We have previously identified a novel cell cycle-regulated
DNA-binding protein termed p92, which interacts with multiple p92
recognition sites within the enhancer of human papilloma virus type 18
(HPV18)(
)(1) . HPV18 belongs to the group of human
papilloma viruses with high oncogenic potential(2) . p92 is a
sequence-specific nuclear factor that specifically binds to the
5`-AATTGCTTGCATAA motif and other related motifs in the HPV18 enhancer (1, 3) . p92 was originally identified by binding site
blotting experiments(1) , and retarded p92s containing
DNA-protein complexes were identified by preparative electrophoretic
mobility shift experiments and analysis of eluted proteins by binding
site blotting (4) . In addition, the 5`-AATTGCTTGCATAA motif is
recognized by p92-related factors as was shown by UV cross-linking
experiments(3) . p92, however, is the most prominent retarded
DNA-protein complex in electrophoretic mobility shift
assays(4) . The HPV18 p92 recognition sequence
5`-AATTGCTTGCATAA consists of two partially overlapping octamer-related
motifs(1, 3) . We have shown by electrophoretic
mobility shift assays (EMSAs) that in vitro translated Oct-1
protein interacts with the 5`-AATTGCTTGCATAA motif. p92, however, is
not related to Oct-1, as was shown by immunoblotting using an Oct-1
antiserum(1) . In addition, the intracellular distribution of
p92 is regulated by growth factors, because p92 was found in the
cytoplasm of serum-starved nontumorigenic HeLa-fibroblast hybrid cells
(444 cells) and was translocated to the nucleus upon serum stimulation
at the beginning of S phase(1, 4) . We have shown by
centrifugal elution experiments that during the cell cycle p92 DNA
binding activity is restricted to S phase of 444 cells that contain
integrated human papillomavirus type 18(4) . In crude nuclear
extracts, we have identified an uncharacterized activity, termed I-92,
which inhibits DNA binding of p92(4) . In cell cycle
populations of 444 cells, p92 DNA binding activity could not be
detected in nuclear extracts from G
phase and from G
phase cells. It was, however, possible to detect p92 after
treatment of these extracts with the detergent
deoxycholate(4) , suggesting that p92 was complexed to an
inhibitor in G
and in G
phase of the cell
cycle.
In the present communication we characterized I-92
biochemically and functionally. We have delineated a molecular
mechanism underlying p92 cell cycle regulation and identified four
nuclear inhibitors that link cell cycle regulation with p92 regulation.
In normal diploid human fibroblasts (NHDFs), p92 DNA binding is
restricted to S phase. In addition, we have determined that p92 may act
as a transcription factor because a p92 binding site from the HPV18
upstream regulatory region conferred enhancer activity on a
heterologous promoter. The p92/I-92 system, therefore, may represent a
novel mechanism of phase-specific gene regulation.
EXPERIMENTAL PROCEDURES
Cell Culture, Cell Synchronization, and Preparation of
Extracts
Normal human fibroblasts (PromoCell) and the
nontumorigenic HeLa-fibroblast hybrid cell line 444 (5) were
maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Life Technologies, Inc.).
Nuclear extracts were prepared as described (6) with some
modifications(1) . In brief, cells were scraped from plates,
washed with phosphate-buffered saline, and then lysed with 0.65%
Nonidet P-40 (Sigma), and nuclei were prepared by low speed
centrifugation at 4 °C. Nuclear proteins were eluted with 520
mM NaCl and slight agitation followed by dialysis against a
buffer containing 50% glycerol, 50 mM NaCl, 10 mM Hepes (pH 7.9), 0.5 mM phenylmethylsulfonyl fluoride, and
0.5 mM dithiothreitol. Lovastatin (a generous gift of Merck,
Sharp and Dome) was used to prepare G
phase 444 cells (7, 8) for the extraction of nuclear proteins and the
analysis of I-92 during the cell cycle. Semiconfluent fibroblasts and
444 cells were cultivated for 24 h in the presence of 20 µM lovastatin and then processed for the extraction of nuclear
proteins. The efficiencies of G
phase synchronization were
determined by nuclear staining with propidium iodide and
fluorescence-activated cell sorting (FACS) measurements of the DNA
content. FACS analysis showed that 70% of cells had a diploid DNA
content (2n), S phase cells were 14%, and 4n cells were 15%. The drug
aphidicolin blocks the activity of DNA polymerase
(9, 10, 11) . We used this reagent to
synchronize 444 in S phase of the cell cycle. In brief, we treated
fibroblasts and 444 cells in culture for 24 h with 3 µM aphidicolin followed by a 3-h release in fresh medium. Under those
conditions FACS analysis showed that 65% of cells were in S phase, 28%
were 4n, and 6% were 2n. We used an aphidicolin release strategy to
obtain 444 cells in G
phase of the cell cycle. We
determined empirically that after a 24-h aphidicolin treatment (3
µM) and a 10-h release, 87% of the cells were 4n as was
shown by FACS analysis. In addition we controlled this synchronization
event by Western blot analysis using anti-cyclin B antibodies, because
cyclin B expression is restricted to G
phase of the cell
cycle(12, 13, 14) . We found strong
expression of cyclin B in G
phase-synchronized 444 cells,
whereas no signal or very little signal was evident in G
phase and in S phase 444 cells. The latter result is another good
control for S phase synchronization because cyclin B is already
expressed early in G
phase. The drug nocodazole blocks the
formation of microtubules, which mediate chromosome segregation during
mitosis(15, 16) . Treatment of 444 cells with
nocodazole (10 µg/ml) for 18 h yields floating cells that no longer
attach to plastic. These shake-off cells are in mitosis, and they were
used as a source for the preparation of ``mitotic'' nuclear
extracts. This preparation contained 86% 4n cells as was measured by
FACS analysis. Deprivation of growth factors or cultivation in low
serum leads to a reversible exit from the cell cycle into the G
phase(17, 18) . In order to obtain G
phase 444 cells, we grew these in 0.05% fetal calf serum for 48
h. FACS analysis, however, will not be able to discriminate between
G
phase and G
phase cells because both have a
2n DNA content. We used the nuclear proliferation marker Ki67 (19) as a measure to assess G
synchronization,
because Ki67 is expressed in cycling but not in resting G
cells.
