Involvement of RFX1 Protein in the Regulation of the Human Proliferating Cell Nuclear Antigen Promoter*

Mingsong LiuDagger §, Benjamin H. LeeDagger , and Michael B. MathewsDagger parallel **

From the Dagger  Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, the  Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11790, and the parallel  Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The proliferating cell nuclear antigen (PCNA) is an essential eukaryotic DNA replication factor that is transcriptionally regulated by the adenovirus oncoprotein E1A 243R. Inducibility of the human PCNA promoter by E1A 243R is conferred by the cis-acting PCNA E1A-responsive element (PERE), which associates with the ATF-1, cAMP response element-binding protein (CREB), and RFX1 transcription factors and is modulated by cellular proteins such as the coactivator CREB-binding protein (CBP) and tumor suppressor p107 (Labrie, C., Lee, B. H., and Mathews, M. B. (1995) Nucleic Acids Res. 23, 3732-3741; Lee, B. H., and Mathews, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4481-4486; Lee, B. H., Liu, M., and Mathews, M. B. (1998) J. Virol. 72, 1138-1145). RFX1 also forms a complex with sequences in the PCNA promoter of mouse and rat that share homology with the RFX1 consensus site. To explore the role of RFX1 in regulating the PCNA promoter, we examined the effects of mutations in the human PERE on RFX1 binding and gene expression. Mutations within the RFX1 consensus binding site reduced RFX1 binding, whereas mutations upstream of the site, or on its border, increased RFX1 binding. These mutations also affected the transcriptional activity of PCNA-chloramphenicol acetyltransferase reporter constructs in transient expression assays. The relative transcriptional activity of mutant PCNA promoters, both in the presence and absence of E1A 243R, was inversely related to their ability to complex with RFX1. These findings suggest that the binding of RFX1 is influenced by sequences outside its consensus binding site and that this transcription factor plays an inhibitory role in the regulation of PCNA gene expression.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Strict control of the cell cycle and DNA replication is critical for maintaining normal cellular growth and differentiation. As an integral component of the eukaryotic DNA replication machinery and cell cycle complexes, the proliferating cell nuclear antigen (PCNA)1 plays a crucial role in these cellular processes. Serving as an auxiliary factor of DNA polymerase delta , PCNA functions by increasing the processivity of DNA synthesis (1-3) and is required for both leading and lagging strand DNA synthesis in a cell-free SV40 DNA replication system (2, 4, 5). Furthermore, PCNA is required for cell cycle progression and growth of yeast (6) and mammalian cells (7), and has been implicated in the repair of mutagen-damaged DNA (8). Consistent with its role in cell growth and DNA repair, PCNA expression is induced by serum (9), growth factors such as epidermal growth factor and platelet-derived growth factor (7), interleukin-2 (10), and wild-type p53 (11, 12).

The adenovirus E1A oncogene encodes two major proteins containing 289 and 243 amino acid residues (hereafter referred to as E1A 289R and E1A 243R), which are translated from two alternatively spliced transcripts, 13S and 12S, respectively (13, 14). The multifunctional E1A 289R and E1A 243R proteins are identical in sequence with the exception of a 46-amino acid domain, conserved region 3 (CR3), which endows the larger protein with potent transactivation properties (13-16). Even though it lacks the CR3 region, E1A 243R is as capable as E1A 289R of inducing PCNA expression either in the context of a viral infection or in transient expression experiments in HeLa cells (17, 18).

Induction of PCNA expression by E1A 243R occurs at the transcriptional level through an increase in PCNA promoter activity (18). A cis-acting PCNA E1A-responsive element (PERE) resides between nucleotides -59 and -45 relative to the transcription start site of the PCNA promoter (19) and can confer induction by the E1A 243R oncoprotein upon a normally E1A-unresponsive heterologous promoter (20). The PERE contains a sequence homologous to the activating transcription factor (ATF) motif (5'-TGACGTCG-3') at its 3' end, and sequences within the PERE including the ATF site are conserved between the rat, mouse, and human PCNA promoters (19). It also contains sequences that match a consensus RFX1 binding site (see below). In electrophoretic mobility shift assays, the PERE forms three major complexes (P1, P2, and P3) with proteins in HeLa cell nuclear extracts (21). While transcription factors ATF-1 and CREB are the major components of complexes P2 and P3, RFX1 is the primary constituent of the P1 complex (21, 22).

