfectants of pCMVRb in K1 cells show an increase in both mRNA and DHFR activity. It is concluded that Sp1 is physically associated with Rb, and that this association increases Sp1-mediated transcription of the hamster dhfr gene. Keywords: DHFR; Sp1; Rb

Introduction It has been suggested that Sp1-mediated transcription is stimulated by Rb (Kim et al., 1992a; Udvadia et al., 1993; Chen et al., 1994). Here we examine a possible association of Rb with Sp1 in the control of the expression of the dhfr gene, which is needed for the synthesis of purines, thymidylate and glycine required for the replication of DNA in S phase of the cell cycle. The dhfr gene promoter contains cis ± acting elements for the transcription factor Sp1, and only the most proximal GC box, at least in hamster cells, is needed to correctly initiate transcription at the major proximal Correspondence: CJ Ciudad Received 23 January 1997; revised 11 November 1997; accepted 11 November 1997

site (minimum promoter). Deletion of this GC box practically abolishes transcription of the dhfr gene (Ciudad et al., 1992) despite the presence of the E2F binding element located just downstream of the major transcriptional start site. On the one hand, Sp1 interacts with other proteins such as p107 (Datta et al., 1995), p53 (Borellini and Glazer, 1993) and E2F (Karlseder et al., 1996; Lin et al., 1996); the interaction with p107 represses Sp1dependent transcription whereas the interaction with E2F leads to synergistic activation of dhfr transcription. On the other hand, the tumor suppressor Rb is involved in the control of transcription of several genes. It can repress positive transcription factors such as E2F, either by sequestering this factor (Helin et al., 1992; Kaelin et al., 1992) or by turning E2F into an active repressor (Weintraub et al., 1992, Weintraub et al., 1995); but Rb can also stimulate the IGF-II (Kim et al., 1992a), c-fos, c-myc, TGF-b1 (Kim et al., 1991; Udvadia et al., 1993), ATF-2, TGF-b2 (Kim et al., 1992b), c-jun (Chen et al., 1994), cyclin D1 (MuÈller et al., 1994), IL-6 (Santhanam et al., 1991), neu (Yu et al., 1992), TK and DHFR (Udvadia et al., 1993) promoters through stimulation of Sp1-mediated transcription. However, although there appears to be a functional interaction between Rb and Sp1, their physical interaction has not been demonstrated. Here, we show that Rb associates with Sp1 in solution, and that both proteins are present together, throughout the cell cycle, in the transcriptional complex that binds to the hamster dhfr promoter. Transient overexpression of Rb leads to an increase in DHFR expression in a homologous system, which is mediated through the Sp1 binding site. The e€ect of Rb on DHFR expression is also observed in permanently transfected cells.

Results Interaction of Rb and Sp1 in binding to the hamster dhfr minimal promoter First, to test whether there was an interaction between Sp1 and Rb, we performed supershift experiments using a probe from the hamster dhfr promoter, nuclear extracts from CHO K1 cells, and antibodies against Rb. As DNA probes we used the two fragments 410f and sp1f described in the Materials and methods section. Gel shift with 410f probe gave rise to three retarded bands. The upper and lower bands corresponded to Sp3 and the central one to Sp1, as identi®ed by speci®c antibodies against Sp1 (PEP 2) and Sp3 (D20) (Figure 1b). The binding of these two Sp proteins was abolished (Figure 1c) when using the

Association of Rb with Sp1 V Noe et al

gc probe containing a single point mutation (G to C) in the ®fth position of the Sp hexanucleotide core binding sequence in the proximal GC box. Polyclonal antibody N9 (against aminoacids 330-610) (Shirodkar et al., 1992), and monoclonal antibody XZ77 (against aminoacids 444-535 and 620-665) (Hu et al., 1991), produced a supershift of the three 410f shifted bands (Figure 1a). N9 antibody also supershifted the binding of Sp1 observed with probe sp1f (Figure 1d). A nonimmune serum (NIS) did not produce the supershift with either probe (Figure 1a and d). As a negative control, the antibodies were incubated in the same experimental conditions with a radiolabeled Oct-1 probe and nuclear extracts from K1 cells. In no case was a supershift observed with this probe (Figure 1e). We also tested the lack of reactivity of antibody N9 with a non-related binding, that produced by the interaction of PPAR/RXR with the PPRE from the mitochondrial HMG-CoA synthase promoter. The addition of N9 antibody did not alter the mobility of the shifted band (Figure 1f).

