©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Inverted Repeat Motif Stabilizes Binding of E2F and Enhances Transcription of the Dihydrofolate Reductase Gene (*)

Michael Wade (1) (2) (3), Michael C. Blake (2) (3)(§), Robert C. Jambou (2)(¶), Kristian Helin (4), Ed Harlow (4), Jane Clifford Azizkhan (1)(**)

From the (1) Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, the (2) UNC-Lineberger Comprehensive Cancer Center and (3) Curriculum in Genetics and Molecular Biology, the University of North Carolina, Chapel Hill, North Carolina 27599-7295, and the (4) Cancer Center, Massachusetts General Hospital, Charlestown, Massachusetts 92129

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular and viral genes. This element, 5`-TTTCGCGCCAAA-3`, is comprised of two overlapping, oppositely oriented sites which match the consensus E2F site (5`-TTT(C/G)(C/G)CGC-3`). Recent work has shown that E2F binding activity is composed of at least six related cellular polypeptides which are capable of forming DNA-binding homo- and heterodimers. We have investigated the binding of cellular E2F activity and of homo- and heterodimers of cloned E2F proteins to the inverted repeat E2F element. We have demonstrated that mutations in this element that abolish its inverted repeat nature, while preserving a single consensus E2F site, significantly decrease the binding stability of all of the forms of E2F tested. The rate of association of E2F-1/DP-1 heterodimers with the inverted repeat wild type site was not significantly different from those with the two single site mutated probes. Furthermore, the mutations decrease in vitro transcription and transient reporter gene expression 2-5-fold, an effect equivalent to that of abolishing E2F binding altogether. These data suggest a functional role that may explain the conservation of inverted repeat E2F elements among the DHFR promoters and several other cellular and viral promoters.


INTRODUCTION

E2F is a eukaryotic transcription factor that binds to the consensus sequence element 5`-TTT(C/G)(C/G)CGC-3`. It was first discovered as a protein in adenovirus-infected HeLa cells that binds to and regulates transcription from the adenovirus E2a early promoter (1, 2, 3, 4, 5) . DHFR() and c- myc were the first cellular genes shown to be subject to regulation through E2F (6, 7, 8) . More than a dozen cellular and viral genes have known or potential E2F-binding sequence elements in their promoter regions (9, 10, 11, 12, 13, 14) . A link between E2F and growth control has been suggested by a large number of studies demonstrating the direct binding of E2F to proteins implicated in the regulation of cellular growth control, including cyclins and cyclin-dependent kinases, as well as tumor suppressor-like proteins which can repress transcription through an E2F-binding site (11, 14, 15) .

Recent data show that the E2F binding activity in mammalian cells consists of a family of proteins which bind DNA as both homo- and heterodimers. Three groups independently cloned a protein termed E2F-1 (16, 17, 18) , and closely related proteins, E2F-2 and E2F-3, have also been characterized (19, 20) . E2F-4 has also been recently cloned and characterized (59) . Additionally, the proteins DP-1, DP-2, and DP-3 have also been shown to be a part of cellular E2F binding activities (14, 21, 22, 23) . Each of these cloned E2F polypeptides contain amino acid sequences that potentially mediate dimerization, and E2F-1/DP-1 heterodimerization has been directly demonstrated (14, 17, 18, 19, 20, 22, 24) . Interactions between E2F family members appears to contribute to regulation of gene expression since E2F-1 and DP-1 cooperatively activate an E2F-responsive promoter (23, 24, 25) , and E2F proteins may differ in their ability to bind to the Rb and Rb-related p107 tumor suppressor proteins (19, 24, 26, 59) .

Binding affinity could represent an important way in which expression of E2F-regulated genes is modulated. This notion is supported by the unique example of the adenovirus E2 promoter, which contains two oppositely oriented E2F sites spaced 16 base pairs apart (1, 2, 3, 4, 5) . Both the spacing and orientation of these sites were found to be critical for proper control of this promoter by the product of the adenovirus E1A immediate early gene (28) . E1A dissociates E2F from other cellular proteins and allows it to bind the 19-kDa E4 gene product, which appears to stabilize cooperative binding of E2F molecules to the two sites in the E2 promoter (29, 30, 31, 32, 33, 34, 35, 36, 37, 38) . E2F binding to the E2 promoter was shown to be very unstable in the absence of E4 protein, and E4 did not stabilize E2F binding to a single E2F site (28, 39) .

We have investigated the role of a conserved overlapping inverted repeat E2F sequence motif found in the promoters of the mammalian DHFR genes (40, 41, 42, 43, 44) . Studies have shown that E2F is required for efficient basal transcription of the DHFR gene (8) , for cell cycle-regulated activation (44) , and/or repression() of DHFR, and for adenovirus- and human cytomegalovirus-mediated activation of the gene (10, 45) . In one of these previous reports, we suggested that the E2F sequence found near the major transcription start site of the three characterized mammalian DHFR genes, 5`-TTTCGCGCCAAA-3`, was a ``full site'' for optimal E2F binding and function, and that the sites found in the E2 promoter, 5`-TTTCGCGC-3`, represented a less stable site for binding of a dimeric transcription factor (10) . A functional role for inverted repeat E2F elements is further supported by the presence of identical or similar sequence elements in a number of other genes (Fig. 1 C) and by two studies which show that inverted repeat E2F elements are selected from among populations of random oligonucleotides by Rb-binding proteins (46, 47) .


