MOLECULAR AND CELLULAR BIOLOGY, Apr. 1996, p. 1659–1667 Vol. 16, No. 4 0270-7306/96/$04.00ϩ0 Copyright ᭧ 1996, American Society for Microbiology

Interaction of Sp1 with the Growth- and -Regulated

JAN KARLSEDER, HANS ROTHENEDER, AND ERHARD WINTERSBERGER* Institut fu¨r Molekularbiologie, Universita¨t Wien, A-1030 Wien, Austria

Received 18 August 1995/Returned for modification 6 October 1995/Accepted 8 January 1996

Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of , a domain also present in E2F2 and but absent in and , were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind Sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was established that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the Sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin- organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle- regulated tethering factor between Sp1 and the basic transcription machinery.

When quiescent cells are stimulated to grow, expression of 24, 72) to a defined DNA motif together with a member of a several coding for DNA synthesis and precursor-produc- small family of proteins, the DP proteins (4, 13, 72, 74). E2F is ing enzymes is induced at the boundary of the G1 to S phase of negatively regulated in G0 and G1 by the underphosphorylated the cell cycle (reviewed in references 47, 52, and 70). This form of the (pRB) or by another mem- induction is at least in part regulated at the transcriptional ber of the so-called pocket proteins (31), p107 or p130 (re- level. One of the intensively studied enzymes of this class is viewed in references 17, 33, 37, 63, and 68). Phosphorylation of cytoplasmic thymidine kinase (TK), an enzyme involved in the the pocket protein by a -dependent kinase relieves the fine-tuning of the precursor pool for nuclear DNA replication. inhibition of E2F activity. Interestingly, E2F1 to E2F3 bind The promoters of the TK genes of several mammalian organ- only to pRB, while E2F4 and E2F5 appear to be regulated by isms are known in some detail and exhibit remarkable differ- p107 and/or p130 (5, 9, 12, 21, 22, 38, 59). The pocket proteins ences, although the genes are very similar with respect to were also shown to be the target of the gene products of several transcriptional regulation (71). Thus, whereas the hamster and DNA tumor viruses (adenovirus E1A, papillomavirus E7, and human TK promoters contain reversed CCAAT boxes and a simian virus 40 and polyomavirus large T antigens) which TATA box, the mouse promoter lacks both of these elements. transactivate E2F-regulated promoters by binding to the un- Accordingly, transcription of the mouse gene starts at several derphosphorylated pocket protein, thereby removing it from points within a region of about 80 nucleotides (15). Common its binding to E2F, which then becomes transcriptionally active to all of the promoters are GC boxes as binding sites for (reviewed in references 37, 43, 48, and 49). transcription factor Sp1. In addition, these promoters carry The TK promoter of the mouse (41, 60) is a very simple one sequences which more or less resemble binding sites for the with respect to binding motifs for known transcription regula- transcription factor E2F, a protein which is involved in the tors. Earlier work had shown that a sequence about 150 bp control of a variety of growth- and cell cycle-regulated genes upstream of the ATG suffices for growth-regulated expression (reviewed in references 36, 37, and 48). Of these, the E2F site of a reporter gene (11, 41, 67). This sequence encompasses a of the murine TK promoter almost perfectly conforms to the single Sp1 site and the E2F motif. Plasmids carrying the gene proposal for an E2F consensus sequence. It closely resembles for chloramphenicol acetyltransferase (CAT) under the con- that of the dihydrofolate reductase promoter, and these two trol of the wild-type TK promoter or of promoters mutated at genes were the first ones among those coding for DNA syn- critical positions within the Sp1 or E2F binding site were stably thesis and precursor-producing enzymes for which evidence for introduced into Swiss 3T3 cells for studies of growth-regulated a role of E2F in growth regulation was obtained (6, 20, 50, 64, expression of the CAT gene. We first confirmed earlier obser- 66). Transcription factor E2F denotes a family of helix-loop- vations with transiently transfected cells which showed that helix proteins (28), five members of which have been cloned (5, mutation of either the Sp1 site (11) or the E2F site (50) 12, 18, 22, 26, 30, 38, 59, 62). E2F binds as a heterodimer (3, 19, abolishes promoter activity. This finding indicates that both Sp1 and E2F are indispensable for transcription from this * Corresponding author. Mailing address: Institut fu¨r Molekularbi- promoter. Since the absolute requirement of both transcrip- ologie, Universita¨t Wien, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria. tion factors for promoter activity could mean that the two Phone: 43-1-79515-2117. Fax: 43-1-79515-2901. Electronic mail ad- proteins interact, we were seeking evidence for such an inter- dress: [email protected]. action. We found that Sp1 and E2F bind to each other in