The hepatitis B virus (HBV) enhancer-associated protein RFX1, also known as enhancer factor-C (23), is a ubiquitous protein that is able to transactivate the HBV enhancer and is required for major histocompatibility complex class II gene expression (23, 24). Conversely, there is evidence that RFX1 can subserve a transcriptional silencer function (25), and recent observations show that it contains both activation and suppression domains (26). To determine whether RFX1 contributes to the regulation of PCNA gene expression, we designed a series of mutations within and around the PERE and conducted both gel mobility shift analysis and transient transfection assays to evaluate the effects of these promoter mutations. The results show that RFX1 binding is sensitive to mutations both within and upstream of its consensus binding site. Moreover, the ability of RFX1 to form a complex on the PCNA promoter is inversely related to the transcriptional activity of the promoter with respect to both basal and E1A-induced expression. These data imply that the RFX1 protein may play an inhibitory role in regulating E1A-transactivated PCNA gene expression, and provide a basis for understanding the role of RFX1 in the p107-mediated down-regulation of the PCNA promoter (27).

    MATERIALS AND METHODS
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Plasmids-- Wild-type PCNA-87 CAT and mutant constructs -46/-39, ATF-BAM, -56GA, and -59/-56 CAT were described previously (19, 28). Reporter plasmids -44/-40 and -53GT CAT contain single or multiple mutations in the PCNA promoter and were created by using an oligonucleotide-directed mutagenesis kit (Amersham Pharmacia Biotech). Plasmids expressing the E1B 19-kDa protein (pCMV19K), beta -galactosidase (pCMVbeta -gal), E1A243R (pCMV12S), and a truncated form of E1A 243R product (pCMV12S.FS), under the control of the cytomegalovirus promoter have been described previously (18). Plasmid pCH110 (29) expressing beta -galactosidase under the control of SV40 promoter was obtained from M. Gilman (Ariad Pharmaceuticals).

Preparation of Nuclear Extract-- Spinner cultures of HeLa cells (ATCC CCL2) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 µg/ml each of penicillin and streptomycin. Nuclear extracts were prepared as described previously (21, 30). Briefly, cells were harvested and rinsed with ice-cold phosphate-buffered saline and homogenized in two packed-cell volumes of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). Nuclei were pelleted by centrifugation and homogenized in 2.5 ml of Buffer C/109 cells. Buffer C contains 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.42 M NaCl, 0.5 mM DTT, 0.5 mM PMSF, and 25% (v/v) glycerol. The supernatants were collected and dialyzed against two changes of Buffer D (20 mM HEPES, pH 7.9, 0.1 M KCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, and 20% (v/v) glycerol). Samples were quickly frozen in liquid nitrogen and stored at -70 °C.

Oligonucleotides-- Oligonucleotides were synthesized in the DNA core facility center at Cold Spring Harbor Laboratory and contain a 5' overhang (5'-GATC-3') for labeling. Double-stranded oligonucleotides were prepared by annealing the complementary single-stranded oligonucleotides together in oligo buffer (10 mM Tris at pH 8.0, 100 mM NaCl, and 0.1 mM EDTA). The oligonucleotides are divided into two groups.

1) Short PERE probes contain sequences from -60 to -40 relative to the transcription initiation site of the human PCNA promoter. This group includes the wild-type PCNA sequence PERE(S), point mutant -53GT, and mutant -59/-56 (mutations are underlined).
<AR><R><C><UP>        PERE</UP>(<UP>S</UP>)<UP>:</UP></C><C><UP>5′-CAGCGTGGTGACGTCGCAACG-3′</UP></C></R><R><C><UP>           −53GT:</UP></C><C><UP>5′-CAGCGTG<UNL>T</UNL>TGACGTCGCAACG-3′</UP></C></R><R><C><UP>       −59/−56:</UP></C><C><UP>5′-C<UNL>CAGA</UNL>TGGTGACGTCGCAACG-3′</UP></C></R></AR>
<UP><SC>Sequences</SC> 1–3</UP>