Figure 1: A, control elements in the hamster DHFR promoter. The open boxes are binding sites for the transcription factor Sp1. The hatched boxes are structural control elements (12). The thin arrow represents the minor transcription start site at position 107 relative to the ATG translation initiation codon at +1. The bold arrow represents the major transcription start site cluster at positions 63, 64, and 66. The overlapping inverted repeat E2F site at position 62 to 51 is depicted by the shaded boxes. The promoter construct used for functional studies contained all of the sequence represented here, except for mutations in the E2F element as indicated. Promoter fragments used in binding studies contained only the wild type or mutated E2F element. B, sequences of wild type and mutant DHFR E2F sites used in binding and functional studies. C, examples of inverted repeat E2F sites in mammalian and viral genes and sequences selected from degenerate oligonucleotides by Rb-associated proteins for subsequent polymerase chain reaction amplification (see text for references).



This paper reports the effect of mutations in the DHFR inverted repeat E2F sequence element on the binding stability of cellular form(s) of E2F and defined homo- and heterodimers of cloned E2F proteins. Because this element consists of two overlapping, oppositely oriented high affinity E2F binding sequences, we were able to use site-specific mutagenesis to destroy the inverted repeat while leaving intact one or the other of the two E2F sites within the element. We demonstrate that all the forms of E2F dimers tested bind significantly more stably in vitro to the inverted repeat sequence than to ``single'' E2F sites that do not possess inverted symmetry. We also show that the rates of association are the same among the three sites. Finally, altering the inverted repeat results in a 2-5-fold decrease in transcription from the DHFR promoter, equivalent to the functional consequence of mutationally abolishing E2F binding (8) . These data support the hypothesis that the inverted repeat overlapping E2F-binding sites in the DHFR and other promoters represents a novel means by which E2F binding and gene transcription are modulated.


MATERIALS AND METHODS

Plasmids and Oligonucleotides

The wild-type and E2F double site mutant DHFR-CAT reporter plasmids have been described previously (44) . Construction of the E2F Site 1 and Site 2 mutants also has been described (10, 45) . Plasmids containing wild type and single site mutant DHFR E2F site promoter fragments (restricted to remove the upstream Sp1 sites) and the synthetic oligonucleotide corresponding to the DHFR wild type E2F site also have been described previously (45) . cDNA encoding E2F-1 amino acids 88-437 was cloned into the pBSK vector (Stratagene) by polymerase chain reaction amplification using the p121 primer previously described to produce the pBSK-E2F1-121 construct (16) . A plasmid encoding DP-1, pGC, was a kind gift of R. Girling and N. B. LaThangue (22) .

Cell Culture and Nuclear Extract Preparation

HeLa S-3 cells were grown in Joklik's modified minimal essential media (Life Technologies, Inc.) supplemented with 5% fetal calf serum in suspension at a concentration of 4-8 10cells/ml with daily 1:2 expansion of cells. Transcriptionally competent nuclear extracts were prepared as described previously (8, 48) .

Transient Transfection Assay

HeLa, Chinese hamster ovary and Balb/c3T3 cells were transfected with the appropriate DHFR-CAT reporter plasmids by the calcium phosphate method (49) and assayed for CAT activity as described previously (8, 50) . CAT activity was quantitated by liquid scintillation counting of spots cut from thin layer chromatography plates.

In Vitro Transcription and Translation-Run-off transcription of DHFR-CAT constructs and analysis of start sites by primer extension assay was performed as described previously (51) . Transcription products were quantitated by laser densitometry of autoradiographs. For production of mRNA encoding E2F-1 protein fragments, pBSK-E2F1-121 (described above) was digested to completion with either BamHI (for amino acids 88-437) or PvuI (amino acids 88-292), phenol-chloroform extracted twice, ethanol precipitated, resuspended in diethylpyrocarbonate-treated water and incubated with T7 RNA polymerase (Promega) in standard transcription reactions essentially as recommended by the manufacturer. The RNA was phenol-chloroform extracted twice, precipitated, resuspended in diethylpyrocarbonate-treated water, and added directly to in vitro translation reaction mixtures. RNA was added to 40 µl of rabbit reticulocyte lysate (Promega) in the presence of RNase inhibitor (40 units of RNasin, Promega) and amino acids (1 mM each), in a total reaction volume of 50 µl. Where indicated, the pBSK-E2F-121, pGC or a control luciferase-encoding plasmid were added directly to a coupled transcription/translation system (TNT, Promega) as recommended by the manufacturer. To prepare S-labeled proteins for denaturing (sodium dodecyl sulfate) polyacrylamide gel analysis, translation or transcription-translation reactions were performed in the presence of methionine-minus amino acid mixtures, and[S]methionine was added.

