(Received for publication, July 21, 1995; and in revised form, October 4, 1995)
From the
Interleukin 2 (IL-2) stimulates T lymphocyte proliferation and
induces the expression of proliferating cell nuclear antigen (PCNA), a
processivity factor for DNA polymerase . Previously, deletion
analysis suggested cis-element(s) in the proximal region of the PCNA
promoter (-40 to +143) are required for IL-2 induction in
cloned T lymphocytes. The sequence 5`-TTGCGGGC-3` located at +10
to +17 is similar to the E2F consensus binding site and is
required for optimal PCNA promoter activity. In IL-2-stimulated T
cells, nuclear proteins are induced to bind to this sequence as
demonstrated using electrophoretic mobility shift assay (EMSA),
competition EMSA, and methylation interference analysis. A 180-kDa
polypeptide was detected by UV cross-linking to bind specifically to
the PCNA E2F-like sequence. Our data indicate that the protein bound to
the PCNA E2F-like site is not one of the transcription factor E2F
proteins. Our results demonstrate that the E2F-like sequence and the
protein(s) binding to it are required for optimal PCNA promoter
activity and IL-2 induction of PCNA expression.
PCNA was first described by Miyachi et al.(1978) as a
nuclear antigen restricted to proliferating cells that reacts with sera
from some patients with the autoimmune disorder systemic lupus
erythematosus, hence the name proliferating cell nuclear antigen. PCNA ()is an auxiliary factor for DNA polymerase
(Bravo et al., 1987; Prelich and Stillman, 1988). It is required for
both DNA replication and DNA repair (Shivji et al., 1992). The
gene for PCNA has been highly conserved throughout the course of
evolution (Mathews, 1989). The ability of PCNA-specific antibodies to
inhibit inducible DNA synthesis in isolated nuclei (Wong et
al., 1987), and of PCNA antisense oligonucleotides to inhibit
proliferation of Balb/c 3T3 cells (Jaskulski et al., 1988),
supports the importance of this protein in cell cycle progression.
However, to date all of the cellular functions of PCNA during the cell
cycle have not been elucidated. In addition to its role as a
processivity factor in DNA replication, PCNA may function in the
regulation of cell cycle progression (McAlear et al., 1994;
Xiong et al., 1993). Recently, PCNA has been found in
complexes with a variety of cyclins, cyclin-dependent kinases, and p21,
one of the inhibitors of cyclin-dependent kinases, also known as Waf1
or Cip1 (Xiong et al., 1992; Waga et al., 1994). Also
p21 controls DNA replication by direct interaction with PCNA (Waga et al., 1994; Flores-Rozas et al., 1994). Therefore
it is important to understand the regulation of PCNA expression and the
function(s) of PCNA within the cell cycle.
Previous studies have shown transcriptional regulation of PCNA (Morris and Mathews, 1990; Shipman-Appasamy et al., 1990). The promoter region and the introns of the PCNA gene are involved in the transcriptional regulation of PCNA (Alder et al., 1992; Huang et al., 1994). Post-transcriptional regulation also seems to play a role in its regulation. During growth factor stimulation of lymphocytes or 3T3 cells, there is an increase in PCNA mRNA stability (Chang et al., 1990; Shipman-Appasamy et al., 1990; Baserga, 1991). Increased PCNA transcription has been demonstrated in IL-2-stimulated T cells (Shipman-Appasamy et al., 1990), but it could not be found in serum-stimulated 3T3 cells (Baserga, 1991). This suggests that lymphocytes and fibroblasts use different mechanisms for regulating levels of PCNA mRNA. We have shown transcriptional regulation of PCNA during IL-2 induced proliferation of T lymphocytes. Tandem CRE binding sites in the murine PCNA promoter are required for optimal promoter activity and the IL-2-induced PCNA transcriptional activation (Huang et al., 1994; Orten et al., 1994; Feuerstein et al., 1995). Morris and Matthews(1991) have shown that the corresponding CRE-binding site in the human PCNA gene is required for adenovirus E1A-driven transactivation.