2) Long PERE probes contain sequences from -70 to -40 relative to the transcription start site of the human, rat, and mouse PCNA promoters. The nucleotide sequence between -70 and -60 is identical among different probes and combines several nonspecific mutations found in the linker-scanning mutants published previously (19). This group includes the wild-type human PCNA sequence PERE(L), and mutant oligonucleotides -44/-40, ATF-BAM, -50/-40, and -56GA (mutations are underlined).
   <AR><R><C><UP> PERE</UP>(<UP>L</UP>)<UP>:</UP></C><C><UP>5′-AGATCTGTCTCAGCGTGGTGACGTCGCAACG-3′</UP></C></R><R><C><UP> −44/−40:</UP></C><C><UP>5′-AGATCTGTCTCAGCGTGGTGACGTCG<UNL>ATCGA</UNL>-3′</UP></C></R><R><C><UP>ATF-BAM:</UP></C><C><UP>5′-AGATCTGTCTCAGCGTGGTG<UNL>GATC</UNL>CGCAACG-3′</UP></C></R><R><C><UP> −50/−40:</UP></C><C><UP>5′-AGATCTGTCTCAGCGTGGTG<UNL>GATCCCATCGA</UNL>-3′</UP></C></R><R><C><UP>    −56GA:</UP></C><C><UP>5′-AGATCTGTCTCAGC<UNL>A</UNL>TGGTGACGTCGCAACG-3′</UP></C></R></AR>
<UP><SC>Sequences</SC> 4–8</UP>

Electrophoretic Mobility Shift Assays (EMSA)-- EMSA were performed as described previously (21) in a 20-µl reaction mixture containing 1× EMSA buffer (12 mM HEPES, pH 7.6, 50 mM NaCl, 1 mM DTT, and 5% (v/v) glycerol), 2 µg of poly(dI-dC)-poly(dI-dC) as nonspecific DNA competitor, 5 µg of HeLa cell nuclear extract, and 20,000 cpm of oligonucleotide probe. Oligonucleotides were labeled with [alpha -32P]dATP (3000 Ci/mmol) and cold dNTPs using DNA polymerase I (Klenow fragment). All probes were purified through native 6% polyacrylamide gels, eluted from gel slices in oligo buffer containing 0.5% SDS, and dissolved in oligo buffer after phenol extraction and ethanol precipitation. In some cases, the nuclear extract was replaced by protein synthesized in vitro (see below). Incubations were conducted on ice for 15 min in the absence of probe, followed by incubation at 37 °C for 15 min after addition of probe. When indicated, antibody was added to the binding reaction at least 1 h prior to the addition of probe and incubated at 4 °C. Anti-RFX1 antibody was a generous gift from P. Hearing (SUNY, Stony Brook, NY). Protein-DNA complexes were resolved in 5% polyacrylamide (29:1 acrylamide:bisacrylamide), 0.5× Tris borate-EDTA gels.

In Vitro Transcription and Translation-- Plasmid pHRFX1 (23) for in vitro transcription-translation (IVT) of RFX1 protein was provided by P. Hearing and W. Reith. RFX1 mRNA was synthesized using HindIII-digested plasmid as template, and then translated in a rabbit reticulocyte lysate. Binding assays were performed under conditions similar to those described above with the addition of 5 µg of bovine serum albumin (Roche Molecular Biochemicals) to each reaction.

Transfections-- Transient transfection assays were performed in ATCC HeLa cells (passage 6-13) as described previously (18). Briefly, duplicate cultures at 40-50% confluence were transfected by the calcium phosphate coprecipitation technique (31). Unless otherwise indicated, each 6-cm plate received a total of 20 µg of DNA including 10 µg of reporter constructs, 0.5 µg of either pCMV12S or pCMV12S.FS, 0.5 µg of pCMV19K, 1 µg of pCMVbeta -gal or pCH110 as a control for transfection efficiency, and salmon sperm carrier DNA. The cells were washed twice with phosphate-buffered saline and fresh medium added 16 h after the transfection. Cells were harvested 48 h after the transfection.