Gel Mobility Shift Assays

For the analysis of E2FDNA complexes formed in the presence of HeLa nuclear extract, gel mobility shift reaction were performed essentially as described previously (8) . For off-rate experiments, 40 µl of reaction mixtures were equilibrated on ice in the presence of 12 µg of nuclear extract. DHFR wild type or mutant promoter fragment probes were fill-in labeled by incubation with the Klenow fragment of DNA polymerase I and [P]dATP; 40,000 cpm (0.2-0.4 ng) was added to each reaction mixture, and binding reactions were incubated for 20 min at room temperature. (Longer incubation times of up to 1 h had no effect on the intensity or mobility of E2F complexes.) The reaction mixtures were then mixed with 3 µl of loading dye (50% glycerol, 0.5 Tris borate (TBE) buffer, 0.0001% xylene cyanol, 0.0001% bromphenol blue) and transferred to a 4 °C cold room. A 6-µl aliquot was immediately removed and applied to a 4% native polyacrylamide gel that was already running at 100 V in a 4 °C cold room (zero time point). Unlabeled competitor oligonucleotide (40 ng) was added to the remaining reaction mixture with thorough mixing by repeated pipetting, and aliquots were removed and loaded onto the running gel at the indicated time points. Addition of probe to each reaction mixture was timed so that each probe was incubated with nuclear extract for 20 min before competitor oligonucleotide was added. Gels were run at 250 V for 1.5-2 h after the loading dyes had migrated into the gel.

For the E2F-1 and DP-1 gel shift experiments, programmed rabbit reticulocyte lysates were added to reactions instead of nuclear extract, and the reactions were done in the presence of 1.5% Nonidet P-40 and 3 mg/ml of bovine serum albumin. A labeled E2F site oligonucleotide probe (see above) was used as the probe in the cotranslation experiments depicted in Fig. 3. For the off-rate experiments, the amount of labeled wild type or single site mutant promoter fragment probe DNA was increased to 80,000 cpm (0.2-0.4 ng), and the amount of competitor oligonucleotide to 80 ng. For the E2F-1 experiments, five µl of programmed lysate (TNT, Promega) was added to the reaction mixture; for the E2F-1/DP-1 experiments, 1 µl of lysate was added and the amount of cold competitor oligonucleotide increased to 400 ng. Native polyacrylamide gels (6% for E2F-1 and 4% for E2F-1/DP-1) were run at 250-400 V for 4 h for E2F-1 and 2 h for E2F-1/DP-1. Otherwise, the off-rate experiments were performed exactly as described above.


Figure 3: Analysis of E2F-1 binding activity. A, reducing 8% SDS-polyacrylamide gel electrophoresis analysis of in vitro translated E2F-1 protein fragments. Lane 1, E2F-1 amino acids 88-437. Lane 2, E2F-1 amino acids 88-292. The migration of molecular weight markers (in kDa) run in parallel is shown at the left. B, gel mobility shift assay of showing dimerization of in vitro translated unlabeled proteins using a labeled oligonucleotide consisting of the wild type DHFR E2F site as a probe. Aliquots of in vitro translation reaction mixtures were added directly to gel shift reaction mixtures. mRNAs encoding E2F-1 (88-292) ( lane 1), E2F-1 (88-437) ( lane 3), or both mRNAs ( lane 2) were added to the in vitro translation reactions, which were then analyzed by gel shift. Non-denaturing 6% gels were run at 300 V for 4 h to adequately separate the three different E2F-1 binding activities from one another and from the lysate. Lane 4 shows gel shift analysis of a control in vitro translation reaction to which brome mosaic virus RNA (1 µg) was added, i.e. the endogenous E2F binding activity in the rabbit reticulocyte lysate. The arrows show the migration of proteinDNA complexes. As indicated, the lysate binding activity is seen in all the samples. The bold arrow in the position intermediate between the two protein fragments depicts the migration of a complex consisting of an E2F-1 dimer composed of protein fragments of different sizes in lane 2. The lysates in which only a single mRNA was translated (those analyzed in lanes 1 and 3) were programmed with 2 µl each out of 10 µl mRNA recovered from the in vitro transcription reaction mixtures. The lysate analyzed in lane 2 was programmed with 1 µl of E2F-1 (88-437) and 1 µl of E2F-1 (88-292) mRNA. C, the half-life of binding of E2F-1 to wild type, Site 1 mutant, and Site 2 mutant DHFR E2F site promoter fragment probes. Gel shift reactions using lysates programmed with E2F-1 were subjected to off-rate analysis by addition of unlabeled E2F DNA-binding site as described for Fig. 2. Densitometry analysis of the E2F-1 complex was used to derive a half-life of binding to the three probes as described under ``Materials and Methods'' and in the legend to Fig. 2; the average half-life of the E2F-1 complex bound to each of the probes in the presence of unlabeled competitor oligonucleotides is shown. The error bars depict standard error, n = 3.



The association rate for E2F-1/DP-1 heterodimers was measured in three separate experiments. Probes consisted of end-labeled DNA fragments (1.5 nmol DNA, 10cpm/µg) containing the inverted repeat E2F site or each of the single E2F sites as used in the off-rate experiments; all probes had the same specific activity. Reactions (80 µl) were initiated with each of the three probes by the addition of 2 µl of reticulocyte lysate programmed with E2F-1 and DP-1 in vitro transcribed mRNA, and aliquots were removed and applied to running gels at 30-s intervals for 3 min.