Proteins and enzymes involved
in DNA synthesis are coordinately regulated with growth in mammalian
cells: they are induced in late G to early S phase of the
cell cycle. These include PCNA, dihydrofolate reductase (DHFR),
thymidine kinase, and DNA polymerase
among others (Hofbauer and
Denhardt, 1991). The molecular basis of this regulation has yet to be
established. Promoter sequences known for some of these genes differ
markedly. Recent studies of these promoters have shown that the binding
site for transcription factor E2F is important for DHFR and thymidine
kinase expression (Blake and Azizkhan, 1989; Dou et al.,
1994). Similar motifs are known to be present in the promoter of other
DNA synthesis genes, namely, those of DNA polymerase
and possibly
thymidine synthase (Pearson et al., 1991). The presence of an
E2F binding site in promoters of several genes encoding DNA synthesis
proteins suggests that growth regulation of these proteins may take
place by a common mechanism (Nevins, 1992). This is similar to the
regulation of the DNA synthesis genes in the yeast Saccharomyces
cerevisiae, in which a regulatory element, identical in sequence
with the recognition site for restriction enzyme MluI
(ACGCGT), plays a central role (Lowndes et al., 1991). One
hypothesis for the mechanism of coordinate regulation of DNA synthesis
genes in mammalian cells is that the transcription factor E2F plays a
central role in activating DNA synthesis genes during growth
stimulation (Nevins, 1992). E2F represents a group of related
transcription factors with similar DNA-binding specificity that
interacts with pRB-related pocket proteins (La Thangue, 1994). Sequence
comparison indicates that an E2F-like binding site is present between
+10 and +17 of the murine PCNA promoter. In addition to the
tandem CRE binding site, our previous results suggested that the
proximal PCNA promoter containing the E2F-like site is critical for
IL-2-induced transcriptional activation (Huang et al., 1994).
Thus, we analyzed the requirement of this E2F-like site for optimal
promoter activity and for protein-DNA interactions in the PCNA promoter
proximal region and demonstrate a functional role for the E2F-like site
in PCNA transcriptional regulation.
Freshly prepared
splenocytes were from CBA/J mice, separated from erythrocytes using
density gradient centrifugation through Ficoll-Hypaque (1.090 g/ml).
Splenocytes at a concentration of 6 10
/ml in
Dulbecco's modified Eagle's medium with additives
containing 500 ng/ml ConA were incubated for 72 h prior to extraction
of proteins.
Figure 1: Nucleotide sequence of PCNA promoter proximal region and schematic representation of the oligonucleotides used in EMSA and competition experiments. Sequence of murine PCNA promoter nt -20 to +30 is shown. The putative binding sites for PEA3, Inr element, and E2F-like binding site are indicated with boxes. The position of oligonucleotide ST and the three short oligonucleotides, ST-1, ST-2, and ST-3, in the PCNA promoter are indicated. PCNA transcription start site is at +1.
Sequence of oligonucleotides (sense strand) used for EMSA are listed below with numbers indicating the positions of start and end bases of the oligonucleotides within the PCNA promoter. The binding sites for PEA3, Inr, and E2F are underlined. Mutated nucleotides are indicated as lower case letters. ST, 5`-TAGGAAGCCGCGGCATTAGACGGTTGCGGGCGCAGA-3` (-14 to +22); ST-1, 5`-CGCGCCTAGGAAGCCGCGGC-3` (-20 to -1); ST-1m, 5`-CGCGCCTAaGAAGCCGCGGC-3` (-20 to -1); ST-2, 5`-AAGCCGCGGCATTAGACGGTTG-3` (-10 to +12); ST-2m, 5`-AAGCCGCGGggTTAGACGGTTG-3` (-10 to +12); ST-3, 5`-GACGGTTGCGGGCGCAGAGGGTTGGT-3` (+5 to +30); ST-3m1, 5`-GACGGTTGCtaGCGCAGAGGGTTGGT-3` (+5 to +30); ST-3m2, 5`-GACGGaaGCGGGCGCAGAGGGTTGGT-3` (+5 to +30); DHFR-E2F, 5`-TGCAATTTCGCGCCAAACTTG-3`; and DHFR-E2Fm, 5`-TGCAATTTCGtaCCAAACTTG-3`.