Enzyme Assays-- Cell extracts were prepared by freezing and thawing cells in 0.25 M Tris-HCl (pH 8.0). Chloramphenicol acetyltransferase (CAT) assays were performed so that they yielded values within the linear range of the assay. Usually, up to 50 µl of the cell extracts were added to the reaction in CAT assay. Thin-layer chromatograms were quantified with a Betascan System (AMBIS, San Diego, CA), and CAT activity was expressed as the percentage of chloramphenicol acetylated by 50 µl of cell extract incubated at 37 °C for 1 h. beta -Galactosidase assays were performed as described previously (32), and beta -galactosidase activity was expressed as the optical density at 420 nm obtained with 50 µl of extract incubated at 37 °C for 1 h. CAT activity was normalized to the levels of beta -galactosidase activity and is expressed as the mean ± S.D. relative to the level of CAT activity obtained by cotransfection of wild-type PCNA-CAT with pCMV 12S.FS.

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Conservation of PERE Sequence in Mammalian PCNA Promoters-- We showed previously that an oligonucleotide probe comprising sequences from -60 to -40 of the human PCNA promoter (thus encompassing the PERE site from -59 to -45) forms three principal complexes, P1-P3, with proteins from HeLa nuclear extracts in an EMSA assay (Fig. 1A, lane 1). The formation of complexes P2 and P3 is dependent upon an ATF/CREB consensus site contained at the 3' end of the PERE between nucleotides -52 and -45 (21), and these complexes contain the ATF-1 and CREB cellular proteins (21, 22). Transcription factor RFX1 is a component of complex P1, and its binding to the PCNA promoter appeared to be dependent upon sequences overlapping the ATF/CREB consensus motif and extending immediately downstream of the PERE to position -40 of the promoter (21). Consistent with the PERE's importance in PCNA promoter function, there is a considerable degree of conservation of PERE sequences among the human (28, 33), rat (34), and mouse (35) PCNA promoters (Fig. 1B). As noted previously (19), the homology extends both upstream and downstream of the ATF/CREB consensus site.


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Fig. 1.   Species-specific formation of complex P1 with PCNA promoter sequences. A, radiolabeled oligonucleotide probes corresponding to the human PERE (lanes 1-3) and homologous sequences from the rat (lanes 4-6) and mouse (lanes 7-9) PCNA promoters were incubated with 5 µg of HeLa cell nuclear extract. DNA-protein complexes were resolved on a 5% native polyacrylamide gel. Complexes P1-P3 are denoted by arrowheads. Anti-RFX1 antibody was added to reactions loaded in lanes 3, 6, and 9. B, PERE sequences from the human, mouse, and rat PCNA promoters. Nucleotides conserved in all three species are shown by boldface capital letters, those present in two species are capitalized, and lowercase letters denote bases that are unique to individual species (adapted from Ref. 19).

To determine whether complexes could also form with PERE sequences from these other mammalian PCNA promoters, we repeated the EMSA with rat and mouse probes. Fig. 1A shows that the three probes all formed complexes P1, P2, and P3, although complex P1 formation was considerably weaker with the rat PERE probe (lanes 1, 4, and 7). In all cases, antibody to RFX1 caused a supershift of the P1 complex whereas normal rabbit serum was without effect (compare lanes 3, 6, and 9 with lanes 2, 5, and 8). Inspection of sequences within and directly downstream of the human PERE revealed a good match of the nucleotides located between positions -53 and -41 of the human PCNA promoter with a recently published RFX1 consensus binding site (Fig. 2A). This consensus sequence consists of two somewhat degenerate half-sites, 5'-GTNRCC/N-3' and 5'-RGYAAC-3', forming an imperfect palindrome usually separated by one or two nucleotides (36). Similar matching sequences are present in the equivalent positions of the rat and mouse PCNA genes (Fig. 2A), although the three sequences are not identical over this region. There is no obvious relationship between their conformity to the consensus RFX1 site and the intensity of P1 complex formation by each species, however. This disparity is not surprising in view of the degeneracy of the consensus sequence. Nevertheless, these observations suggest that variations in complex P1 stability are due to discrete nucleotide changes in the RFX1 binding site.