Western blotting. Nuclear extracts from K1 cells were incubated with anti-Rb N9 antibody, or non immune serum in the primary immunoprecipitation; then the washed immunoprecipitates were subjected to Western blot and detection of Sp1 using the speci®c antibody PEP 2. It can be observed in Figure 2a that Sp1 coimmunoprecipitates with Rb (lanes N9), while no signal was detected when the immunoprecipitation was performed with nonimmune serum (lane NIS). PEP 2 antibody speci®c for Sp1 recognized hamster Sp1 protein (Figure 2b). N9 antibody against Rb does not cross-react with Sp1 protein (Figure 2c). We also performed the complementary immunoprecipitation using anti-Sp1 antibody in the primary immunoprecipitation, followed by detection of Rb in the Western blot, using C-15 antibody. Figure 2d shows that the speci®c anti-Sp1 antibody PEP2 co-immunoprecipitates Rb in the hypo- and hyperphosphorylated forms. These results demonstrate that the two proteins, Sp1 and Rb, form part of the same complex and that the 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 with anti-Rb antibody for the presence of Sp1 using

As a further test of the presence of Sp1-Rb complexes, we measured the binding of Sp1 to the dhfr promoter

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Figure 1 Interaction of Rb with Sp1 in the binding to the dhfr promoter. (a) Gel retardation analysis using 410f probe and Rbantibodies 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 ml of N9 or 10 ml 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 V Noe et al

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Figure 2 Sp1 coimmunoprecipitates with Rb. (a) Coimmunoprecipitation using Rb-antibody. Immunoprecipitates were obtained from 30 mg of K1 cell nuclear extract by incubation with the indicated amounts of NIS, PEP 2 or N9 antibodies. After washing, the bound proteins were resolubilized and Sp1 protein 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 hamster Sp1 by Western blot using 5 mg of K1 nuclear extract (lane KlNE) or 50 ng of recombinant Sp1 protein (lane rSp1) and PEP2 antibody against Sp1. (c) Detection of hamster Rb using 15 mg of K1 nuclear extract and N9 antibody against Rb. The absence of cross-reactivity of N9 antibody was tested with recombinant Sp1 protein (lane rSp1). (d) Coimmunoprecipitation using Sp1-antibody. Twenty mg of K1 cell nuclear extract was immunoprecipitated with anti-Sp1 antibody PEP2 or NIS. The washed immunoprecipitates were subjected to Western analysis using anti-Rb antibody C-15. The ®rst lane (Ext) shows the signal corresponding to Rb obtained directly from 10 mg of K1 nuclear extract

after immunoprecipitation with Rb antibody. Nuclear extracts from exponentially growing cells were exposed to various concentrations of N9 antibody, followed by incubation with insoluble protein A. After centrifugation, the supernatants were used for gel shift analysis of either 410f or sp1f radiolabeled probes. Immunoprecipitation with Rb antibody eliminated the Sp1 shift (Figure 3a and b). As a control we performed gel shift analysis with Oct-1 probe and supernatants from the immunoprecipitation with antibodies against Rb. The binding to Oct-1 probe was unchanged in these conditions, con®rming the speci®city of the immunodepletion (Figure 3c). Interaction of Rb and Sp1 throughout the cell cycle At this point, we were interested in knowing whether the observed interaction between Rb and Sp1 was restricted to a particular phase of the cell cycle. To check this idea, and considering that Rb switches between hypo- and hyperphosphorylated forms during the cell cycle, we performed supershift analyses using nuclear extracts prepared from populations of synchronized CHO K1 cells in each phase of the cell cycle. These nuclear extracts were incubated with radiolabeled 410f as the probe in the absence or the presence of anti-Rb antibody N9. We have checked that this antibody is able to recognize the di€erent states of phosphorylation of Rb. As shown in Figure 4, the anti-Rb antibody is able to produce a supershift of Sp1 in all the phases of the cell cycle. Therefore, Rb and Sp1 interact throughout the cell cycle.