To determine the complexity of the protein(s) interacting with these sequences, a 36-base pair double-stranded oligonucleotide (ST) spanning all three sites (PCNA -14 to +22) was radiolabeled and used in an EMSA (Fig. 1). A series of DNA-protein binding complexes were formed when this probe was incubated with nuclear extracts from the cloned T lymphocyte L2 and mouse splenocytes (Fig. 2). Notably, IL-2 and ConA induce the formation of several complexes with ST in stimulated L2 cells (Fig. 2B, lanes 1 and 2) and in splenocytes (Fig. 2A), respectively. In contrast to PCNA ST, when the same extracts from L2 cells were used with a probe containing a USF binding motif (Gregor et al., 1990; Huang et al., 1994), USF binding activity did not change significantly during IL-2 stimulation (Fig. 2B, lanes 3 and 4). The binding pattern of these induced complexes is very similar in IL-2 stimulated L2 cells and ConA-stimulated splenocytes (Fig. 2A). Initial competition experiments using unlabeled ST as well as a non-related oligonucleotide as competitors of complex formation indicated that complexes I, II, and III are specific for PCNA ST (data not shown). Among them, complex I and II were consistently present in all nuclear extracts we prepared but complex III binding activity varied with different nuclear extract preparations. In addition, three fast migrating bands (Fig. 2B) are not detected reproducibly in other experiments.
Figure 2:
IL-2 and ConA induce nuclear proteins
binding to PCNA ST. A, EMSA of nuclear factors bind to PCNA
ST. P-Labeled oligonucleotide ST containing PCNA nt
-14 to +22 sequence was incubated with increasing amounts of
nuclear extracts prepared from quiescent splenocytes (spln Q),
ConA-stimulated splenocytes (spln S), or IL-2-stimulated L2
cells (L2S). The three prominent bands which were induced in
stimulated cells, complex I (I), complex II (II), and complex III (III)
are indicated. B, EMSA showing IL-2 induction of nuclear
proteins binding to ST.
P-Labeled ST were incubated with
nuclear extracts prepared from either quiescent (lane 1) or
24-h IL-2-stimulated L2 cells (lane 2).
P-Labeled
oligonucleotide containing the µE3 motif was used as a control. USF
binding activity detected by µE3 probe was constitutively present
in both quiescent (lane 3) and 24-h IL-2-stimulated L2 cells (lane 4). Free probe (free) runs at the bottom of the
gel.
To determine where the various IL-2-inducible nuclear proteins bind to the PCNA promoter, a series of short oligonucleotides (Fig. 1) each containing only one of the three putative protein binding sites was made: ST-1 (PEA3), ST-2 (Inr), and ST-3 (E2F). Complex formation with radiolabeled ST-1, ST-2, ST-3, and ST were compared by EMSA using a 24-h IL-2-stimulated L2 cell nuclear extract (Fig. 3). Results in Fig. 3A demonstrated that only ST-3 retains the ability to form all three complexes (complexs I, II, and III). The fast migrating band present in this analysis was not found reproducibly in other experiments.
Figure 3:
Nuclear
factors binding to PCNA ST. A, EMSA of nuclear factors binding
to PCNA promoter oligonucleotides. ST-1, ST-2, and ST-3 containing
PEA3, Inr, and E2F-like sequence, respectively, were P-labeled and incubated with IL-2-stimulated L2 cell
nuclear extract. B, EMSA competition experiment was performed
as described under ``Materials and Methods.'' ST was
P-labeled and incubated with IL-2-stimulated L2 cell
nuclear extract. ST-1, ST-2, and ST-3 and their corresponding binding
site mutations (ST-1m, ST-2m, and ST-3m1) were used as competitors.