Determination of the Cellular DNA Content by FACS
Analysis
5
10
cells were treated with
ethanol for 1 h at -20 °C. Subsequently, cells were washed
with phosphate-buffered saline, suspended in 500 µl of
phosphate-buffered saline containing 40 U/ml of RNase A and 100
µg/ml propidium iodide, incubated for 30 min at 37 °C, and
analyzed by the fluorescence-activated cell sorter using the Cellquest
program (Beckton Dickinson).
Transient Transfection Assays
The p92 recognition
site present in RP3 was cloned in pGL-2 enhancer tester construct;
likewise, a p92 binding mutant RP3-
was cloned in pGL-2 (Promega).
As a control for transfection efficiencies pSV-gal (Promega) and
pCMV-gal (Promega) were used. 1.8
10
444 cells were
transfected using the calcium phosphate technique and 10 µg of
either pRP3-GL-2 or pRP3-/RP3-
and in combination with 5 µg of
pSV-gal or 5 µg of pCMV-gal as internal control. After 24 h, fresh
Dulbecco's modified Eagle's medium was added, and the cells
were incubated for an additional 48 h. Cell lysis and processing of
lysates for luciferase measurements was according to the recommended
procedures by the producer of the luciferase kit (Promega) and a
luminometer (Berthold). The internal control for transfection
efficiency was measured using a Galactolight kit (Tropix).
Preparation and Biochemical Characterization of
I-92
Crude I-92 was prepared following a published protocol (20, 21) with modifications (3, 4) from fibroblasts and 444 cells. In brief,
nuclear extracts were treated with the detergent deoxycholate (DOC),
passed over heparin-Sepharose after the addition of Nonidet P-40 and
the flow-through fraction (crude I-92) was collected. Crude I-92 (200
µg) was size-separated by Sephacryl S300 HR chromatography (500
7-mm inner diameter) using electrophoretic mobility shift
(EMS)-DNA binding buffer as column buffer (1) and a flow rate
of 0.1 ml/min at 4 °C. In each experiment 80 fractions (200 µl)
were collected and assayed for I-92 activity (50 µl). In other
experiments crude I-92 (5 mg) was fractionated by Mono Q-Sepharose.
After loading, the column was washed with 1 column volume (2 ml) of
column buffer, 5% glycerol, 20 mM Hepes (pH 8), 50 mM NaCl, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. Subsequently, bound proteins were
eluted with a NaCl step gradient as indicated in the figures. Each
eluate consisted of 2
0.5-ml fractions, and for the
determination of I-92 activity 25 µl were used. All eluates were
dialyzed using EMS-DNA binding buffer. I-92 activity in all experiments
was monitored by a p92 electrophoretic mobility shift inhibition assay
(EMSIA).
Renaturation of I-92 after SDS-PAGE
Crude I-92 (1
mg) from 444 cells was size-separated by SDS-PAGE (10%). After
electrophoresis the gel was cut in slices, and proteins were eluted
overnight using an elution buffer (750 µl) containing 10 mM Tris, pH 7.9, 100 mM NaCl, 5 mM dithiothreitol,
0.1 mM EDTA, 0.1% SDS, and 0.1 mg/ml insulin as carrier.
Eluted proteins were precipitated with acetone and resuspended in
EMS-DNA binding buffer containing 6 M urea. Denatured proteins
were subsequently renatured after an overnight dialysis against 3 M urea followed by dialysis against EMS-DNA binding buffer. The
procedure followed published
protocols(20, 21, 22) . I-92 activity was
monitored by a p92 EMSIA.
EMSA, Immunoshift, p92 EMSIA, and I-92 Unit
Definition
EMSA was performed as previously
published(1) . For the detection of AP-1 a consensus sequence
was used (Santa Cruz), for the detection of E2F a consensus
oligonucleotide was used(23) , and for the detection of
NF-
B a consensus oligonucleotide was used(24) .