1659 1660 KARLSEDER ET AL. MOL.CELL.BIOL. solution, requiring specific regions within the two proteins, and body. Beads were collected by gentle centrifugation and washed three times with that they form complexes in vivo, as evidenced by coimmuno- 500 ␮l of IP buffer for 5 min. Protein was removed from the beads by boiling in sample buffer for 5 min and subjected to sodium dodecyl sulfate (SDS)-poly- precipitation. The distance between the binding sites is critical acrylamide gel electrophoresis (PAGE) on a 7.5% gel. Western blot (immuno- for optimal promoter activity. In vivo footprinting experiments blot) analysis was carried out with an anti-Sp1 antibody (Santa Cruz). As a showed that both transcription factors are bound to their sites positive control, 2 ␮g of protein from the cell extract or 5 ng of purified Sp1 was at the promoter in arrested cells as well as in growth-stimulated applied to the gel for direct immunoblot analysis. KH95 is an anti-E2F mono- clonal antibody specifically directed against the pRB binding domain. cells and that mutation of either one of the binding sites elim- Construction and synthesis of GST fusion proteins and protein binding as- inates binding of both factors, which is indicative of coopera- says. Plasmids carrying E2F cDNA or the information for glutathione S-trans- tive binding. E2F may thus function as a regulated tethering ferase (GST) fusion proteins were kindly provided by different colleagues. The factor acting to transmit an activation signal from Sp1 to the plasmid for GST-E2F1 expression (pGex-2T-E2F1) was constructed by cloning the BamHI-to-EcoRI fragment of E2F1 cDNA (30) into the pGex-2T vector basic transcription machinery. (Pharmacia). The truncated proteins GST-E2F1(1-122), GST-E2F1(1-208), and GST-E2F1(1-284) were created by cutting plasmid pGex-2T-E2F1 with EcoRI and with SmaI, PpuMI, and BglII, respectively, and religating the plasmid. GST- MATERIALS AND METHODS E2F1(123-437) was generated by inserting the SmaI-to-EcoRI fragment into Plasmids. Plasmids carrying the CAT gene under a wild-type or mutated TK pGex-3x (Pharmacia). A GST-DP1 expression plasmid was constructed by in- promoter were constructed by using standard procedures (58) and are schemat- serting the DP1 cDNA (13) into pGex-3x. For the construction of Sp1 expression ically shown in Fig. 1. For the preparation of the CAT constructs, the gpt gene plasmids, the oligonucleotide 5Ј GATCGATCGCGAGGCCTCGAGCCATGG from plasmid pMSG-CAT (Pharmacia) was removed and replaced by the neo ATCCCCGGG 3Ј was inserted into the EcoRI- and BamHI-digested vector gene, and the mouse mammary tumor virus promoter was removed and replaced pGex-2TK (30), thereby creating vector pGex-2TK-MCS, which allows cloning of by a fragment encompassing the sequence from the EcoRI site to the ATG of the Sp1 plasmids in the correct reading frame for expression. Sp1-cDNA (29) was mouse TK promoter (60). Mutations in the Sp1 and E2F sites were introduced cloned into the EcoRI- and BamHI-digested vector. pGex-2TK-MCS-Sp1(1- by oligonucleotide-directed in vitro mutagenesis, using an Amersham system as 293), pGex-2TK-MCS-Sp1(1-621), and pGex-2TK-MCS-Sp1(622-788) were gen- recommended by the vendor. erated by cutting pGex-2TK-MCS-Sp1 with EcoRI and PpuMI, with EcoRI and To generate the plasmid with an additional 6 bp between the E2F and Sp1 BamHI (partially), and with BamHI, respectively, and religating the plasmid. sites, the promoter fragment between the SacI site and the XmnI site was For the experiment shown in Fig. 2C, E2F1 coding sequence was transcribed with T7 polymerase (Promega). The RNA was translated with reticulocyte lysate replaced by an oligonucleotide carrying an additional BglII site between the E2F 35 and Sp1 motifs. The mutant with an additional 10 bp was created by cutting the (Promega) and radiolabeled with [ S]methionine. newly introduced BglII site of the 6-bp mutant, filling in the site with the Klenow Fusion proteins were synthesized in Escherichia coli BL21 and purified as polymerase, and religating the plasmid. To generate the mutant with an addi- described previously (10). Expression was induced by isopropythiogalactopy- tional 20 bp, the newly introduced BglII site of the 6-bp mutant was cut, and a ranoside (IPTG; 0.4 mmol/liter). One hundred micrograms of protein from 14-bp oligonucleotide was ligated into the site. All mutations were verified by whole cell extracts of 3T3 cells (prepared as