Increasing amounts of each competitor oligonucleotide (5 or 20 ng) were
used. The three complexes are indicated as I, II, and III. Free probe (free) runs at the bottom of the
gel.
To determine whether the
putative binding sites are required for complex formation, a series of
binding site mutations (see ``Materials and Methods'') was
made in these oligonucleotides based on previously defined consensus
sequences of PEA, Inr, and E2F sites and on mutation studies (Yoo et al., 1991; Smale and Baltimore, 1989; Mudryj et
al., 1990). Fig. 3B shows a competition binding
experiment in which ST was P-labeled and incubated with
IL-2-stimulated L2 cell extract. Excess amounts of unlabeled
oligonucleotides containing the non-mutated or mutated binding site
were used as competitors of complex formation. Among them, ST-3 was the
most efficient competitor. In contrast, ST-3m1, an oligonucleotide with
a mutation in the E2F-like site (see Fig. 3B) failed to
inhibit any of the binding activities. This result indicates that the
E2F-like site is essential for complex formation.
It is interesting that both ST-1 and ST-2 show some dose-dependent inhibition of the binding activities (Fig. 3B). Since there is no obvious common sequence among ST-1, ST-2, and ST-3, this result suggests that the binding protein(s) may have multiple contact sites along the PCNA promoter sequence. It is more striking that ST-2m which has a mutated Inr element failed to inhibit complex formation, suggesting that the protein complexes may also contact the Inr element. To reconcile these data with Fig. 3A where there was no detectable binding to radiolabeled ST-1 and ST-2, we propose that the interaction of the three complexes with sequences from ST-1 and ST-2 (including the Inr element) is of low enough affinity that the interactions can only be inferred in the presence of high concentrations of oligonucleotides used during competition. On the basis of the data presented in Fig. 3, we conclude that the E2F-like site is required for initiating complex formation and the protein complexes may contact other sequences within the PCNA promoter.
Figure 4:
Specificity of nuclear factors binding to
PCNA E2F-like site. A, EMSA of nuclear factors binding to PCNA
E2F-like site. PCNA ST-3 (wt), ST-3m1 (m1), and
ST-3m2 (m2) were P-labeled and incubated with L2
cell nuclear extract. B,
P-labeled ST-3 was
incubated with 24 h IL-2-stimulated L2 cell nuclear extract. Unlabeled
oligonucleotides ST-3, ST-3m1, and ST-3m2 were used as competitors.
Increasing amounts of each competitor (0, 1, 5, and 20 ng) were used in
the binding reactions. Complex I and II are the most prominent bands.
Low amounts of complex III can also be found. The faster migrating
bands (a1, a2, a3, b, and c) were variable in
repeated experiments. Free probe (free) runs at the bottom of
the gel.
The results of a
competition binding experiment are shown in Fig. 4B.
Consistent with the results from Fig. 4A, mutations in
the E2F-like site, both ST-3m1 and ST-3m2, reduce the inhibition of
complex formation with the wild type ST-3 probe. The relative binding
affinity for the wild type and mutated E2F-like sequences is: ST-3
ST-3m2 > ST-3m1. In addition to complexes I, II, and III, a series
of fast migrating complexes could be detected with the radiolabeled
ST-3 probe. The two bands immediately below complex II (a1 and a2) and
the fastest migrating band (a3) show a competition pattern similar to
complex II suggesting that they may also form on the E2F-like site.
However, the nature of these binding complexes is not ascertained
because of their low abundance and because they are only present in
some of our nuclear extract preparations. The relatively abundant fast
migrating bands (b and c) were judged to be nonspecific for the
E2F-like site, band c might represent an SP-1 related binding factor
since band c is competed by ST-3 m2 but only very poorly by ST-3m1. The
functional relevance of these binding activities is unknown.