Double-stranded oligonucleotides (BioTez) were radioactively labeled as
published(25) . For immunoshift analysis an anti-Fos rabbit
polyclonal antiserum (Medac) was used at the concentrations indicated
in the legend to Fig. 3. As a control an unrelated rabbit
polyclonal antiserum directed against a human endogenous retrovirus
antigen (a gift of R. Kurth, Paul Ehrlich Institute, Langen) was used
at the concentrations indicated in the legend to Fig. 3. For the
quantitation of I-92 activity we used partially purified p92 as a
target and an electrophoretic mobility shift as a read-out system. A
nuclear extract of 444 cells was treated with deoxycholate and passed
over heparin-Sepharose. p92 was eluted with 500 mM NaCl and
dialyzed against EMS-DNA binding buffer, 10 mM Tris (pH 7.5),
50 mM NaCl, 50 mM dithiothreitol, 4% glycerol. In all
electrophoretic mobility shift inhibition assays, 0.4 µg from the
same p92 preparation were used. For EMSIA I-92, preparations were
incubated with 0.4 µg of partially purified p92 at room temperature
(20 min), and end-labeled RP3 double-stranded oligonucleotide (15,000
cpm) and 200 ng of poly(dI-dC)(dI-dC) were added and incubated for 15
min. Subsequently DNA-protein complexes were analyzed on a 4% native
polyacrylamide gel using 0.25% TBE (25) as a running buffer.
Dried gels were exposed at -70 °C using Kodak X-Omat AR
films.
Figure 3:
Selectivity of I-92 inhibition. EMSA with
a [
-
P]ATP end-labeled AP-1 consensus
recognition site (Santa Cruz) as a probe and a nuclear extract from 444
cells. Free AP-1 oligonucleotide and retarded AP-1 complexes are
indicated by arrows (lane 1). As a specificity
control for the identification of the retarded AP-1 DNA-protein complex
a polyclonal anti-Fos antiserum (Medac), 1 and 5 µg was included in
the binding reaction (lanes 2 and 3). Lanes 4 and 5 contained the same amounts of an unrelated rabbit
polyclonal anti-Gag antiserum directed at a human endogenous retrovirus
antigen. In order to test whether I-92 affects DNA binding of AP-1 (lane 6), crude I-92 was included in the binding reaction
prior to the addition of the probe: 0.5 units of crude I-92 (lane
7), 1 unit of crude I-92 (lane 8), and 2 units of crude
I-92 (lane 9). EMSA was performed as described under
``Experimental Procedures.''
Western Blot Analysis
SDS-polyacrylamide gel
electrophoresis and Western immunoblot analysis were performed
according to standard procedures(25) . Samples (30 µg) were
mixed with an equal volume of twice concentrated Laemmli sample buffer (26) and boiled for 5 min. Samples were subjected to SDS-PAGE
(10% polyacrylamide), and the proteins were transferred onto
nitrocellulose (Bio-Rad). The blots were blocked with 10% dried milk
(Carnation) in TTBS buffer (25) for 2 h. Rabbit polyclonal
antiserum directed against a peptide of the cyclin B protein was used
at 1:200 dilution, and a monoclonal antiserum directed against the Ki67
antigen (Dianova) was used at 1:300 dilution. Detection of
immunoreactive bands was by the enhanced chemiluminiscence method
(Amersham Corp.).
RESULTS
Our previous work suggested that the DNA binding activity of
nuclear factor p92 is cell cycle-regulated by an inhibitor termed I-92 (4) . It was the aim of this study to characterize I-92 and p92
biochemically and functionally.
Cell Cycle Regulation of p92 in Normal Human Diploid
Fibroblasts
As shown previously, p92 DNA binding activity is
cell cycle-regulated in 444 cells, which are immortalized,
nontumorigenic HeLa-fibroblast hybrid cells harboring integrated human
papillomavirus type 18 DNA(1) . First we wanted to address the
question of p92 regulation in a nontransformed normal human cell
population. We therefore analyzed p92 activity in NHDFs. NHDFs were
synchronized in tissue culture using lovastatin for G
phase
cells, aphidicolin and a 3-h release for S phase cells, aphidicolin and
a 7-h release for G
phase cells, nocodazole for mitotic
cells, and serum starvation for G
phase cells. From each
synchronized cell population nuclear extracts were prepared and
examined for p92 DNA binding activity by EMSAs. p92 was not detectable
in serum-starved G
phase cells (Fig. 1, lane
1), almost undetectable in lovastatin-treated G
phase
cells, and likewise almost undetectable in G
phase cells (Fig. 1, lanes 2 and 4). In S phase cells,
however, large amounts of p92 were present (Fig. 1, lane
3). In addition, p92 was almost undetectable in mitotic cells (Fig. 1, lane 5). We conclude from these results that
p92 is a factor that operates in normal human diploid fibroblasts and
that p92 activity is restricted to S phase of the cell cycle. These
results were also confirmed by centrifugal elution of NHDF cells. (
)
Figure 1:
Cell cycle
regulation of p92 in synchronized human fibroblasts. Electrophoretic
mobility shift analysis of p92 in nuclear extracts from serum-starved
fibroblasts (lane 1), from lovastatin-treated fibroblasts (lane 2), from aphidicolin-treated fibroblasts and a 3-h
release (lane 3), from G
phase fibroblasts after
aphidicolin treatment and a 7-h release (lane 4), and from
nocodazole treated fibroblasts (lane 5). EMSA was performed as
described under ``Experimental Procedures.'' Free RP3
oligonucleotide and retarded p92-RP3 complexes are indicated by arrows. The probe used was a
[
-
P]ATP end-labeled RP3 double-stranded
oligonucleotide containing a p92 recognition
sequence.