Computer densitometry analysis of video-captured autoradiographs was performed using the Image program (NIMH Research Services Branch). The half-life of E2F-1-DP-1 complex and the rates of association were determined by direct quantitation of radioactivity in bands on dried gels using a Molecular Dynamics PhosphorImager system.


RESULTS

The 12-nucleotide inverted repeat E2F sequence near the major transcription start site is 100% conserved among the DHFR promoters in mouse, hamster ( shaded boxes, Fig. 1A), and human (40, 41, 42, 43, 44) . Additionally, inspection of E2F sequences in a number of other gene promoters, including the E1A promoters of at least eight serotypes of adenovirus and seven different cellular promoters, including both the human and mouse E2F-1 promoters (12, for review; 52, 53), reveals inverted repeat-like sequences (Fig. 1 C). Finally, in two studies several E2F sites selected from among random oligonucleotide populations by proteins associated with Rb have inverted repeat character (Fig. 1 C; 46, 47). Therefore, we set out to determine whether the DHFR inverted repeat sequence conferred any changes in E2F binding relative to binding to sequences that contained only a single site. Using site-directed mutagenesis, we created mutations in the DHFR E2F site that allowed us to abolish either the 5` site or the 3` site separately; each of the these mutations preserved only a single site. The sites that remain (5`-GCGCCAAA-3` in the Site 1 mutant and 5`-TTTCGCGC-3` in the Site 2 mutant) each represent single consensus E2F sites. An additional mutation at the center of the inverted repeat (the double site mutant) abolished both E2F sequences (Fig. 1 B); this double site mutation completely eliminates E2F binding activity (8) . E2F binding to the single site mutant probes produced gel mobility shift complexes indistinguishable in mobility from those produced by binding to the wild type inverted repeat element (Fig. 2, lanes 1, 6, and 10). Extensive investigation of the adenovirus E2 promoter, which consists of two oppositely oriented E2F sites, had established that extracts from adenovirus-infected cells gave a slower mobility gel shift complex characteristic of simultaneous binding of E2F molecules to two separate sites (29, 30, 31, 32, 33, 34, 35, 36, 37, 38) . However, titration experiments showed that E2F complexes with identical gel mobility formed with the wild type and single site mutant DHFR E2F sites even at high protein concentration (data not shown).


Figure 2: Off-rate assay of HeLa cell E2F binding activity. A, a representative autoradiograph showing the effect of addition of unlabeled competitor oligonucleotides on E2F binding to labeled wild type E2F site, Site 1 mutant, or Site 2 mutant promoter fragment probes. Competitor oligonucleotide (100-fold molar excess) was added ( t = 0), and aliquots of gel mobility shift reaction mixtures were removed at the indicated times after the addition ( min.) and immediately applied to a continuously running gel. The major E2F band is indicated by the arrow. The dark band just above the E2F band represents nonspecific DNA binding by extract protein. The free probe is the wide band at the bottom of the gel. B, the half-life of E2F binding activity in the presence of cold competitor oligonucleotides. The half-life of binding for off-rate experiments was computed by plotting the disappearance of the major E2F band as measured by densitometry analysis of autoradiographs of gels. The graph depicts the average half-life value for the major E2F band. The error bars represent standard error, n = 3.



Having demonstrated that the inverted repeat sequence apparently did not serve as two independent sites, we undertook a series of experiments to determine whether it enhanced the stability of E2F binding relative to binding to a single E2F site. E2F in HeLa cell extracts was allowed to bind to radiolabeled promoter fragment probes containing the wild type, Site 1 mutant or Site 2 mutant E2F sites (Fig. 1 B). The binding reaction was allowed to reach equilibrium (15-30 min at room temperature), and an excess of a competitor oligonucleotide consisting of the wild type DHFR E2F site was added to the reaction mixture. Aliquots of the mixture were then applied to a gel that was already running at time points after addition of the competitor oligonucleotide.

Initial results confirmed earlier studies of the E2 promoter (28, 34) indicating that E2F binding to DNA in vitro is unusually unstable: binding of HeLa cell E2F to all three of the probes used in our study was virtually abolished within 5 min of addition of the competitor. However, our more detailed examination of binding revealed that the DHFR inverted repeat site conferred a significant increase in binding stability. In three separate experiments, the half-life of HeLa cell E2F binding to the inverted repeat sequence was found to be an average of 35.3 s. The half-life of binding to the Site 1 mutant sequence was 18 s, while the half-life of binding to the Site 2 sequence was 19 s (Fig. 2). Furthermore, the single site mutant sequences compete for binding to the wild type probe (data not shown), indicating that the the same factor(s) binds all three sites. The amount of binding to the three probes in this experiment does not appear to be significantly different. In contrast, in the experiment presented in Fig. 5, the amount of E2F bound at equilibrium is different (see Fig. 5 legend) between the wild type and mutant probes under the conditions of that experiment. The difference in these results is due to differences in the concentrations of probe and E2F in the binding reactions; probe is clearly in at least 5-fold excess in the binding reactions presented in Fig. 2, whereas, probe is limiting in the binding conditions of the experiment shown in Fig. 5. With probe in excess, the dissociation rate difference does not result in a different amount of binding at equilibrium because the reaction is driven by the rate of association.