Fig. 5shows that mutations in the E2F-like site (M-1 and M-2) cause partial reduction in PCNA promoter activity. The relative promoter strengths of the wild type promoter and the two promoter mutants are consistent with the results of the relative binding affinity determined by EMSA (Fig. 4C), wt > M-2 > M-1. These data demonstrate that the E2F-like binding activity is required for optimal PCNA promoter activity and transcriptional activation of the PCNA gene in IL-2-stimulated T cells.
Figure 5: Effect of mutations at the E2F-like site on PCNA promoter activity in L2 cells. pPCNA5-LUC construct containing -182 to +143 genomic sequence of the mouse PCNA gene and two E2F-like site mutation constructs (M-1 and M-2) were transiently transfected into L2 cells. The promoterless vector pSVOA was also transfected as a negative control. To assure efficient transfection, L2 cells were stimulated with IL-2 (10-20 h) prior to transfection. Luciferase activities are presented as the percentage of luciferase activity derived from transfection of the wild type PCNA construct, pPCNA5-LUC (pPCNA5). All results were normalized to luciferase DNA content as measured by DNA slot blotting. Results represent the average of duplicates. The experiment was repeated three times with similar results. Standard deviations of luciferase activity are indicated.
Figure 6: Methylation interference analysis of nuclear proteins binding to PCNA E2F-like binding site. A, coding strand of the PCNA ST was end-labeled at the 5`-end. The probe DNA was partially methylated with dimethyl sulfate before addition to the binding reaction. Labeled DNA in the free (F) and bound (I and II) bands were recovered, cleaved at sites of methylation, and analyzed by urea-PAGE. The methylated guanine residues that interfere with binding of proteins are depicted at the side (G). Maxam and Gilbert sequencing, G, G + A, T + C, and C reactions were performed and loaded in parallel to confirm the positions of each guanine residue in the PCNA sequence. B, analysis of the non-coding strand of the PCNA probe (5`-end labeled), the same procedure was performed as above. C, sequence of PCNA ST and its methylation interference pattern are summarized.
Methylation interference analysis for both of the DNA strands is shown in Fig. 6. Maxam and Gilbert DNA sequencing reactions were performed to confirm the positions of the G-residues in the PCNA promoter sequence. Methylation interference analysis of the shifted bands corresponding to complex I and complex II resulted in identical interference patterns centered at the E2F-like site on both strands. The pattern shows specificity for the E2F-like site on both strands with the top (coding) strand results more clearly overlapping with the E2F-like motif. The top strand contains three G-residues within the E2F site that interfere with E2F binding when methylated. On the bottom strand, one of the two G-residues within the E2F-like site shows methylation interference. The second G residue in the center of the E2F-like motif is very poorly cleaved in the free DNA and, thus, it is impossible to determine whether there is methylation interference at this position.
This pattern is consistent with the results obtained from both EMSA and competition experiments (Fig. 4). Notably, the Inr sequence CATT located at -2 to +2 is not protected in the methylation interference analysis suggesting that the nuclear protein is in close contact with DNA at the E2F-like site. Our data show variable and weak methylation interference at the PEA3 site which we believe is inconclusive and requires further experimentation to determine if there is protein binding at this site. Taken together, we conclude that the major binding complexes form at the E2F-like sequence between +10 and +17.
Figure 7:
Comparison of complex formation with the
PCNA E2F-like site and DHFR-E2F motif. PCNA ST-3 (PCNA), hamster
DHFR-E2F oligonucleotides (E2F), and DHFR-E2F with mutation at the E2F
motif (E2Fm) were P-labeled and incubated with 24-h
IL-2-stimulated L2 cell nuclear extract. Poly(dI-dC) or salmon sperm
DNA was used as nonspecific competitor DNA. They each gave different
binding complexes. In addition to nonspecific DNA, various
oligonucleotides (50 ng) containing PCNA ST-3 (PCNA), DHRF-E2F, or
DHFR-E2F mutation (DHFR-E2Fm) were used as competitors in the binding
reactions. The specific PCNA binding complexes (I and II) and specific
E2F binding complex are indicated.