The p92 Recognition Motif Confers Activation
of a Heterologous Promoter
One goal of this study was to
characterize p92 functionally. In order to do so, we wanted to
determine whether p92 might act as a transcription factor. We have
previously characterized multiple p92 recognition sites in the enhancer
of HPV18 (1) and presented evidence that the 5`-AATTGCTTGCATAA
motif, consisting of two partially overlapping octamer-related
sequences specifically interacted with p92 and other p92-related
proteins, as was shown by UV cross-linking(3) . EMSAs revealed,
however, that in S phase the retarded RP3-p92 complex is always the
most prominent DNA-protein complex (Fig. 1, lane
3)(4) . The 5`-AATTGCTTGCATAA motif is part of the longer
oligonucleotide RP3, which was previously identified as a p92 binding
sequence element(1, 3) . In oligonucleotide RP3-
the 5`-TTGCTTGCATAA sequence of the 5`-AATTGCTTGCATAA motif was
deleted, and we showed that p92 no longer bound to the RP3-
mutant (3) . In addition, we have shown previously that the deleted
5`-TTGCTTGCATAA sequence binds p92 in a sequence-specific
manner(3) . For functional analysis oligonucleotides RP3 and
RP3-
were cloned in the enhancer tester luciferase construct pGL-2
and assayed in the nontumorigenic HeLa-fibroblast hybrid cell line 444.
RP3 mediated activation of the luciferase reporter gene to a level of
12.9% when compared with the cotransfected pSV-
-gal control,
whereas the deletion mutant RP3-
did not confer significant
luciferase gene activation (Fig. 2). Reporter gene assays were
repeated 6 times with two different DNA preparations and were
reproducible. The error bars in the control experiments pGL-2 and
RP3-/delta are not visible due to low luciferase activities of these
constructs. These experiments showed that the p92 recognition site
conferred transcriptional activation, and suggests that proteins that
specifically bind to this sequence act as transcription factors. We
assume that p92 was responsible for transcriptional activation in these
experiments because it is the major retarded complex detectable by EMSA
in nuclear extracts of 444 cells(4) . It was not part of this
work to analyze in detail the sequence requirements for p92 DNA
binding.
Figure 2:
p92-mediated activation of the SV40
minimal promoter in pGl-2 enhancer tester construct. RP3
oligonucleotide containing a p92 recognition site was cloned in pGl-2
enhancer tester vector. RP3-
is a p92 binding mutant where the p92
binding sequence was deleted. Transfection efficiencies were controlled
by cotransfection of a pSV-
-gal reporter construct. All transient
expression assays were done in triplicate using two different DNA
preparations as described under ``Experimental Procedures.''
Luciferase activity was measured in a luminometer, and the values are
indicated in the figure as relative light units (RLU)
of the cotransfected pSV-
-gal control. The error bars of the
control and the p92 binding mutant RP3-
are not visible due to low
luciferase activities of these constructs.
Functional and Biochemical Characterization of
I-92
A key question is how gene expression is coordinated with
cell cycle progression. In this report we have shown that a p92 binding
site conferred activation of the SV40 minimal promoter present in
reporter gene vector pGL2. We have established previously that in the
HeLa-fibroblast hybrid cell line 444, which contains integrated human
papillomavirus type 18, p92 DNA binding was restricted to S phase of
the cell cycle, and we have presented evidence that the DNA binding
activity of p92 could be regulated by a putative nuclear
inhibitor(4) . This assumption was based on our observation
that p92 could be released from putative I-92-p92 complexes by
treatment with the detergent DOC (4) . Based on the observation
that p92 acts as a transcription factor and that p92 might be cell
cycle-regulated by I-92, we hypothesized that I-92 could link cell
cycle regulation with transcription factor regulation. In order to
characterize I-92 we have accomplished a biochemical and functional
analysis of I-92 activities. In the following we summarize several
features that candidate I-92 proteins must show in order to be
considered as I-92. 1) The DNA binding activity of p92 should be
inhibited by partially purified I-92. 2) I-92 inhibition of p92 DNA
binding should be selective. 3) I-92 inhibition of p92 DNA binding
should be cell cycle-regulated. 4) I-92 inhibition of p92 DNA binding
should be reversible after treatment with the detergent DOC.
Selectivity of I-92 Inhibition
p92 binds to
octamer-related sequences from the HPV18 enhancer(1) . We have
previously shown that ``crude'' I-92 did not inhibit DNA
binding of the Oct-1 transcription factor(3) . In order to
prepare crude I-92, nuclear extracts of 444 cells were treated with the
detergent DOC following a published procedure (4, 20, 21) and passed over
heparin-Sepharose. The flow-through contains crude I-92. In order to
further define selectivity of I-92 inhibition we chose to analyze other
transcription factors whose DNA binding activities are regulated by
specific inhibitors. We examined the transcription factor AP-1, which
is regulated by IP-1(27) . In this experiment an AP-1 consensus
oligonucleotide was used in EMSA. The identity of Fos-Jun retarded
complexes was verified by immunoshift analysis using a polyclonal
rabbit anti-Fos antiserum (Fig. 3, lanes 2 and 3). As a control an unrelated polyclonal rabbit antiserum was
used directed against the Gag protein of a human endogenous retrovirus
HERV-K (Fig. 3, lanes 4 and 5). The addition
of increasing amounts of crude I-92 did not affect AP-1 retarded
complex formation, suggesting that AP-1 is not regulated by I-92 (Fig. 3, lanes 7, 8, and 9). The I-92
unit definition was described(3) . In addition we found that
DNA binding of neither NF-
B, which is regulated by
I-
B(25) , nor of E2F(23, 28) , which is
regulated by pRB(29) , was regulated by I-92. (
)These results demonstrate that I-92 inhibition is
selective for p92.