0 NIS 10 12 15 20 30 100 RB-Ab (ng) in IP c

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oct-1 GS 100 10 100 Ab (ng) in IP Figure 3 E€ect of immunoprecipitating Rb or Sp1 on nuclear protein binding to dhfr promoter sequences. Thirty micrograms of nuclear extract from K1 exponentially growing cells were immunoprecipitated with the indicated amounts of anti-Rb antibody N9. After removal of the immune complexes with the aid of insoluble Protein A, the supernatants were used in gel-shift analyses with probes 410f (a), sp1f (b) or Oct-1 (c). NIS, a gel shift performed after immunoprecipitation under the same conditions with 100 ng of nonimmune serum

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Hours after serum stimulation Figure 4 Interaction of Rb with Sp1 throughout the cell cycle. 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 Rbantibody. Four ml of anti-Rb antibody (N9) were added to the gel shift binding mixture and the reaction was incubated for a further 15 min period before electrophoresis. 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. Exp, exponentially growing cells.

Co-transfection of dhfr minigenes plus expression vectors for Sp1 and Rb We then examined the e€ect of co-transfecting Sp1 and/or Rb, driven by eukaryotic expression vectors, on DHFR expression using an homologous system. We transfected plasmid p410 (hamster dhfr minigene driven by its minimal promoter) into hamster DG44 cells (deletion mutant of the dhfr locus ) and the resulting DHFR activity was determined by incorporation of tritiated deoxyuridine into DNA. This assay provides a

Association of Rb with Sp1 V Noe et al

1934

rapid and convenient means of testing DHFR expression when DHFR activity is the limiting factor 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% and 100%, respectively, the DHFR activity provided by the expression of plasmid p410 in DHFR-de®cient recipient cells. Then, we proceeded to co-transfect, along with p410, increasing amounts of either pCMVSpl or pCMVRb. Overexpression of either protein resulted in an increased DHFR transient activity (Figure 5a and b). The combined overexpression of Sp1 and Rb together with p410 had a synergistic e€ect on DHFR transient activity (Figure 5c). However, overexpression of Sp1 plus Rb using pGC as DHFR minigene failed to increase DHFR transient activity (Figure 5c) indicating that the e€ect exerted by Rb is mediated through Sp1. Levels of Rb protein, dhfr mRNA and DHFR activity in CHO cells stably transfected with Rb Finally, we examined whether the e€ect of Rb on DHFR activity observed in co-transfection experiments is reproduced in cells stably transfected with Rb. First, a pool of permanent transformants (K1-RbO3) overexpressing Rb was developed by transfecting CHO K1 cells with pCMVRb (0.3 mg) and further selection with G418. This pool showed a threefold level of overexpression of Rb as determined by Western analysis (Figure 6a). Then, DHFR mRNA and activity were determined. The levels of DHFR mRNA showed a threefold increase (Figure 6b) as determined by RTPCR, and DHFR activity doubled (Figure 6c) as measured by the incorporation of tritiated deoxyuridine. The results attained with a similar pool of

Figure 5 DHFR transient activity in co-transfections of p410 with pCMVSpl and/or pCMVRb. (a) Dose response of cotransfection of p410 with pCMVSp1. DG44 cells (250 000/35 mm dish) were co-transfected with 1 mg of p410 and the indicated amounts of pCMVSp1. After 24 h expression, 2mCi of 6-[3H]deoxyuridine was added for an additional 24 h. The incorporation of radioactivity to DNA was measured in a scintillation counter. The control value corresponds to DG44 cells tranfected with p410 only. (b) Dose response of co-transfection of p410 with pCMVRb. The experiment is the same as in a, but using the indicated amounts of pCMVRb in the co-transfection. (c) Co-transfections of p410 or pGC with pCMVSp1 and/or pCMVRb. DG44 cells were co-transfected with 1 mg of p410 or 1 mg pGC, plus 2 mg of pCMVSp1 and/or 1 mg of pCMVRb. Results are presented as the mean+s.e. (4 exp)