Several labeled bands were obtained with different doses of UV irradiation. The specificity of the photoaffinity labeling was determined by adding either nonspecific or specific DNA to the binding reactions. The labeling of the 180-kDa band was not competed with an excess of nonspecific oligonucleotide in the binding reaction (Fig. 8, lanes 3 and 7) but was efficiently competed with a specific competitor: an oligonucleotide bearing PCNA E2F-like site (Fig. 8, lanes 4 and 8). The binding specificity of the labeled species of 30- and 43-kDa proteins were not clear. These data demonstrated that a 180-kDa protein binds specifically to PCNA E2F-like site. It is distinct from the known E2F-binding proteins because E2F family members have been characterized to be 40-60 kDa.
Figure 8:
Biochemical characterization of the
E2F-like binding activities. UV cross-linking of proteins to P-labeled PCNA ST probe was performed as described under
``Materials and Methods.'' Samples were UV irradiated for
either 30 min (lanes 1-4) or 60 min (lanes
5-8) as indicated. A band of approximately 180 kDa is
labeled (-). Competition experiments were performed in the
presence of different unlabeled oligonucleotides. Lanes 1 and 5, with no nuclear extract; lanes 2 and 6,
with nuclear extracts in the absence of competitor; lanes 3 and 7, with nuclear extracts in the presence of
nonspecific-competitor (NS) oligonucleotides (containing a
CCAAT box); lanes 4 and 8, with nuclear extracts in
the presence of ST as a specific competitor (S).
In mammalian cells, stimulation of quiescent cells by serum
or growth factors causes a marked increase in the mRNA levels of PCNA,
and other genes coding for proteins of the DNA synthesis machinery,
such as DHFR, thymidine kinase, DNA polymerase , and thymidine
synthase (Pardee, 1989; Nevins, 1992).
The growth factor-responsive expression of the mouse PCNA gene in L2 cells requires elements within a 180-base pair region immediately upstream of the transcriptional start site (Huang et al., 1994). Previously we have shown that tandem cAMP response element binding protein/ATF binding sites located at nucleotides from -37 to -52 in the PCNA promoter are critical for IL-2-induced PCNA promoter activity (Huang et al., 1994). In this report, we identify an E2F-like motif with the sequence 5`-TTGCGGGC-3` located between +10 and +17 of the murine PCNA promoter that is required for PCNA transcriptional regulation. It is worth noting that in several other genes, including the hamster and murine DHFR promoters, E2F elements are found within a sequence near transcription start sites and are critical for transcriptional regulation (Blake and Azizkhan, 1989; Means et al., 1992).
Transcription factor E2F was originally identified as a protein binding to a cis-element needed for the activation of the adenoviral early E2 gene promoter (Kovesdi et al., 1986; Berk, 1986). There are at least six similar but not identical E2F-like sites found in promoters of several cellular genes, with the sequence often being TTTCGCGC or TTTGGCGC (Mudryj et al., 1990; Nevins, 1992). Regulation of E2F activity involves its interactions with multiple cellular proteins including pRB, cyclin A, and cdk2 (Nevins, 1992). The retinoblastoma protein pRB as a tumor suppressor forms a specific complex with E2F providing an important mechanism for pRB regulation of cell growth. E2F-1 cDNA was cloned by virtue of its ability to bind to pRB (Helin et al., 1992). Soon after its cloning, it became apparent that the term E2F represents a family of transcription factors including E2F-1, DP-1, E2F-2, E2F-3, E2F-4, and E2F-5 (La Thangue, 1994; Lees et al., 1993; Ginsberg et al., 1994; Sardet et al., 1995). Recent studies have shown E2F-1 and DP-1 normally form heterodimers in binding to the E2F site. Therefore, E2F is an expanding group of related transcription factors with similar DNA-binding specificity that appear to function in cell cycle regulation.