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Fig. 2.   RFX1 site homology and PCNA promoter mutations. A, alignment of the RFX1 binding consensus sequence (36) with the functionally defined RFX1 site of the human PCNA promoter (nucleotides -54 to -41 relative to the transcription start site) and corresponding regions from the rat and mouse genes. Nucleotides that are invariable in the consensus sequence are in boldface type. Nucleotides that deviate from the consensus sequence are in lowercase. N, any nucleotide; Y, pyrimidine. B, structure of wild-type and mutant human PCNA promoter sequences. Sequences from -60 to -39 relative to the transcription initiation site of the wild-type promoter and promoter mutants are illustrated. The PERE (from -59 to -45) is boldface, the ATF/CREB consensus site (from -52 to -45) is underlined, and the RFX1 consensus site (from -54 to -39) is indicated. Base changes in the mutants are shown; conserved bases are indicated by hyphens.

Effects of Promoter Mutations on P1 Complex Formation-- Analysis of the binding of RFX1 to the PERE is complicated because the ATF/CREB binding site is completely contained within the RFX1 consensus motif (Fig. 2B). To differentiate between the actions of these two proteins on the PCNA promoter, we assessed the effects of mutating particular nucleotides (Fig. 2B) on the formation of the RFX1-containing protein complex, P1, in vitro. These experiments were conducted with two forms of the PERE probe: the PERE(L) probes are longer than the PERE(S) probes, which contain sequences from -60 to -40 of the PCNA promoter, by virtue of the addition of 10 nucleotides at the upstream end. Extending the length of the DNA probe increased the stability of complex P1 without greatly affecting complexes P2 and P3 (Fig. 3; compare lanes 5 and 1). The precise composition of the 10 extra nucleotides appeared to be inconsequential, as the P1 band was intensified regardless of whether the extension was composed of wild-type or mutant PCNA sequences (data not shown). Because linker-scanning mutations between -70 and -60 do not affect either basal or E1A-transactivated expression from the PCNA promoter (19), the PERE(L) probe used here contained a 10-base pair sequence devised by combining the mutated nucleotides of several linker-scanning mutations studied previously in PCNA-CAT reporter constructs (19) in an attempt to exclude possible nonspecific interactions between these longer EMSA probes and proteins from HeLa cell nuclear extract.


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Fig. 3.   Differential effects of promoter mutations on complex P1 formation in vitro. Radiolabeled oligonucleotides were incubated with 5 µg of HeLa cell nuclear extract at 4 °C. Protein-DNA complexes were resolved in a native 5% polyacrylamide gel and detected by autoradiography. Lane 1, wild-type PERE(S) probe, corresponding to nucleotides -60 to -40 of the PCNA promoter. Lanes 2 and 3, PERE(S) mutant probes -53GT and -59/-56, respectively. Lane 5, wild-type PERE(L) probe, containing nucleotides from -60 to -40 of the PCNA promoter extended to -70 with mutant sequence. Lanes 6-10, PERE(L) mutant probes -44/-40, ATF-BAM, -50/-40, -56GA, and -59/-56, respectively. Complexes P1-P3 are denoted (arrowheads).

Consistent with previous observations (21), the clustered point mutations between nucleotides -44 and -40, which drastically change the downstream RFX1 half-site (in this case, 5'-CGCAAC-3'), diminished formation of P1 but not of P2 or P3 (Fig. 3; compare lane 6 with lane 5). Mutation of nucleotides between -50 and -47, which comprise the core of both the ATF site, not only abolished complexes P2 and P3 as expected, but also abrogated P1 complex formation (ATF-BAM, lane 7). This suggests that the formation of the P1 complex depends on the integrity of the upstream RFX1 half-site (in this case 5'-GTGACG-3') as well as the downstream half-site. The -50/-40 mutation, which affects both RFX1 half-sites in addition to the ATF site, also eliminated all three bands (lane 8).

On the other hand, several upstream mutations appeared to stabilize the P1 complex. The P1 band, which was barely detectable with the wild-type PERE(S) probe (lane 1), was dramatically intensified by a point mutation at position -53 (a G to T substitution) located at the 5' border of the RFX1 consensus site (-53GT, lane 2). The -53GT mutation also diminished the formation of complexes P2 and P3 slightly, although it lies just outside the ATF/CREB consensus site. The intensity of the P1 band also increased, albeit less strikingly, when the PERE(S) mutant oligonucleotide -59/-56 was used as probe (lane 3). This observation was confirmed in the context of the PERE(L) probe; the clustered mutations between -56 and -59 appreciably increased the intensity of the P1 band without affecting P2 and P3 (compare lane 10 with lane 5). A similar result was obtained with a single G to A point mutation at position -56 (lane 9). These data imply that nucleotide sequences at or near the 5' boundary of the RFX1 consensus binding site contribute to the DNA binding properties of RFX1; mutation of these nucleotides strengthens P1 complex formation, while other mutations in the RFX1 site destabilize the complex.