described in reference 51) or DNA sequencing. purified Sp1 protein (50 ng; Promega) was incubated with glutathione-agarose Cell culture, transfection, and CAT assays. Swiss 3T3 cells were grown in preloaded with 1 ␮g of GST fusion protein for1hat4ЊC in a binding buffer Dulbecco’s modified Eagle’s medium containing antibiotics and 10% fetal calf containing 20 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid serum. Transfection with the CAT constructs was performed by using Polybrene- (HEPES; pH 7.9), 1 mM MgCl2, 40 mM KCl, 0.1 mM EDTA, 0.1% Nonidet assisted gene transfer (2). Stably transfected cells were isolated by selection in P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 100 ␮gof the presence of Geneticin (600 ␮g/ml of medium), and many clones from several ethidium bromide per ml (35). The beads were washed four times with binding petri dishes were pooled for further propagation. The presence and the amount buffer and then boiled in SDS sample buffer for elution of bound proteins. These of each of the integrated TK promoter-CAT genes was determined by Southern proteins were then analyzed by SDS-PAGE followed by Western blotting with blotting whereby a 0.7-kb SalI fragment from the CAT gene was used as a anti-Sp1 antibody (Santa Cruz). hybridization probe. In vivo footprinting. Medium was removed from cell cultures and replaced by Cells were growth arrested by reducing the serum concentration in semicon- fresh medium supplemented with 20 mM HEPES (pH 7.3) and 4 ␮l of dimeth- fluent cultures to 0.2% and keeping cells under these conditions for 72 h. Growth ylsulfate per ml. After 2 min at room temperature, the reaction was stopped by stimulation was achieved by addition of serum to 20%. About 16 h after serum two rapid washes of cells with ice-cold phosphate-buffered saline containing 2% stimulation, cells were in mid-S phase. (vol/vol) mercaptoethanol. Further treatment was as described by Mueller et al. CAT assays were performed as described by Gorman (14), using equal (44). Naked DNA was subjected to the same treatment with dimethylsulfate amounts of protein from cell extracts. followed by cleavage with piperidine. Quantification of CAT mRNA. The CAT gene from pMSG-CAT was cloned DNA fragments were amplified as described previously (45, 54) and analyzed into plasmid pGEM-3Z by using the SalI restriction sites. Thirty-eight base pairs in sequencing gels. The following oligonucleotides from within the TK promoter were removed from the CAT gene by digestion with BalI and NcoI, filling in of were used as primers: Prom1, 5Ј AGACAAGTTCCTCCAGGGATTCTG 3Ј; the recessed end, and blunt-end ligation. The deletion was verified by DNA Prom2, 5Ј GAAGGAAACGCCATGGCCAGATCCG 3Ј; and Prom3, 5Ј sequencing. RNA was synthesized in vitro starting from the T7 promoter of GAAACGCCATGGCCAGATCCGGAGG 3Ј. pGEM and using T7 RNA polymerase from Promega as recommended by the supplier. RESULTS Cytoplasmic RNA was isolated after lysis of cells with Nonidet P-40 and purified as described previously (58), including an incubation with RNAse-free Sp1 and E2F are both essential for TK promoter activity. DNAse. Approximately 5 ␮g of RNA was mixed with a 1:105 dilution of the in vitro-produced shortened CAT mRNA and reverse transcribed, using the CAT- We first determined the contribution of the E2F and the Sp1 specific oligonucleotide 5Ј TGGCCACTCATCGCAGTACTGTTG 3Ј. One-fifth binding motifs to the transcriptional activity of the promoter by (5 ␮l) of the reverse transcription reaction was used for PCR (sense oligonucle- mutating either one or both sites in constructs carrying the otide, 5Ј CTGGCCTATTTCCCTAAAGG 3Ј; antisense oligonucleotide, 5Ј CAT gene as reporter under the TK promoter (Fig. 1A). Using GCATGATGAACCTGAATCG 3Ј). PCR was performed under standard con- ditions as recommended by the supplier of Taq polymerase (Promega), with the synthetic oligonucleotides comprising the Sp1 or E2F binding addition of 0.2 ␮lof[32P]dCTP to each reaction (30 s at 92ЊC,30sat62ЊC, and site of the TK promoter, we found that the mutations abolish 5sat72ЊC for 24 cycles). Products were electrophoretically separated on 6% the capacity of the oligonucleotide to bind the respective pro- polyacrylamide gels and visualized by overnight exposure to Fuji HRH films. tein in band shift experiments (not shown). The plasmids used Coimmunoprecipitation. Cells were washed twice with immunoprecipitation wash buffer (20 mM Tris [pH 8], 135 mM NaCl, 10% glycerol), scraped off the for stable transfection in addition carried the bacterial neo petri dish, and collected by centrifugation. Protein was extracted by gently shak- gene for selection of transfected cells with Geneticin. Combin- ing the cells at 4ЊC in immunoprecipitation buffer (IP buffer; 20 mM Tris [pH 8], ing a great number of Geneticin-resistant