Figure 5: E2F-1 and DP1 were cotranslated as described for Fig. 5. At time 0, a binding reaction was started by the addition of DNA probe (wild type or single site mutant). At the times indicated, an aliquot was removed from the reaction and loaded onto a running polyacrylamide gel. The gel was subjected to analysis by a Molecular Dynamics Phosphorimager to obtain quantitative output. The ratio of the amount of protein bound at each time point ( B) to the amount bound at equilibrium ( B) (3 min) is plotted. The experiment was repeated three times, and a representative experiment is shown. The amount of binding at equilibrium to the three probes is different. In these experiments, binding at equilibrium, expressed as the ratio of the counts bound over the total counts and averaged for three separate experiments is: 0.0285 for the wild type probe, 0.0145 for the site 1 mutant probe, and 0.014 for the site 2 mutant probe.



The cloning of the E2F-1 protein (16, 17, 18) allowed a direct test of E2F dimerization. Using in vitro transcription of E2F-1 cDNA, we synthesized run-off RNA transcripts encoding either E2F-1 amino acids 88-437 or 88-292. These transcripts were then translated separately or together in rabbit reticulocyte lysates, and the resulting protein products, which migrated in a denaturing gel at approximately 45 and 16 kDa, respectively (Fig. 3 A), were analyzed by gel mobility shift analysis using the wild type DHFR E2F site as a probe.

The results of these experiments are shown in Fig. 3B. The protein fragments encoded by these RNA transcripts produced distinct gel shift complexes that could be distinguished from the endogenous E2F binding activity characteristic of reticulocyte lysates (45, 54; Fig. 3 B, lane 4 versus lanes 1 and 3). When the different-sized E2F-1 transcripts were translated simultaneously, a new gel shift complex of intermediate mobility was observed, in addition to those seen with 88-437 or 88-292 alone (Fig. 3 B, lane 2, indicated by bold arrow). This complex most likely results from a dimer consisting of one molecule of each of the two different-sized fragments of E2F-1. There is an extra band in lane 2 which is the result of persistence of a portion of the larger E2F after homodimer formation between the large and small forms. It should also be noted that the E2F binding activity native to reticulocyte lysates is more active than that of the E2F-1 homodimers.

E2F-1 DNA binding was analyzed further in off-rate experiments similar to those described above for HeLa cell E2F activity. Off-rate analysis of the gel mobility shift complex consisting of the E2F-1 (88-437) homodimer (Fig. 3 B) demonstrated that E2F-1 homodimers also bind significantly more stably to the DHFR inverted repeat site than to the single sites. The half-life of binding to the wild type site in three experiments averaged 222 s, compared to 67 s for the Site 1 mutant and 83 s for the Site 2 mutant (Fig. 3 C), suggesting that the inverted repeat sequence confers additional stability on the DNA binding of a defined homodimeric form of E2F-1. It should be noted that while the binding stability difference is significant, the much longer absolute binding half-lives of the complexes bound to each of the three probes (relative to those observed with the HeLa cell E2F activity, Fig. 2 ) probably reflects different binding conditions related to the relatively large amount of reticulocyte lysate required to visualize the E2F-1 homodimers, which bind to DNA much lessavidly than the ``native'' and probably heterodimeric (see below) E2F activity in the lysate or in cellular extracts. As shown in Fig. 3 B, the complex that is due to E2F in the lysate is the slowest mobility band in these gel shifts, and its binding parallels that of the E2F-1 homodimer on the three probes, supporting the notion that binding conditions affect the absolute measurement of the tbut do not affect the relative differences in stability between binding sites.

With the availability of cloned DP-1, we addressed whether heterodimers of E2F-1/DP-1, produced by cotranslation, also displayed enhanced binding to the DHFR inverted repeat E2F sequence. A coupled in vitro transcription-translation system was used to produce E2F-1 and DP-1 proteins in rabbit reticulocyte lysates. The production of proteins was monitored by incorporation of [S]methionine into aliquots of the reaction mixtures and visualized by SDS-polyacrylamide gel electrophoresis followed by autoradiography as shown in Fig. 4 A. A plasmid encoding full-length DP-1 produced a protein that migrated at approximately 60 kDa, while a plasmid encoding the C-terminal 348 amino acids of E2F-1 produced a protein that migrated as a doublet of approximately 45 kDa (Fig. 4 A). The wild type DHFR inverted repeat E2F sequence was used as a probe to analyze the DNA binding characteristics of these proteins by gel mobility shift (Fig. 4 B). The endogenous E2F binding activity contained within the lysate produces three bands in a gel mobility shift ( lane 2). When E2F-1 was translated, an additional band of slightly faster mobility was observed ( lane 3); when DP-1 was translated, no additional bands over those seen with the control lysate were seen ( lane 4), indicating that DP-1 alone binds very weakly to the E2F sites. When E2F-1 and DP-1 were cotranslated, a novel complex of faster mobility than that of the lysate E2F was observed ( lane 5, heterodimer indicated by the arrow). This complex was the strongest signal seen in the three lanes, indicating that heterodimer binding to the E2F site is stronger than that of homodimer. The distinct gel mobility shift complex produced by E2F-1/DP-1 heterodimers was subjected to off-rate analysis. A representative experiment is shown in Fig. 4 C; due to the shorter exposure of the gel and less material/lane as compared to the gel in panel B, complexes resulting from endogenous E2F activity in the lysate are only weakly visible and the only prominent complex is that of the E2F-1/DP-1 heterodimer. The average of five experiments is shown in Fig. 4 D; an E2F-1/DP-1 heterodimer dissociates from the inverted repeat site with an average half-life of 43.9 s in the presence of excess competitor oligonucleotide, compared to an average half-life of 21 s for binding to the Site 1 mutant and 22.1 s for binding to the Site 2 mutant. The data demonstrate that E2F-1 and DP-1 heterodimers, like the predominant E2F binding activity in extracts from HeLa cells (Fig. 2) and E2F-1 homodimers (Fig. 3), bind with optimal stability to an inverted repeat sequence, relative to binding to a single E2F site.