Effects of Mutations on PCNA Promoter Activity-- To evaluate the influence of RFX1 binding on basal and E1A-induced PCNA promoter activity, we cotransfected PCNA-CAT reporter constructs harboring these single or clustered promoter mutations into HeLa cells with a plasmid expressing wild-type E1A 243R protein (pCMV12S) or, as a control, with a plasmid expressing an inactive, truncated E1A protein (pCMV12S.FS). The resultant CAT activity was compared with that generated by the wild-type PCNA-87 CAT reporter. Consistent with previous results (19), the -59/-56, -56GA, and -53GT mutations reduced E1A-induced PCNA promoter activity by about 3-fold (Fig. 4). These mutations also lowered basal transcription by about 2-fold. In contrast, the two downstream mutations, -44/-40 and -46/-39, elevated both basal and E1A 243R-induced promoter activity by 30-40%. These results indicate an inverse relationship between PCNA promoter activity and the stability of complex P1; mutations that stabilized the P1 complex (-53GT, -56GA, and -59/-56) reduced basal and E1A-induced transcriptional activity, whereas mutations that abrogated complex P1 (-44/-40 and -46/-39) accentuated PCNA promoter activity (see Table I).


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Fig. 4.   Effect of promoter mutations on basal and E1A-transactivated PCNA-CAT activity. Wild-type PCNA-87 CAT (WT) and the mutant reporters indicated were cotransfected with either pCMV12S.FS (control, black box) or pCMV 12S (E1A 243R, hatched box). CAT activity was corrected for beta -galactosidase activity, generated from a cotransfected reporter plasmid, and is expressed as the mean ± S.D. relative to the CAT activity obtained by cotransfection of wild-type PCNA-CAT with pCMV12S FS. Results represent three independent transfections performed in duplicate.

                              
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Table I
Comparison of gel mobility shift results and promoter activities
Table shows changes relative to data obtained with the wild-type PCNA promoter: 0, no change; +, increase; ++, large increase; -, decrease; - -, large decrease.

Complex P1 Formation Is Characterized by RFX1 Protein Binding-- The foregoing discussion has assumed that changes in the level of complex P1 are determined by the stability of RFX1 binding. To validate this assumption, we examined the effects of mutations introduced into the PCNA promoter on the binding of RFX1 protein synthesized in vitro (IVT RFX1). With the PERE(S) and (L) probes, unprogrammed rabbit reticulocyte lysate formed nonspecific complexes and faint bands with mobilities similar to those of P2 and P3 (Fig. 5, A and B, lanes 2), but did not give rise to complex P1 formed with HeLa nuclear extract (Fig. 5, A and B, lanes 1). The weak complex formed by IVT RFX1 with the wild-type PERE(S) probe migrated with P1 and was dramatically intensified with PERE(S) variants harboring upstream mutations (compare Fig. 5A, lanes 6 and 7 with lane 3). This behavior confirms observations made with HeLa nuclear extract (Fig. 3) and clearly illustrates the effect of the clustered mutations between -59 and -56 (-59/-56) as well as the point mutation at position -53 (-53GT).


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Fig. 5.   Binding of RFX1 protein to PERE mutants. A, full-length RFX1 was synthesized in vitro (IVT RFX1) and incubated with labeled wild-type (lanes 3-5) or mutant (lanes 6 and 7) PERE(S) probe. Normal rabbit serum (NRS) or anti-RFX1 antibody was added to the reactions run in lanes 4 and 5, respectively. Lanes 1 and 2, IVT RFX1 was replaced by HeLa nuclear extract (HeLa NE) or unprogrammed rabbit reticulocyte lysate (RRE), respectively. B, IVT RFX1 protein was incubated with radiolabeled wild-type (lanes 3-5) or mutant (lanes 6-9) PERE(L) probe. Normal rabbit serum (NRS) or anti-RFX1 antibody was added to the reactions run in lanes 4 and 5, respectively. Lanes 1 and 2, IVT RFX1 was replaced by HeLa nuclear extract (HeLa NE) or unprogrammed rabbit reticulocyte lysate (RRE), respectively. Other details are as for Fig. 3.