colonies from several 135 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM phenylmethylsulfonyl petri dishes, we obtained pools of transfected cells, which were fluoride, 20 ␮g of aprotinin per ml) for 15 min, and cell debris was removed by centrifugation. Two hundred micrograms of protein was incubated with 10 ␮lof then tested for the presence and expression of the CAT gene. anti-E2F1 antibody-agarose conjugate (KH95; 2 ␮g/␮l; Santa Cruz Biotechnol- Southern blot analyses (not shown) indicated that Geneticin- ogy) in 500 ␮l of IP buffer under gentle shaking for1hat4ЊC. Control exper- resistant cells harbor two to five copies of the CAT gene. iments were carried out by using (i) 5 ng of purified Sp1 protein for the immu- The CAT activity in extracts of cells transfected with the noprecipitation reaction with anti-E2F antibody and (ii) 200 ␮g protein from cell extracts for incubation with 10 ␮l of agarose-protein A beads carrying an unre- various CAT constructs, as expected, revealed strong expres- lated antibody (antihemagglutinin) of the same subclass as the anti-E2F anti- sion of the CAT gene from the wild-type TK promoter in VOL. 16, 1996 E2F BINDS TO Sp1 1661

logarithmically growing cells. In contrast, mutation of either the Sp1 site or the E2F site reduced promoter activity to very low levels (Fig. 1B). That the failure to express CAT is in fact due to a failure to transcribe the gene was proven by measuring mRNA levels for CAT. Because CAT mRNA is of low abun- dance and hence difficult to quantitate, we used a PCR method for determining the amounts of CAT mRNA in the various transfected cell lines. mRNA was reverse transcribed, and the product was amplified by using CAT-specific oligonucleotides. For quantitation, a shortened version of the CAT gene was constructed in a pGEM vector, and RNA was synthesized in vitro from this truncated gene. Equal amounts of this RNA were added to each one of the cellular RNA preparations and processed together with the mRNA as an internal control for the PCR. Under conditions which led to equal amplification of the added truncated RNA, there was almost no mRNA de- tectable in cells carrying either one of the mutated TK pro- moters, but a significant amount of CAT mRNA was produced in cells carrying the wild-type promoter, in full agreement with results of the enzymatic CAT assays (Fig. 1C). These results suggest that the activity of the TK promoter may depend on an interaction between Sp1 and E2F. Sp1 and E2F bind to each other. To gain support for the assumption that Sp1 and E2F interact directly at the TK pro- moter, we first tried to determine whether the two transcrip- tion factors bind to each other in solution. To this end, GST fusion proteins of E2F1 and fragments thereof were produced in E. coli, purified, and then fixed to glutathione-agarose, and cell extracts were applied to the column. After extensive wash- ing of the column, bound proteins were eluted and analyzed by Western blotting with an antibody against Sp1. As can be seen in Fig. 2A, E2F1 and truncated versions thereof retained Sp1 provided that the N-terminal 122 amino acids of E2F1 were contained within the fusion protein. Purified Sp1 could be used in this assay in place of the Sp1-containing cell extract and gave the same results, indicating that the binding of Sp1 to E2F1 is direct and not mediated by additional cellular proteins. It is noteworthy that a large fraction of input purified Sp1 is bound to E2F1, which indicates that E2F1 has a strong binding ca- pacity for Sp1. If cell extracts were used as a source of Sp1, the fraction bound is considerably smaller, most likely because cell extracts contain many proteins (aside of E2F) which bind Sp1, resulting in competition for this transcription factor. The bind- ing assays were carried out in the presence of ethidium bro- mide (35) in order to avoid an interference of the DNA bind- ing region of E2F in the assay. The data thus indicate that Sp1 binds to E2F and that a specific region of this transcription factor is required for this interaction (Fig. 2B shows a sche- matic representation of functional domains of E2F1). DP1, which shows no similarity to E2F1 in this part of the molecule (37), failed to bind Sp1. The region within the Sp1 protein which is required for binding to E2F was resolved by using truncated versions of Sp1 as GST fusion proteins and deter- mining their capacity to bind in vitro-synthesized, labeled