colonies obtained from transfection with the empty vector are also shown (K1 CMV). Discussion The dhfr minimal promoter contains regulatory elements for the binding of the transcription factors Sp1 and E2F. The role of E2F in cell growth regulated transcription of the dhfr promoter has been emphasized by the groups of Azizkhan (Blake and Azizkhan, 1989; Wade et al., 1992) and Farnham (Means et al., 1992; Slansky et al., 1993; Li et al., 1994). On the other hand, Sp1 powerfully activates DHFR transcription (Farnham and Shimke, 1986; Swick et al., 1989; Blake et al., 1990; Ciudad et al., 1992), and given this essential role of Sp1 in activating transcription, we have explored the possibility of the interaction of Sp1 with the cell cycle regulator Rb. The main conclusion of this study is that the retinoblastoma gene product Rb forms a complex with Sp1, as indicated by the supershift, co-immunoprecipitation, and immunodepletion experiments. Using CHO nuclear extracts and radiolabeled 410f as a probe, both anti-Rb antibodies (polyclonal N9 or monoclonal XZ77) supershift this probe. Polyclonal antibody N9 against Rb also supershifts the sp1f probe. Sp1 protein is present in the immunoprecipitate obtained with N9 anti-Rb antibody, and both hypoand hyperphosphorylated forms of Rb protein are present in the immunoprecipitate obtained with PEP2 anti-Sp1 antibody. Rb-immunodepletion of the nuclear extracts abolishes the formation of Sp1-gel shifts. The association between Sp1 and Rb is speci®c since nonimmune serum does not produce either supershift, coimmunoprecipitation, 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 phosphorylation 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 cotransfection experiments using wild type or a mutated version of dhfr minigenes containing the minimal promoter for this gene, we demonstrate that overexpression of either Rb or Sp1 activates the dhfr promoter through the Sp1 binding site. Overexpressing both Rb and Sp1 has a synergistic e€ect on transcription. These results agree with those obtained by Udvadia et al., 1993. These authors used a heterologous system cotransfecting, into Drosophila cells, a hamster dhfr promoter driving the CAT gene. In contrast, we used an homologous system transfecting (into dhfr de®cient hamster cells) a hamster dhfr minigene, containing intron-1 which is needed for correct expression, and determining DHFR dependent activity. Also, stable overexpression of Rb results in an increase in both DHFR mRNA and activity.

Association of Rb with Sp1 V Noe et al

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b aprt

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Figure 6 Analysis of stable transfectants of pCMVRb. K1 cells were transfected by the calcium phosphate method with 0.3 mg of 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 enhances Sp1-binding activity (Chen et al., 1994) and that the IGF-II (Kim et al., 1992a), c-fos, c-myc (Udvadia et al., 1993) and c-jun (Chen et al., 1994) promoters can be regulated by Rb through stimulation of Sp1-mediated transcription. Interestingly, cotransfection of Rb with Sp1 in Drosophila Schneider SL2 cells resulted in a marked increase in dhfr transcription, irrespective of the presence of E2F sites within the dhfr promoter (Udvadia et al., 1993). Another tumor suppressor, p53, has also been shown to increase in concentration in response to the growth factor GMCSF and to form heterocomplexes with Sp1 that stimulate the DNA-binding activity of the latter in proliferating TF-1 cells (Borellini and Glazer, 1993). It has been proposed (Chen et al., 1994; Shew et al., 1990) that the mechanism of the increase in the Sp1mediated transcription by Rb is through the capture by Rb of an inhibitor of Sp1. This proposal was based on the lack of evidence of an association between Rb and Sp1 and on the identi®cation of two proteins of 20 KDa (Chen et al., 1994; Shew et al., 1990) and 74 KDa (Murata et al., 1994) with Sp1-inhibitor characteristics. Our evidence of a direct association between Rb and Sp1, as shown by the supershift and co-immunoprecipitation experiments, does not preclude such an inhibitor-mediated mechanism. Two reports describe a novel interaction between Sp1 and members of the E2F1 family, including the superactivation of Sp1-mediated transcription by E2F (Karlseder et al., 1996; Lin et al., 1996). Therefore, Sp1 is bound by both E2F and Rb proteins, each of which can increase Sp1-mediated transcription. This is in keeping with the picture evolving that regulation of transcription results from interactions that produce di€erent combinations of transcription factors. It has to be stressed that indeed there is good evidence that Rb can cause a repression of transcription when it is tethered to a promoter, for instance