Complex formation with IVT RFX1 protein was also accentuated by using the extended wild-type PERE(L) probe (compare Fig. 5B, lane 3, to Fig. 5A, lane 3). Mutations of this probe that alter nucleotides in the RFX1 consensus site, namely -44/-40, ATF-BAM, and -50/-40, abrogated the ability of IVT RFX1 to bind to the DNA probe (Fig. 5B, lanes 6-8), while the -56GA mutation increased IVT RFX1 protein binding (Fig. 5B, lane 9). Evidently, probe mutations that affected the stability of complex P1 formed in HeLa nuclear extract (Fig. 3) all exerted corresponding effects on IVT RFX1 protein binding (Fig. 5; see Table I).

Finally, antibody interference experiments were performed to determine whether the increased intensity of complex P1 brought about by alterations in the PERE probe is due solely to the formation of more RFX1-containing complexes (Fig. 6). HeLa cell nuclear extract was preincubated with anti-RFX1 polyclonal antibody (lanes 2, 3, and 5), and EMSA was performed with the PERE(L) probe (lanes 1-3) or the -53GT probe (lanes 4 and 5). In both cases, the P1 complex was completely supershifted by anti-RFX1 antibody but no changes in P2 and P3 were observed. Thus, the increased formation of complex P1 observed with these probes is attributable to the increased binding of RFX1. These data, taken together with the comigration of complex P1 formed with nuclear extract and IVT RFX1, support the conclusion that complex P1 consists chiefly, if not solely, of RFX1 protein.


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Fig. 6.   Increased complex P1 stability is due to the increased binding of RFX1 protein. Labeled PERE(L) probe (lanes 1-3) or PERE(S) mutant -53GT probe (lanes 4 and 5) was incubated with HeLa cell extract in the absence (lanes 1 and 4) or presence (lanes 2, 3, and 5) of antibody against RFX1. Other details are as for Fig. 3.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous work demonstrated that the RFX1 transcription factor associates with the PCNA promoter (21), but the biological role of RFX1 in the regulation of PCNA expression, if any, remained to be established. Here we show that the RFX1 binding site is conserved in mammals, and that RFX1 binding to the PERE correlates inversely with transcription from the PCNA promoter.

Participation of RFX1 in PCNA Promoter Function-- To address the role of RFX1, we designed a series of mutations surrounding the PERE ATF/CREB site and examined their effects on RFX1 binding and on the basal and E1A-activated expression of the PCNA promoter. As summarized in Table I, the mutations exhibited differential effects on complex P1 formation and RFX1 binding depending on their location. Clustered mutations in the downstream RFX1 half-site reduced P1 complex formation and RFX1 binding, as expected. More surprisingly, mutations at the upstream border of the upstream RFX1 half-site and further upstream had the opposite effect, and extending the length of the PERE at its 5' border also increased P1 complex formation and RFX1 binding. One possible explanation for this finding is that RFX1 binding and P1 complex formation are stabilized by an unidentified protein that binds in this region. However, this possibility is unlikely in view of the nature of the mutations that give the effect. An alternative explanation is that the upstream region influences binding indirectly, perhaps through a change in the local conformation of the DNA.

Despite this mechanistic uncertainty, the mutations provide compelling evidence that RFX1 plays a role in regulating the activity of the PCNA promoter. Transient expression assays revealed an inverse correlation between complex P1/RFX1 binding and PCNA promoter expression (see Table I). This inverse relationship held with both upstream and downstream mutations and for basal and E1A 243R-induced expression. The consistent inverse relationship between RFX1 binding and PCNA promoter activity implies that the RFX1 protein complex P1 plays an inhibitory role in the regulation of human PCNA gene expression, both in the absence and presence of E1A. Judging from the EMSA data obtained with rodent PERE sequences, it is likely that similar controls are exerted over the mouse PCNA gene but possibly not over its homologue in the rat, presenting a potentially interesting case of species-specific PCNA transcriptional regulation.

