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INTRODUCTION |
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
, 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).
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MATERIALS AND METHODS |
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),
-galactosidase
(pCMV
-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
-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).
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).
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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 [
-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 pCMV
-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.
-Galactosidase assays were performed as
described previously (32), and
-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
-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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RESULTS |
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).
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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.
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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).
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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 -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.
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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.
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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.
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DISCUSSION |
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.