when fused to the DNA binding domain of GAL4 (Adnane et al., 1995). According with this result pRb can decrease transcription of the TGF-b1 promoter in NIH3T3 and AKR-2B mouse cells (Kim et al., 1991); also it can turn E2F into an active repressor (Weintraub et al., 1995). However, pRb has also been described to stimulate transcription from the IGF-II (Kim et al., 1992a), c-fos, c-myc, ATF-2, TGF-b2 (Kim et al., 1992b), c-jun (Chen et al., 1994), cyclin D1 (MuÈller et al., 1994), IL-6 (Santhanam et al., 1991), neu (Yu et al., 1992), TK and DHFR (Udvadia et al., 1993) promoters. In addition pRb can increase transcription of TGF-bl (Kim et al., 1991; Udvadia et al., 1993) in CCL-64 mink lung epithelial cells and A-549 human lung adenocarcinoma. Therefore, pRb can both stimulate and repress transcription on di€erent sets of genes, and this may depend on the recipient cell used in the co-transfection. Recently, Strauss and co-workers have reported, using the DDRT-PCR technique, that 16 genes are stimulated by introducing a wild type Rb cDNA into Rb-de®cient human mammary carcinoma cells, and two in a mouse system aside from the 15 that are repressed (Rhode et al., 1996). This behaviour may be inherent to the characteristic of Rb as a key regulator of the cell cycle in the G1 phase. To date, the role of Rb in regulating the dhfr gene has been assumed to be the sequestering of E2F in arrested cells, thus preventing activation of E2Fresponsive promoters. In this model, stimulation of cell proliferation results in phosphorylation of Rb (Buchkovich et al., 1989; DeCaprio et al., 1989) and E2F (Fagan et al., 1994) with the concomitant release of free E2F, which then directly stimulates the transcription of genes containing E2F-binding sites. On the other hand, we now show a physical interaction between Rb and Sp1 which is maintained throughout the cell cycle, regardless the phosphorylation state of Rb. Therefore, Rb can have di€erent behaviours in its

Association of Rb with Sp1 V Noe et al

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interaction with either E2F or Sp1. This does not exclude the possibility that the phosphorylation state of Rb may be important for Sp1-mediated transcription. At present, what seems very clear is that the level of expression of Rb a€ects positively Sp1 transcriptional activity. Given the reported activation of Sp1 by E2F, and our present observation that Rb associates and activates Sp1, Rb protein may act at a higher level of regulation controlling in a dual fashion genes containing cis-acting elements for E2F and Sp1, such as dhfr.

Materials and methods Cell culture Conditions for the monolayer culture of CHO cells were as described previously (Urlaub et al., 1985). CHO K1 and CHO DG44 cells were grown in Ham's F12 medium supplemented with 7% fetal calf serum (FCS, above two from GIBCO) and maintained at 378C in a humidi®ed 5% CO2-containing atmosphere. For synchronization experiments, 36105 cells were seeded in 100 mm-diameter dishes in F12 medium with 0.5% fetal calf serum for 7 days. Following serum starvation, cells were refed with medium containing 7% FCS for periods of time which corresponded to the di€erent phases of the cell cycle (Noe et al., 1997). Flow cytometry analysis The progression of cells through starvation and the cell cycle after serum stimulation was monitored by ¯ow cytometry. For this analysis, nuclei were stained with 25 m/ml of propidium iodide (Sigma Quõ mica, Madrid) and analysed on a Becton Dickinson ¯ow cytometer. Synchronized CHO cells, upon addition of 7% FCS reenter the cell cycle, reaching the G1/S border in 11 h, and G2/M after 19 h (Noe et al., 1997). Therefore, populations of cells were harvested at 0 h (Go), 9 h (G1), 11 h (G1/S), 15 h (S) and 19 h (G2/M), to prepare nuclear extracts. DNA probes DNA probe 410f was prepared by PCR ampli®cation from plasmid DNA p410. This is a BAL-31 deletion mutant minigene that includes just 48 bp upstream of the major transcriptional start site of the hamster dhfr promoter (Ciudad et al., 1988). The oligonucleotides used in the PCR reaction hybridized with sequences ¯anking the Sp1 binding site and the translational start, producing a fragment of 150 bp. The forward primer was 5'GTTCTAGTCAGCCAGGCAAG-3', and the reverse primer 5'-GTTCAGCGGTCGAACCAT-3'. The ampli®ed DNA, 410f, was digested with HpaII (at 774 relative to the translational start), and the 59-bp fragment (splf), which contains the recognition sequence for Sp1, was gelpuri®ed. Probe gc was generated in the same manner that 410f but from plasmid pGC (Ciudad et al., 1992). A ds DNA fragment containing the Oct-1 consensus sequence (5'-TGTCGAATGCAAATCACTAGAA-3', Promega) was used as a control for the shift and supershift experiments. PPAR and RXR (peroxisomal proliferator activated receptor/retinoic acid receptor) were synthesized in vitro and used as heterodimers to bind to HMG-CoA synthase PPRE (peroxisomal proliferator response element) in supershift experiments to test the cross-reactivity of Rbantibody N9. The probes were end-labeled with T4 polynucleotide kinase (BRL) using g-[32P]-ATP (3 000 Ci/ mmol, Amersham).