Figure 4: Analysis of specific E2F-1/DP-1 DNA binding activity. A. radiolabeled proteins produced in coupled in vitro transcription-translation reactions were resolved on an 8% SDS-polyacrylamide gel electrophoresis gel under reducing conditions. Lane 1, luciferase control protein. Lane 2, E2F-1 amino acids 88-437. Lane 3, DP-1. Lane 4, E2F-1 and DP-1. The migration of molecular mass markers in kDa is shown at the left. B, gel shifts. The transcription-translation reactions were performed as for panel A in the presence of unlabeled amino acids. The reticulocyte lysates were programmed with RNA encoding E2F-1 ( lane 3), DP-1 ( lane 4), or E2F-1 and DP-1 ( lane 5) and were incubated with labeled wild type DHFR E2F site probes gel in mobility shift reactions. In lanes 6 and 7, 400 ng of an oligonucleotide corresponding to the wild type DHFR E2F site was added to the reaction mixtures analyzed in lanes 3 and 5, respectively; in lanes 8 and 9, 400 ng of an oligonucleotide corresponding to the double site mutant DHFR E2F site was added to the reaction mixtures analyzed in lanes 3 and 5, respectively. C, off-rate analysis of E2F-1/DP-1 DNA binding activity. One µl of the E2F-1/DP-1 lysate was incubated with the wild type, Site 1 mutant, or Site 2 mutant DHFR E2F site probes in a 40-µl reaction mixture, and aliquots of the mixture were applied to a running gel at the indicated time points as in Fig. 2 after addition of competitor oligonucleotide (400 ng). The predominant DNA binding activity is a single band from the E2F-1/DP-1 heterodimer which is different from that observed in lane 5 of panel C because one-fifth of the reaction was loaded on the gel and the exposure is shorter. D, bands corresponding to the E2FDP-1 complex as shown in the autoradiography in panel C were directly counted using a Molecular Dynamics Phosphorimager system, and the radioactivity of each complex measured to derive a half-life of binding to each of the three probes, as in Figs. 2 and 3. The average half-life of E2F-1/DP-1 binding to each of the three probes is shown. Error bars depict standard error, n = 5.



To address whether the different E2F sites have different affinities for E2F, the rates of association of E2F-1/DP-1 to the same sites were measured. Binding reactions were initiated by addition of probe, and aliquots were removed at 30-s intervals and immediately applied to a gel that was already running. Equilibrium binding was achieved by 2.5 min. Data at each time point were expressed as the amount bound over the amount bound at equilibrium (B/B) (Fig. 5). This experiment was repeated three times and no significant difference was observed in B/Bamong the probes at any time point. The amount of binding at equilibrium was different; the wild type sequence bound approximately twice as much protein as did the two single site mutants. These data support the hypothesis that there is little difference between the rate of association and that the difference in affinity between the inverted repeat site and the single E2F site is due to the different rates of dissociation.

Finally, we tested the effects of our Site 1 and Site 2 mutations that reduced E2F binding stability in in vitro transcription and reporter gene expression assays. Fig. 6, A and B show in vitro transcription analysis in a HeLa cell extract of wild type and mutant DHFR promoter constructs driving expression of the bacterial CAT gene. This experiment showed that the effect on in vitro transcription of the Site 1 and Site 2 mutations is roughly the same as the effect of a double site mutation that completely abolishes E2F binding; in all three mutants, transcription was reduced approximately 5-fold. Therefore, two separate mutations which leave intact single E2F sites, and which clearly allow E2F binding, have virtually the same effect on basal transcription of the DHFR gene as a double site mutation, which completely abolished detectable E2F binding.