cells growing logarithmically. The control was a cell line carrying a promoterless CAT construct. Spots of acetylated chloramphenicol were scanned to calculate relative CAT activities. wt, wild type. (C) Analysis of CAT mRNA produced in logarithmically growing cells carrying the TK promoter-CAT constructs indi- cated. Lane 0 denotes the cell line carrying the promoterless CAT construct. The analysis involves PCR to amplify the signal as described in Materials and Meth- FIG. 1. (A) Schematic representation of the wild-type and mutated promoter ods. The faster-migrating 152-bp fragment derives from a truncated version of region of the murine TK gene used for the production of TK promoter-CAT the CAT gene, which was transcribed in vitro. This RNA was then added in equal constructs used in this work. Mutations introduced into the binding sites for amounts to all RNA samples as an internal standard for reverse transcription and transcription factors E2F and Sp1 are also shown. (B) CAT activities in cell lines PCR. The 190-bp fragment corresponds to the fragment expected from reverse carrying various TK promoter-CAT constructs. Extracts were prepared from transcription and PCR amplification of CAT mRNA. 1662 KARLSEDER ET AL. MOL.CELL.BIOL.

FIG. 2. E2F and Sp1 bind to each other. (A) GST-E2F1 and truncated forms thereof were bound to glutathione-agarose and incubated with total cell extracts from 3T3 cells or with purified, commercially available human Sp1. The beads were then washed extensively, and bound proteins were eluted with sample buffer, separated by electrophoresis, and visualized by immunoblotting with an antibody against Sp1. Input means that 1/10 of the amount of cellular extract or of Sp1 protein that was used for binding to GST-E2F was added directly onto the gel and visualized by immunostaining without binding to and elution from GST-E2F beads. wt, wild type. (B) Domain structure of E2F1 indicating the region required for binding to Sp1. (C) GST-Sp1 and truncated forms thereof were fixed to glutathione-agarose and incubated with in vitro-synthesized, labeled E2F1. After electrophoresis, bound protein was visualized by fluorography. IVT, in vitro-transcribed. (D) Domain structure of Sp1 indicating the region required for binding to E2F. (E) GST-E2F1, GST-E2F2, and GST-E2F3, bind Sp1 whereas GST-E2F4 and GST-E2F5 do not. As a negative control, GST-E2F1(123-437), which lacks the binding region, was included. Also shown is that GST-DP1 does not bind Sp1. Input denotes the signal of Sp1 given by 1/50 of the amount of cell extract that was used for the GST-E2F binding experiments. The numbers at the sides in panels A, C, and E are relative molecular weights (in thousands) of marker proteins.

E2F1. This experiment (Fig. 2C) revealed that the binding conceivable that interaction of domain D of Sp1 with E2F is domain for E2F within Sp1 lies within the C-terminal quarter necessary to transmit an activation signal in this TATA-less of the protein (Fig. 2D shows the domain structure). This promoter. region of Sp1 contains the zinc fingers and domain D, which A comparison of the sequences of the five E2F proteins was previously found to be dispensable for transmitting an described so far (59) reveals some similarity between E2F1, activation signal from an Sp1 tetrameric complex on a single E2F2, and E2F3 within that part of the molecules which is GC box but to be essential for a synergistic interaction of required for binding to Sp1. E2F4 and E2F5, on the other tetramers at adjacent GC motifs to form higher-order com- hand, lack this region almost completely. We therefore exam- plexes, with the consequence of considerably stronger activa- ined whether E2F2, E2F3, E2F4, and E2F5, in addition to tion of transcription from a promoter carrying a TATA box E2F1, could interact with Sp1. GST fusion proteins of all of (53). Since the TK promoter has only a single GC box within these E2Fs were bound to glutathione-agarose, and elution the region responsible for growth-regulated expression, it is experiments were carried out as for Fig. 2A. From the result VOL. 16, 1996 E2F BINDS TO Sp1 1663

FIG. 3. Coimmunoprecipitation of Sp1 and E2F from extracts of mouse cells. Proteins from cellular extracts were immunoprecipitated with an antibody against E2F1 bound to protein A-agarose. After separation and washing of the precipitate, proteins were eluted, separated by SDS-PAGE, and analyzed by using an antibody against Sp1. Two control experiments are included to prove the specificity of the immunoprecipitation. In control 1, purified Sp1 (5 ng, giving the signal shown under ‘‘input for control 1’’ when directly applied to the gel) was used instead of the cell extract for immunoprecipitation by anti-E2F antibody. In control 2, protein A beads carrying antihemagglutinin antibody instead of anti- E2F antibody were used. In both cases, Sp1 could not be detected. The numbers on the left are relative molecular weights (in thousands) of marker proteins. To exclude participation of DNA in this assay, the experiments were repeated in the presence of 100 ␮g of ethidium bromide per ml in IP buffer. The result was identical to that shown here.