Biochemical Characterization of I-92 Proteins
As a
source of crude I-92 we used the nontumorigenic HeLa-fibroblast hybrid
cell line 444(5) , because the limited life span and slow
growth rates of normal diploid human fibroblasts preclude large scale
cell culture, which is required for I-92 preparations. Crude I-92
prepared from 444 cells was size-separated by Sephacryl S300
chromatography. Individual Sephacryl fractions were assayed for I-92
activity by a p92 EMSIA using a defined amount of partially purified
p92 (0.4 µg) and the p92 recognition site RP3 as a probe. These
experiments reproducibly revealed I-92 activities in fractions
46-53 of the S300 column, corresponding to a molecular mass range
from 15 to approximately 50 kDa (Fig. 4A), suggesting
that either multiple I-92 proteins exist or that I-92 proteins form
oligomers.
Figure 4:
Identification of diverse I-92 proteins. A, a nuclear extract from 444 cells was treated with the
detergent DOC following published
procedures(4, 20, 21) , and passed over
heparin-Sepharose. The flow-through fraction termed ``crude
I-92'' was size-fractionated by chromatography on Sephacryl S300
HR. Individual Sephacryl S300 fractions were analyzed for I-92 by an
EMSIA, as described under ``Experimental procedures'' using
partially purified p92 and [
-
P]ATP
end-labeled RP3 double-stranded oligonucleotide as a probe. Previous
experiments established that all detectable I-92 activities from 444
cells were present between fractions 46 and 52 in our Sephacryl S300
column. B, crude I-92 was size-fractionated by SDS-PAGE, after
electrophoresis the gel was dissected into small gel slices. Proteins
were eluted from individual slices in elution buffer and renatured.
I-92 activity from individual gel slices was monitored by EMSIA. C, crude I-92 was fractionated by Mono Q chromatography and
elution with a salt step gradient. Each salt elution was in two steps
of 0.5 ml. I-92 was monitored by the EMSIA: p92 control (lane -), Mono Q flow-through (lane FT), 100 mM salt (lanes 1 and 2), 200 mM salt (lanes 3 and 4), 300 mM salt (lanes 5 and 6), 400 mM salt (lanes 7 and 8), 500 mM salt (lanes 9 and 10),
600 mM salt (lanes 11 and 12), 700 mM salt (lanes 13 and 14), 800 mM salt (lanes 15 and 16), and 900 mM salt (lanes 17 and 18). Free RP3 and the p92 retarded
complex are indicated by arrows. The faster migrating
RP3-protein complexes in lanes 2, 3, 5, and 6 are derived from the I-92
preparation.
Identification of Diverse I-92 Proteins
In order
to address the issue whether diverse I-92 proteins exist we have
size-fractionated crude I-92 on reducing 10% SDS-polyacrylamide gels
with the aim of recovering I-92 for EMSIAs. After electrophoresis, a
gel area corresponding to an individual lane was cut in small segments,
and proteins were eluted, renatured, and assayed for I-92 activities by
EMSIA (Fig. 4B). In order to determine molecular
weights of I-92 and to monitor protein recovery, eluted proteins were
analyzed by SDS-PAGE and silver staining.
I-92 activities
were recovered from gel slices 2, 11, 12, 13, and 14 (Fig. 4B). The apparent molecular masses range from 15
(slice 2) to 50 kDa (slice 14). In Fig. 4B eluates from
slices 6-8 are not shown because they did not contain detectable
I-92 activity. Given the fact that a reducing SDS-PAGE was used in this
experiment we conclude that diverse I-92 proteins exist. In order to
characterize I-92 further, we have analyzed crude I-92 by Mono Q
chromatography. After loading crude I-92 onto the Mono Q column the
flow-through fraction was collected. Subsequently, bound proteins were
eluted with a NaCl step gradient, and from each salt step two 0.5-ml
fractions were collected. All I-92 activities were retained on the
column, because the flow-through fraction did not contain any
detectable I-92 activity (Fig. 4C, lane FT).
The Mono Q fractionation yielded four different salt fractions with
I-92 activity: 100 mM NaCl, 200 mM NaCl, 700 mM NaCl, and 800 mM NaCl, as was shown by EMSIA (Fig. 4C, lanes 2, 4, 13, 14, 15, and 16). These results strongly
support the observation that diverse I-92 proteins exist. Note that the
faster migrating complexes in lanes 2, 3, 5,
and 6 were derived from the crude I-92 preparation, because
they could be detected by EMSA without added p92, suggesting that they
are not p92 degradation products that could have been generated during
the electrophoretic mobility shift inhibition assay.