Although there is sequence similarity between the PCNA promoter +10 to +17 region and the consensus E2F binding site, we concluded that distinct proteins bind to the PCNA E2F-like site based on the following observations: 1) there is no cross-competition between the PCNA E2F-like site and an oligonucleotide containing the E2F consensus sequence from the DHFR promoter (Fig. 7) or the E2F site from the adenoviral E2 promoter (data not shown). 2) The optimal condition for complex formation differs, when used as a nonspecific competitor DNA in the binding reactions, poly(dI-dC) favors complex formation with the PCNA E2F-like site while salmon sperm DNA favors complex formation with the DHFR-E2F site. 3) Results from a UV cross-linking experiment indicate that a protein of 180 kDa binds to the PCNA E2F-like site. In contrast E2F-1 is a 54-kDa nuclear protein previously identified in cross-linking experiments by several groups (Mudryj et al., 1990; Chellappan et al., 1991). The other E2F proteins have molecular mass ranging from 40 to 60 kDa. Therefore, this 180-kDa protein binding to the PCNA E2F-like site is distinct from all known E2F family proteins. However, we cannot rule out the possibility that this 180-kDa protein is related to the E2F family of transcription factors.
RNA polymerase is known to bind to DNA at the transcription start site to form an initiation complex with general transcription factors such as TFIID. Three forms of RNA polymerase II large subunit have been described, each differing in apparent molecular mass, 180, 210, and 240 kDa (Sawadogo and Sentenac, 1990; Young, 1991). Is it possible that the 180-kDa protein binding next to the initiation site represents RNA polymerase? Our results argue that this protein is not RNA polymerase because: first, methylation interference data indicate that the 180-kDa protein contacts a small region (nt +12 to +19) within or adjacent to the E2F-like site, while previous analyses have revealed that RNA polymerase protects a much larger region of DNA on both sides of the initiation site (Sawadogo and Sentenac, 1990); second, the interaction of the 180-kDa protein with DNA requires the E2F-like site, while RNA polymerase is thought to bind to DNA in a nonspecific manner.
A similar situation exists with studies of the DHFR promoter and our findings with the mouse PCNA promoter. E2F proteins have been shown to bind to the E2F site in the murine DHFR promoter between nt -10 and -3 (Slansky et al., 1993) but Means et al. (1992) have shown that HIP1, a 180-kDa protein also binds near this site at -9 to -1. This sequence has been shown to function as the initiator element that controls the start site for murine DHFR transcription (Means and Farnham, 1990). The protein binding to the PCNA E2F-like site that we characterized in this report is similar to HIP1 protein. 1) Both proteins have molecular mass of approximately 180 kDa. 2) Both bind to a sequence at or adjacent to the transcription initiation site. 3) Both proteins contact sequences which share homology to the E2F site. Our data suggest that the 180-kDa protein does not bind to the hamster DHFR E2F site (Fig. 7). We noticed that the murine DHFR E2F motif contains a G as the second base 5` of the E2F site, in contrast, the hamster E2F motif contains an A at the -2 position. Previously it has been shown that the sequence adjacent to the murine DHFR E2F core sequence is required for complex formation of HIP1 (Means et al., 1992). Therefore a difference of the sequences in the -2 position of the DHFR E2F elements may account for the observation that this 180-kDa protein binds to the PCNA E2F-like site as well as the murine DHFR E2F motif but not to the hamster DHFR E2F motif.
Mutations that abolish complex formation on this E2F-like site only reduced PCNA promoter activity moderately, to about 60-80% of the wild type promoter activity. One of the explanations for this is that other cis-elements including the tandem CRE/ATF binding sites in the PCNA promoter also contribute substantially to optimal promoter activity. Further experiments making double or multiple mutations of these cis-elements in the PCNA promoter should help to determine the cooperation between these sites for PCNA promoter activity.
The identification of an E2F-like binding site in the PCNA promoter and of nuclear proteins interacting with this E2F-like site in this report provides a basis for further study on the mechanism of PCNA transcriptional regulation and contributes to our understanding of the mechanisms by which IL-2 activates T lymphocyte proliferation.