shown in Fig. 2E, it is evident that, as expected, E2F1, E2F2, and E2F3 bound Sp1 whereas E2F4 and E2F5 failed to do so. It should be mentioned that because of the availability of the respective cDNAs and GST constructs, we used human E2F and human Sp1 for these experiments; our other studies in- volved mouse fibroblasts. However, it was shown recently that murine and human E2F1 are 86% identical (40), and a similar homology can be expected for murine and human Sp1 since rat Sp1 is 97% identical with the human protein (25). In agree- FIG. 4. Promoter activity depends on the distance between the binding sites ment with this assumption, we did not see any difference be- for Sp1 and E2F. The distance between the Sp1 and E2F sites in the murine TK tween purified human Sp1 and that present in mouse cell promoter was increased by introduction of extra nucleotides as shown. These extracts in band shift experiments (not shown). modified TK promoter-CAT constructs were stably introduced into Swiss 3T3 Coimmunoprecipitation of E2F1 and Sp1 from cell extracts. cells, and CAT activity was measured. The control carried no promoter. wt, wild To verify that the interaction between the two proteins which type. we observed in vitro resembles the situation in vivo, coimmu- noprecipitation experiments were carried out with an antibody against E2F1 immobilized on protein A-agarose to bind the significant reduction of CAT activity with increasing distance complexes. Proteins bound to the antibody were then eluted between the transcription factor binding sites. and analyzed by Western blotting for the presence of Sp1. As Binding sites for Sp1 and E2F at the TK promoter are shown in Fig. 3, Sp1 was bound by antibody against E2F1, occupied in arrested cells and at different times after growth indicating that complexes of the two proteins exist in vivo. In stimulation. Since E2F is known to be a growth- and cell control experiments, it was verified that the antibody against cycle-regulated transcription factor whereas Sp1 may be E2F1 did not precipitate purified Sp1 and that an unrelated present and active constitutively, we carried out in vivo foot- antibody (antihemagglutinin) did not result in any signal. printing experiments to determine whether there is a differ- When the Western blot was reprobed with DP1, it was found ence in the occupation of the binding sites for the two tran- that this protein can be detected as well (not shown). This scription factors depending on the growth condition. As shown result does, however, not allow us to determine whether DP1 in Fig. 5B for the lower strand (compare with the sequence of is present in a ternary complex consisting of E2F-Sp1 and DP1 the respective region of the promoter shown in Fig. 5A), this is or merely in the well-known dimeric complex with E2F. not the case. Binding sites for both transcription factors exhib- Promoter activity depends on the distance between the bind- ited a clear footprint in all cases, indicating that proteins bind ing sites for Sp1 and E2F. If Sp1 and E2F interact at the to the promoter in serum-starved, quiescent cells as well as in promoter in vivo, it could be assumed that the distance be- cells growth stimulated for different periods of time. Fluores- tween their binding sites plays an important role for promoter cence-activated cell sorting (FACS) analysis (Fig. 5C) carried activity. To test this, we produced TK promoter-CAT con- out in parallel showed that cells were in G0/G1 at the beginning structs in which the distance between the binding sites was of the experiment and moved into the S phase after addition of increased by 6, 10, or 20 bp. To facilitate the formation of the serum. The observation that there is no difference in the oc- correct chromatin structure at the TK promoter, these con- cupation of the Sp1 and E2F binding sites under the conditions structs were again stably introduced into Swiss 3T3 cells, and that we have studied is in accord with in vitro footprinting CAT activity was measured. As shown in Fig. 4, there is a experiments (57) which showed identical binding, regardless of 1664 KARLSEDER ET AL. MOL.CELL.BIOL. VOL. 16, 1996 E2F BINDS TO Sp1 1665 whether the cell extracts were obtained from quiescent or from significant reduction of the promoter activity in vivo. As bind- growing cells. This result agrees well with the current view of ing between GST-E2F1 fusion protein and Sp1 was observed the regulation of E2F whereby cell cycle-dependent binding of not only with Sp1 from cellular extracts but also if purified Sp1 pRB or another pocket protein to E2F interferes with its tran- was used, it is most likely that this binding is direct and does scriptional activation potential. This type of regulation, there- not involve other cellular proteins. It is of particular interest fore, would not