Some
of the fast moving complexes in fraction 2 of the 200 mM NaCl
eluate, however, may have been generated, at least in part, during the
electrophoretic mobility shift inhibition assay (see below). In
contrast, none of these faster moving complexes could be detected
either in SDS-PAGE eluates containing the four I-92s (Fig. 4B, lanes 2, 11, 12, 13, and 14) or in Sephacryl S300 fractions with I-92
activity (Fig. 4A).
Cell Cycle Regulation of I-92 Proteins
Next we
analyzed whether the four different I-92 activities that were
identified by Mono Q chromatography are subject to cell cycle
regulation. The most important feature of I-92 candidate proteins is
that their activities should be cell cycle-regulated. More
specifically, they should be active either in G
phase
and/or in G
phase but not in S phase. We have used
biochemical cell synchronization approaches as outlined under
``Experimental Procedures'' in order to obtain sufficient
amounts of crude I-92 for further characterization by Mono Q
chromatography and EMSIAs. The efficiencies of cell synchronization
were controlled by propidium iodide staining of cellular DNA and FACS
analysis. Western blot analysis using an antibody against the
proliferation marker Ki67 (19) was used for the assessment of
G
phase synchronization, and cyclin B antibodies were used
for the determination of the G
phase synchronization of 444
cells (Fig. 5A). We conclude that our experiments were
efficiently synchronizing G
, S, G
, M, and
G
phase cell populations.
Figure 5:
Cell cycle regulation of I-92:
identification of G
phase, G
phase, and G
phase inhibitors. A, assessment of cell synchrony of 444
cells: FACS analysis of propidium iodide-stained DNA from G
phase cells that were treated with lovastatin (top, left). Shown are FACS analysis of propidium iodide-stained DNA
from aphidicolin treated S phase cells (top, middle)
and FACS analysis of G
phase cells that were treated with
aphidicolin and a 10-h release (top, right). The
efficiency of G
phase synchronization was verified by the
detection of G
phase cyclin B in nuclear extracts of
G
phase 444 cells (bottom, right). FACS
analysis of propidium iodide-stained DNA from nocodazole-treated
mitotic cells (bottom, middle) is shown. G
synchronization (bottom, left), was verified by
analysis of Ki67 expression in serum-starved 444 (G
) cells
by Western blotting using a polyclonal rabbit antiserum (DAKO) directed
against the proliferation marker Ki67. Nuclear extract from
serum-starved 444 cells (lane G
) and from
exponentially growing 444 cells (lane C, bottom
left). As a detection system we used the ECL kit (Amersham).
Details of the biochemical cell synchronization conditions are
described under ``Experimental Procedures.'' B,
crude I-92 from lovastatin-treated 444 cells (G
phase) was
fractionated by Mono Q chromatography (as described in Fig. 4),
and I-92 was monitored by the EMSIA. Each salt elution was in two steps
of 0.5 ml. p92 control (lane -), 100 mM salt (lanes 1 and 2), 200 mM salt (lanes 3 and 4), 300 mM salt (lanes 5 and 6), 400 mM salt (lanes 7 and 8),
500 mM salt (lanes 9 and 10), 600 mM salt (lanes 11 and 12), 700 mM salt (lanes 13 and 14), 800 mM salt (lanes 15 and 16), and 900 mM salt (lanes 17 and 18) are shown. Free RP3 and the p92 retarded complex are
indicated by arrows. C, crude I-92 from
aphidicolin-treated 444 cells (S phase cells) was fractionated by Mono
Q chromatography as above, and I-92 was monitored by EMSIA. Each salt
elution was in two steps of 0.5 ml. p92 control (lane -), 100 mM salt (lanes 1 and 2),
200 mM salt (lanes 3 and 4), 300 mM salt (lanes 5 and 6), 400 mM salt (lanes 7 and 8), 500 mM salt (lanes 9 and 10), 600 mM salt (lanes 11 and 12), 700 mM salt (lanes 13 and 14),
800 mM salt (lanes 15 and 16), 900 mM salt (lanes 17 and 18) are shown. Free RP3 and
the p92 retarded complex are indicated by arrows. D,
444 cells were treated with aphidicolin and a 10-h release. G
phase was confirmed by a cyclin B Western blot and by FACS
analysis of propidium iodide-stained cells. Crude I-92 was prepared
from nuclear extracts of G
phase 444 cells and analyzed by
Mono Q chromatography as above, and I-92 was monitored by EMSIA. Each
salt elution was in two steps of 0.5 ml. p92 control (lane -), 100 mM salt (lanes 1 and 2),
200 mM salt (lanes 3 and 4), 300 mM salt (lanes 5 and 6), 400 mM salt (lanes 7 and 8), 500 mM salt (lanes 9 and 10), 600 mM salt (lanes 11 and 12), 700 mM salt (lanes 13 and 14),
800 mM salt (lanes 15 and 16), and 900
mM salt (lanes 17 and 18) are shown. Free
RP3 and the p92 retarded complex are indicated by arrows. E, crude I-92 from nocodazole-treated 444 cells was analyzed
by Mono Q chromatography as above and EMSIA. Each salt elution was in
two steps of 0.5 ml. p92 control (lane -), 100 mM salt (lanes 1 and 2), 200 mM salt (lanes 3 and 4), 300 mM salt (lanes 5 and 6), 400 mM salt (lanes 7 and 8), 500 mM salt (lanes 9 and 10),
600 mM salt (lanes 11 and 12), 700 mM salt (lanes 13 and 14), 800 mM salt (lanes 15 and 16), and 900 mM salt (lanes 17 and 18) are shown. Free RP3 and the p92
retarded complex are indicated by arrows. F, crude
I-92 was prepared from nuclear extracts of serum-starved 444 cells.