Nuclear extracts Nuclear extracts were prepared as described (Ciudad et al., 1992) from exponentially growing CHO K1 cells, or cells throughout the cell cycle. Protein concentrations were determined using the Bio-Rad protein assay based on the method of Bradford M, 1976, using BSA as standard (Sigma), and extracts were frozen in liquid N2 and stored at 7808C. 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, 60 mM KCl, 2 mg 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 labeled probe and then for a further 30 min in its presence. Immediately afterwards, the samples were subjected to polyacrylamide gel electrophoresis (5% polyacrylamide: bis 30 : 1, 5% glycerol in 0.56TBE (16TBE is 90 mM Tris borate, pH 8, 2 mM EDTA) for 3 ± 4 h at a maximum of 20 mA. The gel was dried and the radioactive bands were visualized by autoradiography. In supershift experiments, N9 rabbit polyclonal serum raised against a Trp-Rb fusion protein, XZ77 mouse monoclonal antibody raised against bacterially expressed Rb, PEP 2 rabbit polyclonal antibody against Sp1 non cross-reactive with Sp2, Sp3 or Sp4, and D20 polyclonal antibody 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 generously provided by JA DeCaprio (N9), and E Harlow and N Dyson (XZ77), or purchased from Santa Cruz Biotechnology (PEP 2, D20). Co-immunoprecipitations Nuclear extracts from K1 cells were immunoprecipitated using either N9 anti-Rb antibody, PEP 2 anti-Sp1 antibody or nonimmune serum (NIS) with the aid of Protein ASepharose as above. Then, the pellets were washed twice in 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. Immunodepletions Immunoprecipitations were performed on ice for 1 h using 30 mg of nuclear extracts from exponentially growing CHO K1 cells, and amounts ranging from 10 ± 100 ng, of anti-Rb antibody N9, in 0.1% NP40 (Sigma Quõ mica). The immunecomplexes were removed by centrifugation with 3 ml of insoluble Protein A (Sigma Quõ mica) and the supernatants were used in gel shift assays with either 410f and sp1f radiolabeled probes. Western blot analysis Di€erent amounts of nuclear extract (stated in the legends) from CHO K1 cells, 50 ng of recombinant puri®ed Sp1 (Promega), or the washed pellets from the immunoprecipitations, were resolved on SDS-7% polyacrylamide gels (Laemmli, 1970), and transferred to a PVDF membrane (Immobilon P, Millipore) using a semidry electroblotter. The membranes were probed with either anti-Rb antibodies (N9, 1 : 1500 dilution; or C-15 from Santa Cruz Biotechnology, 1 : 100 dilution), or anti-Spl antibody (PEP 2, 1 : 1500 dilution). Signals were detected by secondary horseradish peroxidase-conjugated antibody (1 : 12000 dilution) and enhanced chemiluminiscence, as recommended by the manufacturer (Boehringer Mannheim).