necessitate a removal of the transcription fac- that the N-terminal region of E2F1 to E2F3, which was found tor in situations in which the promoter is inactive. to be required for the interaction with Sp1, is absent from Evidence for cooperative binding of Sp1 and E2F to the E2F4 and E2F5 (59). Accordingly, both E2F4 and E2F5 did murine TK promoter. Having available cells with stably inte- not bind Sp1. Considering that it is not known which member grated transgenes under the control of wild-type or mutated of the E2F family functions in the regulation of TK gene version of the mouse TK promoter, we were able to examine expression, these observations make it unlikely that E2F4 or the occupancy also of these promoters. Lanes 1 and 2 of Figure E2F5 is involved. As E2F4 and E2F5 bind p107 and/or p130 5D show that cells carrying the wild-type TK promoter-CAT whereas E2F1 to E2F3 bind pRb (5, 9, 12, 21, 22, 38, 59), it is construct exhibited the same in vivo footprints as untransfected furthermore unlikely that p107 (as suggested in reference 8) or 3T3 cells (Fig. 5B), in which the footprint resulted from the TK p130 is implicated in the control of TK expression at the G1/S gene only. As our transfected cells carry on average about border. It should also be mentioned that the N-terminal region twice as many copies of the transgene compared with the of E2F1 (and because of sequence similarity in this region endogenous TK gene, this is strong indication that within the probably also of E2F2 and E2F3) carries a binding site for limits of this assay, the promoter of the transgene is organized cyclin A (reviewed in reference 36). Whether the regions of identically to that of the TK gene. In the next experiment, we interaction with Sp1 and cyclin A overlap, with possible func- examined the effect of a mutation in either the Sp1 (Fig. 5D, tional consequences, or whether they are distinct has yet to be lanes 3 and 4) or the E2F (lanes 5 and 6) motif on binding of determined. the two transcription factors. The results clearly show that mutation of either binding motif almost totally eliminates pro- Within the Sp1 protein, the C-terminal part, containing re- moter occupancy. Lanes 4 and 6 reveal that the footprints at gion D, was found necessary for E2F binding. This region is not both sites were greatly reduced compared with the situation at required for Sp1 action from a single GC box but appears to be the wild-type promoter (lane 2). That they did not disappear involved in the formation of higher-molecular-weight Sp1 com- completely is due to the contribution of the promoter of the plexes at multiple consecutive GC boxes (53). Being one of the endogenous TK gene, which, of course, is wild type for both transcription factors for which binding sites exist in a great transcription factor binding sites. These data can be inter- variety of promoters, Sp1 was previously found to connect with preted to indicate that binding of Sp1 and E2F to the chroma- several other proteins involved in the regulation of transcrip- tin-organized TK promoter is cooperative. tion. For example, Sp1 was found to interact cooperatively with OTF-1 (27) and to bind other transcription regulators, notably the E2 protein of human papillomavirus (39), the abundant DISCUSSION transcription factor YY1 (61), and a negative regulator of Sp1 Promoters of genes which are regulated at the border of the transcriptional activity (46). Of particular interest is the recent

G1 to S phase of the cell cycle frequently carry binding motifs observation that Sp1 interacts with the erythroid transcription for E2F and for Sp1. The murine TK promoter is a simple factor GATA, whereby, like in the interaction with E2F de- example of such a promoter; it carries only a single Sp1 motif scribe here, the C terminus of Sp1, and in particular the zinc and the E2F site in the relevant part of the upstream region of finger domain, is involved (42). In TATA box-containing pro- the gene. It was found (references 11 and 50 and this work) moters, Sp1 appears to transmit a signal via the coactivators that both the Sp1 site and the E2F site are absolutely required TAF110 and TAF250 (23, 69) whereby at least the former pro- for promoter activity. Our findings suggest that Sp1 and E2F tein seems to bind directly to Sp1. interact with each other, and this inference is supported by The effect of mutations of the E2F site or the individual GC several observations: (i) E2F and Sp1 bound to each other in boxes in the dihydrofolate reductase promoter (6, 7, 20) like- solution, depending on particular regions of the two transcrip- wise suggest an interaction between E2F and Sp1 bound to the tion factors, (ii) the two proteins could be coimmunoprecipi- GC box nearest to the E2F site; direct evidence for this is tated from cell extracts, and (iii) increasing the distance be- reported in the accompanying paper (41a). In this case, evi- tween the binding sites for Sp1 and E2F resulted in a dence for a biological function of the E2F-Sp1 interaction was