Figure 6: The functional effects of mutations abolishing either one or both of the E2F sites in the DHFR inverted repeat motif. A, denaturing gel analysis of the products of in vitro transcription reactions using HeLa nuclear extract and CAT reporter gene templates under the control of the wild type DHFR promoter or the same templates bearing Site 1, Site 2, or the E2F double site mutations. A 494-base Sp6 transcript was included as an internal control for sample recovery and is indicated by an asterisk in the figure. The amount of DHFR major and minor transcripts were quantitated in three separate experiments by laser densitometry of the autoradiograms; the difference in transcript produced by the three mutants as compared with the wild type construct was 4.9 ± 0.83-fold. B, in vitro transcription products assayed by primer extension to map initiation sites. The transcription products analyzed are in the same order as shown in panel A, and were electrophoresed alongside a sequencing ladder run in parallel, which was used to map the start site positions. For primer extension, in vitro transcription reactions were performed in the presence of unlabeled ribonucleotides, and the resulting transcripts were subjected to reverse transcription in the presence of [P]-ATP-labeled primer. One-third of the radiolabeled DNA resulting from the wild type template was analyzed on the gel, while all of the radiolabeled DNA resulting from the mutant templates was analyzed. C, the promoter-CAT constructs that were run-off transcribed in the experiments shown in panels A and B were transfected into HeLa cells by calcium phosphate coprecipitation, and CAT activity in cell lysates was analyzed by measuring acetylation of [C]chloramphenicol after 48 h. An autoradiograph of a representative thin layer chromatography plate is shown. The experiment was repeated four times, with quantitation by direct liquid scintillation counting of plates, and the range of activity of the double-stranded and single-stranded mutants was 2-5-fold less than the wild type. The three mutants were not significantly different from one another ( p < 0.05), but were significantly from the wild type ( p < 0.01).



The reduced efficiency of the single E2F site was confirmed by the CAT assay shown in Fig. 6 C. The same promoter-CAT constructs used for the in vitro transcription analysis in Fig. 6A were transfected into HeLa cells, and CAT gene expression was monitored by thin layer chromatography of [C]chloramphenicol converted to the acetylated form by the expressed CAT gene product. The assay demonstrates that CAT gene expression was reduced by approximately equivalent amounts by the Site 1, Site 2, and double site mutations in the inverted repeat E2F site, relative to expression driven by the wild type promoter. Within each of four separate transfection experiments, the CAT activities of the mutated promoters (Site 1, Site 2, and double site) were not significantly different from one another ( p < 0.05) but were significantly less than the wild type ( p < 0.01), The difference observed was 2-5-fold reduction in CAT activity from the mutants as compared to the wild type construct. Very similar results were obtained in Chinese hamster ovary and Balb/c 3T3 cells (data not shown). The relatively wide range of difference is likely a reflection of variability in the growth state or confluence of the transfected cells in spite of efforts to plate exactly the same number of cells in each experiment; the plating efficiency and degree of dispersion of the cells will certainly affect growth state. The effect of E2F on transcription varies according to the growth state of the cell. If a cell is in late log phase, E2F acts as a repressor, whereas if in mid-log cells, E2F stimulates transcription.() In a 48-h experiment, some of the cells may be reaching late log in some areas of the tissue culture dish, which would abrogate the stimulatory effect of E2F on DHFR transcription and reduce the difference between the mutant and wild type constructs.

Because the inverted repeat E2F sequence is located just 3` of the major transcription start site of the hamster DHFR promoter, we also tested whether the mutations we created in the sequence affected the start site. The transcripts produced in the in vitro transcription assay were subjected to primer extension analysis. The results showed that none of the mutations introduced in the inverted repeat E2F-binding site affected the nucleotides at which transcription was initiated. As with the wild type construct (51) , approximately 80% of the transcripts originated from a major start site cluster at positions 63, 64, and 66, while approximately 20% originated from the minor initiation site at 107 for each of the mutated promoters (Fig. 6 B). We conclude, therefore, that neither completely abolishing binding, reducing the stability of E2F binding to this site, nor eliminating its palindromic nature affects the specificity of transcription initiation, but instead affects the efficiency of transcription from the DHFR promoter. E2F binding at this sequence thus appears to function to enhance the efficiency of transcription, not to influence the selection of the start sites.


DISCUSSION

In the promoters of three mammalian DHFR genes, the inverted repeat sequence 5`-TTTCGCGCCAAA-3` is absolutely conserved. As shown in Fig. 1C, similar or identical sequences are found in the E1A promoters of at least eight serotypes of adenovirus and in several cellular gene promoters, including those of the human and mouse E2F-1 genes (52, 53) . This sequence conservation indicates the functional significance of the inverted repeat E2F binding sequence. We demonstrate in this report that this element confers significantly enhanced stability on the binding of the cellular transcription factor E2F, relative to binding to slightly shorter sequences which match the consensus E2F-binding site (5`-TTT(C/G)C(/G)CGC-3`). This finding applies to HeLa cell E2F binding activity, as well as to homo- and heterodimers of cloned E2F proteins expressed in vitro. Interestingly, inverted repeat-like E2F sequences also are selected from among random populations of oligonucleotides by Rb-associated proteins (46, 47) . Since Rb binds E2F, this indicates that these sequences confer the most stable DNA binding for E2F.

Previous studies of the adenovirus E2 promoter have strongly suggested that enhancing the stability of E2F binding can provide a means of activating gene expression. During adenovirus infection, the adenovirus E4 ORF 6/7 protein acts as a molecular clamp to stabilize the binding of E2F heterodimers to separate sites, an effect that is correlated with enhanced E2 gene expression (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) . Interaction with the E4 protein can increase the half-life of E2F binding to sites in this particular configuration from a period of less than 5 min to at least 30 min (28, 34) . In this study, we have shown that the overlapping inverted repeat E2F sites, which are found in the DHFR and many other cellular and viral promoters, provides a more stable binding site for E2F homo- and heterodimers than the single E2F sites found in many other promoters. The difference in affinity is due to the difference in dissociation rate since the association rate is the same. While the stability difference conferred in vitro on E2F binding by the inverted repeat sequence investigated in this report is much more subtle than that in the E2 gene in adenovirus infected cells, the functional effects of mutations altering the repeat structure are significant. At the level of basal transcription, the inverted repeat sequence element is essential for optimal DHFR gene expression. Mutations which merely abolish the inverted repeat but leave intact either of the two overlapping consensus E2F sites in this element have virtually the same functional effect as a mutation that completely eliminates E2F binding.