Association of Rb with Sp1 V Noe et al

Transfections, co-transfections and DHFR transient activity assay Stables transfections of CHO K1 cells with eukaryotic expression vector for Rb (pCMVRb, Qin et al., 1992), or the empty vector, were performed by the calcium phosphate method (Urlaub et al., 1989). After 24 h expression, cells were selected with Geneticin (800 mg/ml) for 3 weeks. The surviving colonies were pooled and used for Western, mRNA and DHFR enzymatic analyses. Co-transfections experiments were carried out in dhfr de®cient cells (CHO-DG44) by the Polybrene method (Chaney et al., 1986) as described in Ciudad et al., 1988. Plasmid p410 (dhfr minigene driven by its minimal promoter), or plasmid pGC (single point mutant from p410 in the Sp1 recognition site, Ciudad et al., 1992), plus eukaryotic expression vectors for Rb (pCMVRb), Sp1 (pCMVSp1), or both, were co-transfected. After 24 h expression, the resulting DHFR activity was determined by incorporation of radiactive deoxyuridine to cellular DNA, as described in Ciudad et al., 1988 with the following modi®cation: after 24 h labeling with 2 mCi of 6-[3H]deoxyuridine, cells were rinsed twice in phosphate-bu€ered saline and lysed in 100 ml of 0.1% SDS. The lysate was collected on Whatman 31ET papers (2 cm62 cm), which were immediately placed onto ice-cold 66% ethanol containing 250 mM NaCl, in order to precipitate DNA. 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. mRNA analysis by RT-PCR Total mRNA was extracted from either CHO K1 cells, K1 cells stably transfected with the expression vector for Rb (pCMVRb), or the empty vector using the Ultraspec TM RNA reagent (Biotecx) in accordance with the manufacturer's instructions. cDNA was synthesized in a 20 ml reaction mixture containing 1 mg of RNA (in DEPC treated water), 125 ng of random hexamers (Promega), 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (Sigma Quõ mica), 4 ml of 56RT bu€er and 200 units of MLV reverse transcriptase (above two from BRL). The reaction mixture was incubated at 378C

for 60 min. Five ml of the cDNA mixture was used directly for PCR ampli®cation. PCR reactions were typically carried out as follows. A standard 50 ml mixture contained 5 ml of the cDNA mixture, 4 ml of 106PCR bu€er (Mg2+free), 1.5 mM MgCl2, 0.2 mM dNTPs, 2.5 mCi of a-[32P]dATP (3000 Ci/mmol, Amersham), 1 unit of Taq polymerase (BRL) and 500 ng of each of four primers. The primers used were: 5'-CGCCAAACTTGGGGGAAGCA-3' and 5'-GAACCAGGTTTTCCGGCCCA-3' for dhfr, and 5'-ATCCGCAGTTTCCCCCGACTT-3' and 5'-TCACACACTCCACCACCTCA-3' for aprt, which was used as an internal control. The reaction mixture was separated in two phases by a solid paran wax layer (melting T=59 ± 608C, Fluka), which prevents complete mixing of PCR reactants until the reaction has reached a temperature at which nonspeci®c annealing of primers to non-target DNA is minimized. The lower solution contained the MgCl2, the dNTPs, the four primers, the a[32P]-dATP and half the bu€er, and the upper solution contained the cDNA, the Taq enzyme and the remaining bu€er. PCR was performed for 25 cycles, after a 1 min denaturation at 948C; each cycle consisted of denaturation at 928C for 1 min, primer annealing at 598C for 75 s, and primer extension at 728C for 110 s. Five microliters of each PCR sample was electrophoresed in a 5% polyacrylamide gel. The gel was dried and the radioactive bands were visualized by autoradiography.

Acknowledgements We thank Mr Robin Rycroft from the Language Advisory Service (SAL) for correcting the English manuscript, and Dr Diego Haro for providing the PPRE probe from the rat mitochondrial HMG-CoA synthase, and the in vitro translated PPAR/RXR heterodimers. This work was supported by grants SAF94-177 and SAF96-74 from CICYT, and SGR96-84 from ClRlT to CJC and NIH grant GM-22529 to LAC. CJC was a recipient of a fellowship from CIRIT of Catalonia. VN was a recipient of a predoctoral fellowship from the Spanish Ministry of Education, and CA is a recipient of a predoctoral fellowship from the Catalonian Department for Scienti®c Research.

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