FIG. 5. In vivo footprinting experiments, involving the Sp1 and the E2F site at the murine TK promoter, demonstrate constitutive occupation of both sites. (A) Sequence of relevant parts of the TK promoter. Sequences chosen for the synthesis of oligonucleotides for PCR as well as the two transcription factor binding sites are written with larger letters. (B) In vivo footprint of the lower strand of the endogenous TK promoter, i.e., the strand complementary to the one shown in panel A, using oligonucleotides (Prom1 to Prom3) for specific amplification of fragments obtained from cells growth arrested by serum withdrawal and those stimulated to grow by the addition of fresh serum for the times indicated. G residues as targets for methylation are indicated; asterisks mark residues which are masked and thus exhibit a footprint. (C) FACS analyses of cells used for the experiment shown in panel B, proving that cells were arrested at time zero and moved into S phase upon addition of serum. (D) In vivo footprint of the lower strand of the TK promoter in cells stably transfected with the TK promoter-CAT construct. For these experiments, cells were serum starved for 72 h, serum was added, and the cells were incubated for 16 h. As the oligonucleotides Prom1 to Prom3 are specific for the TK promoter, all of the lanes represent a mixture of endogenous and transfected promoters. It should be noted, however, that the copy number of the transgenes is at least twice that of the endogenous TK gene. Lanes 1 and 2 show the footprint obtained with the cell line carrying the transfected CAT construct under the wild-type promoter. Comparison of this footprint with that obtained for the endogenous TK gene only (B) indicates that endogenous and transfected wild-type promoters exhibit similar occupancy with respect to Sp1 and E2F. Lanes 3 and 4 show the footprint obtained with a cell line carrying a transfected construct in which the Sp1 motif was mutated. The footprints of both E2F and Sp1 were lost, despite the intactness of the E2F site. Lanes 5 and 6 show the complementary experiment with a promoter in which the E2F motif was mutated. Again both footprints disappeared, although the Sp1 site was wild type. The bands in lanes 4 and 6 are slightly weaker than those in the corresponding in vitro lanes because of the background of the endogenous wild-type promoter, whose sites are always protected. The mutations in the binding sites (indicated by the A’s in the sequences at the left and right of panel D) are not visible in the footprint because two C residues (which do not show up in the footprint) were mutated to A’s in case of the E2F site, while in case of the Sp1 site, a C residue and a G residue were mutated to A. In this case, the lost G is not visible because of band compression. 1666 KARLSEDER ET AL. MOL.CELL.BIOL. obtained by cotransfection experiments. Since E2F was also ACKNOWLEDGMENTS found to interact with TATA-binding protein (16), this tran- We are grateful to Rene Bernards, William G. Kaelin, Willy Krek, scription factor therefore appears to play a decisive role in the Jaqueline A. Lees, Nick B. La Thangue, and Robert Tjian for cDNA establishment of an active transcription complex at these or GST clones. We thank Christian Seiser for helpful discussions and TATA-less promoters. E2F may thus be considered a regu- Jane Azizkhan for communication of results prior to publication. J.K. lated tethering factor (55, 56) transmitting the activation signal is grateful to J. M. Blanchard and his colleagues in Montpellier for from Sp1 to TATA-binding protein in a growth- and cell cycle- instructions on in vivo footprinting. This work was supported by grants from the Fonds zur Fo¨rderung dependent manner. der wissenschaftlichen Forschung and the Austrian Ministry of Science Another point requiring consideration is our observation and Research. J.K. is grateful to EMBO for financial assistance that Sp1 and E2F are constitutively bound to their sites in the through a short-term fellowship. TK promoter. This is seen in the footprints of Fig. 5B, which The first two authors contributed equally to this work. are the same in arrested cells and in cells growth stimulated by serum addition for different times. A similar situation was REFERENCES found at the P2 promoter of the c- gene (54). It has re- 1. Adams, C. C., and J. L. Workman. 1995. Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative. Mol. Cell. Biol. cently been reported that during S phase, E2F1 associates with 15:1405–1421. cyclin A and cdk2 in complexes not containing p107. Through 2. Aubin, R. J., M. 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