RFX1 Is a Versatile Transcription Factor-- RFX1 is a 130-kDa protein that belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins (RFX1-RFX5) and contains a highly conserved DNA binding domain (37). The RFX proteins function in diverse and unrelated systems (37); while the RFX1 binding site functions as a positive element required for the transcriptional activities of both the major histocompatibility complex class II and HBV genes (23, 24), RFX1 has been suggested to play an inhibitory role in other systems. Thus, RFX1 is part of a complex that binds to the MIF-1 binding site, an element which has high homology with the RFX1 consensus sequence (12 out of 13 base pairs) and can function as a transcriptional silencer in both hepatocarcinoma HepG2 and HeLa cell lines (25). Interestingly, the MIF-1 regulatory element has been identified in the intron I region of the c-myc gene, and mutation of the MIF-1 site, which abolishes MIF-1 binding activity, also results in the up-regulation of c-myc gene expression (38). Taken together, the data suggest that the MIF-1 site and presumably its binding protein RFX1 may function to negatively regulate c-myc gene expression as we now propose in the case of the human PCNA promoter. Moreover, dissection of RFX1 has revealed that it is a dual-function regulator, possessing domains with intrinsic transcriptional activation and silencing properties (26). Although these two domains effectively counteract one another in the intact molecule, resulting in transcriptional near-neutrality, it is possible that contextual signals can shift the balance toward activation or repression. The lack of obvious correspondence between the departures of the rodent sequences from the consensus RFX1 site and their abilities to form RFX1-containing complexes, implies that subtle contextual changes may have profound functional consequences.

The Role of RFX1 in the Regulation of E1A 243R-induced PCNA Expression-- In the PCNA promoter, the RFX1-binding site surrounds and overlaps the ATF/CREB binding site. The nature of the interplay between these two sites and their cognate factors remains to be established, however. For example, it is not clear whether the two types of DNA-binding proteins can co-occupy the PERE or, alternatively, compete with one another for occupancy. To date, we have not detected a complex that contains them both, arguing that there is no strongly cooperative binding interaction, but this conclusion must be taken with caution as EMSA was conducted in conditions of DNA excess.

One mechanism by which E1A 243R can target and transactivate the PCNA promoter at the PERE site is via its interaction with CBP and CREB (22). Mutation of the PERE ATF/CREB site completely abrogates both basal and E1A-induction of the PCNA promoter, presumably by interrupting this CBP-CREB-DNA pathway. The requirement for PERE sequences upstream of the ATF/CREB site (from -59 to -53) for optimal E1A induction of the PCNA promoter in vivo (19, 21) can now be explained in terms of RFX1 binding. Results presented here show that these upstream sequences, as well as those immediately downstream, influence the interactions of RFX1 with the PCNA promoter. Because the stability of complex P1 correlates with lower transcriptional activity of the PCNA promoter, we infer that the human PCNA promoter is subject to negative regulation by RFX1. Although the magnitude of this modulation is not dramatic, at least in the model system studied here, recent data suggest that repression by RFX1 can also be relieved by E1A 243R through its interaction with the retinoblastoma-like tumor suppressor protein p107, which also associates with the PCNA promoter (27). We conclude that RFX1 participates in a complex transcriptional regulatory mechanism, involving DNA elements contained within this region of the PCNA promoter together with their cognate binding proteins, to control this essential DNA replication factor.

    ACKNOWLEDGEMENTS

We thank P. Wendel for excellent technical assistance. We are grateful to P. Hearing, W. Reith, R. Agami, and Y. Shaul for helpful discussions and RFX1 reagents.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA 13106.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Cardiovascular Research Inst., University of California, San Francisco, CA 94143-0130.

** Present address. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, New Jersey Medical School, UMDNJ-Newark, 185 S. Orange Ave., Newark, NJ 07103-2714. Tel.: 973-972-4411; Fax: 973-972-5594; E-mail: mathews{at}umdnj.edu.

    ABBREVIATIONS

The abbreviations used are: PCNA, proliferating cell nuclear antigen; PERE, PCNA E1A-responsive element; CR, conserved region; ATF, activating transcription factor; CREB, cAMP response element-binding protein; HBV, hepatitis B virus; CBP, CREB-binding protein; EMSA, electrophoretic mobility shift assay; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; CAT, chloramphenicol acetyltransferase; IVT, in vitro transcription-translation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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