Crude I-92 was fractionated by Mono Q chromatography and elution with
a salt step gradient. I-92 was monitored by EMSIA. Each salt elution
was in two steps of 0.5 ml. p92 control (lane -), 100
mM salt (lanes 1 and 2), 200 mM salt (lanes 3 and 4), 300 mM salt (lanes 5 and 6), 400 mM salt (lanes 7 and 8), 500 mM salt (lanes 9 and 10), 600 mM salt (lanes 11 and 12),
700 mM salt (lanes 13 and 14), 800 mM salt (lanes 15 and 16), and 900 mM salt (lanes 17 and 18) are shown. Free RP3 and the p92
retarded complex are indicated by arrows.
Identification of Two Different G
Phase
Inhibitors
444 cells were synchronized with lovastatin, and
nuclear extracts were prepared. Crude I-92 was isolated after DOC
treatment of the extract and passage over heparin-Sepharose columns.
Subsequently I-92 was fractionated by Mono Q chromatography. I-92 was
eluted with 100 mM increments of NaCl, and two independent
fractions were collected of each eluate. Each salt eluate was assayed
for I-92 activity by EMSIA (Fig. 5B). We discovered
here that I-92 was present in fraction 2 of a 100 mM NaCl
eluate (Fig. 5B, lane 2) and in fractions 1
and 2 of a 700 mM NaCl eluate (Fig. 5B, lanes 13 and 14). For screening I-92 activities in
Mono Q fractions, an arbitrary amount of each fraction was used (25
µl). Note that the 100 mM NaCl I-92 activity seems to be
present in much lower concentrations than the 700 mM NaCl I-92
activity. It is, however, possible to titrate this inhibitory activity
and obtain complete inhibition of p92 DNA binding in EMSIA (see also Fig. 6, lane 2). We conclude that the 100 mM and the 700 mM NaCl Mono Q eluates contain two forms of
I-92 that are active in G
phase of the cell cycle. We call
I-92 from the 100 mM eluate G
I-92a and from the
700 mM eluate G
I-92b. Note that the 300 mM Mono Q eluate contains an activity that generates faster moving
RP3-protein complexes (Fig. 5B, lane 6). This
activity, however, was not further characterized and will be part of
future work.
Figure 6:
Negative regulation of p92 DNA binding by
G
, G
and G
inhibitors is
reversible. Inhibition of p92 DNA binding by the G
inhibitor (G
I-92a) present in the 100 mM salt fraction of a Mono Q I-92 fractionation (lane 2), by
the G
inhibitor (G
I-92) (lane 3), the
700 mM G
by the G
I-92b inhibitor
present in the 700 mM fraction (lane 4) and the
G
inhibitor (G
I-92) (lane 5). p92
without added inhibitor (lane -). Release of p92 from
inactive I-92-p92 complexes after DOC treatment is shown. Treatment of
partially purified p92 with 0.2% DOC (lane 6), DOC treatment
after inhibition with G
I-92a (lane 7), G
I-92 (lane 8), G
I-92b (lane 9) and
G
I-92 (lane 10) are shown. The retarded p92
complex and free RP3 are indicated by arrows.
In S Phase I-92 Is Not Detectable
We had
previously shown that p92 DNA binding activity was restricted to S
phase of eluted 444 cells(4) . For this reason I-92 should not
be active in S phase of the cell cycle. We analyzed crude I-92
preparations from S phase synchronized 444 cells, using aphidicolin and
a 3-h release, by Mono Q chromatography and EMSIA. We found that none
of the four I-92 activities that were identified in exponentially
growing 444 cells (see Fig. 4C) were detectable in Mono
Q fractions of S phase cells (Fig. 5C). This result
demonstrates that all four inhibitors that were identified by Mono Q
chromatography of a crude I-92 preparation from 444 cells are subject
to cell cycle regulation.
Identification of G
Phase I-92
In
order to obtain G
phase 444 cells, 444 cells were
synchronized by aphidicolin treatment followed by a 10-h release, and
subsequently crude I-92 was analyzed as described above. The efficiency
of G
phase synchronization was controlled by FACS analysis
and Western blotting using a cyclin B antiserum (Fig. 5A, bottom right). Analysis of the Mono
Q fractions by EMSIA demonstrated clearly that in G
phase
cells only a single I-92 activity was detectable in fraction 2 of the
200 mM NaCl eluate (Fig. 5D, lane 4).