Neither the findings presented here nor the previous studies of E2 promoter activation necessarily imply that E2F binds to promoters with a half-life of less than 5 min in vivo. Interactions among E2F and other factors on gene promoters very likely would serve to enhance E2F's binding stability. Not unexpectedly, we found that by varying the experimental conditions, such as the total protein concentration in the binding reactions, we could influence the measured half-life of binding, which probably explains the longer overall half-life of binding by the E2F-1 homodimers (and lysate under these conditions) to all three sites as shown in Fig. 3 C. However, we suggest that it is the difference in binding stability between the inverted repeat sequence and the shorter consensus sequences preserved by our Site 1 and Site 2 mutant promoters that is significant; it is reasonable to argue that a conserved sequence element which can effectively double the stability of binding of a transcription factor and apparently increase its ability to activate transcription in vitro could confer the same relative effects in vivo. This is the first study to implicate sequence-related differences in E2F binding stability as a mode of differential gene regulation. Given the considerable variability in the sequences of reported and proposed E2F sites (9, 10, 12, 27) , it will be interesting to determine whether sequence variations contribute to the range of E2F's regulatory capabilities. Expression clones of additional forms of E2F will facilitate such studies.

The DHFR inverted repeat E2F sequence is located immediately 3` of the major transcription initiation site in the hamster and human DHFR promoter (51; Fig. 6B). In most E2F-regulated promoters, the E2F sequences act from a position much farther 5` of the transcription start site(s) (12) . In studies of the mouse DHFR promoter in which the entire initiation region has been deleted or subjected to multiple point mutations, the initiation site can be changed (27, 55) ; however, our study indicates that point mutations that either abolish E2F binding or diminish its stability affect only the efficiency of transcription, not the sites of initiation (Fig. 6 C). This finding, as well as a recent study in which wild type and mutant DHFR initiation region sequences were placed in a heterologous promoter background, suggests that E2F itself does not act as a DNA-binding ``initiator'' protein (56, 57) .

Our footprinting studies of the DHFR inverted repeat E2F sequence element show that the entire region is somewhat resistant to chemical or enzymatic cleavage even in the absence of cellular proteins and that E2F binding creates a distinct footprint that is virtually identical on the wild type, Site 1 mutant, and Site 2 mutant promoters (8 and data not shown). Resistance to DNA-cleaving agents suggests the possibility that the inverted repeat element can form unusual structures. Interestingly, recent work has shown that binding of E2F-1 and HeLa cell E2F produces pronounced DNA bending with a flexure angle of more than 125° (58) . The inverted repeat sequence may favorably influence such bending. Alternatively, the inverted repeat motif, by providing a consensus sequence element on each side of the DNA helix, could provide a double binding site for E2F dimers that rapidly dissociate from and then reassociate with the DNA in this region; however, the finding that the rate of association to the three different sites was the same argue against this interpretation. It would appear that both sites can be occupied by a single E2F dimer.

Finally, oligomeric interactions between E2F family members and a number of other cellular proteins (including pRb, p107, p130, cyclin A-cdk2, and cyclin E-cdk2) point to another area in which the stability of E2F binding could influence gene regulation. Binding of Rb to E2F, which represses E2F-dependent transcription, increases the half-life of E2F-DNA complexes 10-15-fold (58) . The binding characteristics of other multimeric E2F-DNA interactions have not been reported. Future studies will address whether the inverted repeat overlapping sites on the DHFR promoter influence binding of multimeric complexes. Sequences which enhance the binding stability of such complexes would also be expected to influence their regulatory functions.


FOOTNOTES

*
This work was supported by Grant CB42 from the American Cancer Society and March of Dimes Grant 1-1038. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pediatrics, University of Kentucky, Lexington, KY 40536-0284.

Present address: Genetic Therapy Inc., 19 First Field Rd., Gaithersburg, MD 20878.

**
To whom correspondence should be addressed: Tel.: 716-845-3563; Fax: 716-845-8857.

The abbreviations used are: DHFR, dihydrofolate reductase; CAT, chloramphenicol acetyltransferase; cpm, counts/minute.

D. Jensen and J. C. Azizkhan, unpublished data.

D. Jensen and J. C. Azizkhan, unpublished observation.


ACKNOWLEDGEMENTS

We acknowledge Dr. Joseph Nevins and members of his laboratory for helpful discussions, Drs. R. Girling and N. B. La Thangue for the DP-1 plasmid, and Drs. Al Baldwin, Bill Marzluff, and Adrian Black and our laboratory colleagues for comments on the manuscript. We also thank Dr. Adrian Black for helpful suggestions on the kinetic studies, Carol Hoover for technical assistance with the association rate studies, and Dr. Bill Kaelin for sharing data prior to publication.


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