We call the G
phase inhibitor G
I-92. G
I-92 was undetectable in the 200 mM Mono Q salt eluates
of G
phase and S phase cells (Fig. 5, panel
B, lane 4, and panel C, lane 4), very
low levels of G
I-92 could be detected in 200 mM Mono Q salt eluates of M phase and G
phase 444 cells (Fig. 5, panel E, lane 4, panel F, lane 4. Note that there are two faster moving complexes in
EMSIA of the 200 mM NaCl fraction containing G
I-92 (Fig. 5D, lane 4). These complexes
were reproducibly detected by EMSIA experiments using Mono Q-separated
G
I-92 preparations. In contrast, G
I-92 from
Sephacryl S300 fractions did not contain similar complexes.
I-92 Is Not Active in Mitotic 444 Cells
In
NHDFs, p92 DNA binding was restricted to S phase of the cell cycle (Fig. 1), and p92 was undetectable in M phase. We noticed,
however, that in mitotic 444 cells p92 was detectable by EMSA.
In order to examine I-92 in M phase of 444 cells we have used
nocodazole-treated 444 shake-off cells for I-92 analysis. An
examination of a Mono Q fractionation of mitotic 444 cell nuclear
extracts revealed that I-92 was undetectable with the exception of a
low amount of the G
I-92 inhibitor present in the 200
mM NaCl eluate (Fig. 5E, lane 4).
Note that this fraction, however, did not contain any detectable faster
moving RP3-protein complexes (Fig. 5, panel E, lane
4, and compare with panel D, lane 4). This
result suggested that I-92s are not active in M phase of 444 cells and
that for this reason the transcription factor p92 is deregulated in
mitotic 444 cells. We interpret this result to indicate a deficiency of
I-92 in mitotic 444 cells. However, we have not attempted to identify
the putative mitotic inhibitor in NHDFs. It is also possible that p92
is inactivated or degraded during mitosis in normal human fibroblasts.
These questions will be addressed as part of future experiments.
I-92 in G
Phase Cells
We next wanted
to analyze I-92 regulation in G
phase of the cell cycle.
444 cells were serum-starved for 48 h and assayed for the quiescent
state by Western blotting using the Ki67 antibody (19) (Fig. 5A, bottom left, lane
G
). The Ki67 antigen is present at any stage of the
cell cycle but down-regulated in G
phase. The serum
starvation for 48 h of 444 cells was effective because the
proliferation marker Ki67 was undetectable, whereas in exponentially
growing 444 cells the Ki67 signal was readily detectable (Fig. 5A, bottom left, lane C). The
activity of I-92 was analyzed in Mono Q fractions by EMSIA, and we
identified another I-92 activity in fraction 2 of the 800 mM NaCl eluate (Fig. 5F, lane 16). We call
this inhibitor G
I-92. The G
phase inhibitors
were undetectable, and a low amount of G
I-92 was detected (Fig. 5F, lane 4). Further experiments, which
are beyond the scope of this report, are necessary to characterize the
different cell cycle-regulated I-92 activities at the molecular level. Taken together these observations demonstrate cell cycle regulation
of the diverse I-92 forms and suggest that the different I-92
activities are sequentially activated during the cell cycle and
therefore link cell cycle regulation with transcription factor
regulation. The I-92 system is an example of a novel mechanism of
phase-specific transcription factor regulation and, in consequence, of
phase-specific gene control.
Inhibition of p92 DNA Binding by Different I-92 Proteins
Is Reversible
We have identified four different I-92 activities
by SDS-PAGE and Mono Q chromatography. Our previous work suggested that
crude I-92 inhibits p92 DNA binding in a reversible manner(4) .
However, it is also conceivable that other activities exist, unrelated
to I-92, which could degrade p92 or remove or add modifications, which
in consequence could regulate the DNA binding activity of p92. In order
to resolve this issue we tested whether inhibition of p92 DNA binding
by the cell cycle regulated inhibitors present in Mono Q fractions was
reversible by DOC treatment. For this purpose, appropriate amounts of
the 100 mM NaCl (G
I-92a), 200 mM NaCl
(G
I-92), 700 mM NaCl (G
I-92b), and
800 mM NaCl (G
I-92 Mono Q eluates were used in
order to inhibit DNA binding of partially purified p92 (0.4 µg) in
the EMSIA (Fig. 6, lanes 2, 3, 4, and 5). We then tested whether ``free'' p92 could be
recovered after DOC treatment (Fig. 6, lanes 7, 8, 9, and 10). We observed that G
I-92a, G
I-92b, G
I-92, and G
I-92 inhibited p92 binding in a reversible fashion, although
subtle differences were observed. p92 DNA binding inhibition by G
I-92b from the 700 mM eluate was 100% reversible (Fig. 6, lanes 4 and 9), whereas p92 DNA
binding inhibitions by G
I-92a (lanes 2 and 7) from the 100 mM eluate, by G
I-92 from
the 200 mM NaCl fraction (lanes 3 and 8) and
by G
I-92 from the 800 mM NaCl fraction (lanes
5 and 10) were reversible to a lesser extent. These
results demonstrate that the four phase-specific I-92 forms were able
to complex and inactivate p92 in a reversible manner.
DISCUSSION
p92 was previously identified in nontumorigenic
HeLa-fibroblast hybrid cells as a cell cycle-regulated DNA-binding
protein interacting with the enhancer of human papillomavirus type
18(1) . We have established in this report that p92 is also
present in normal human diploid fibroblasts and have shown by cell
synchronization experiments that p92 DNA binding activity was
restricted to S phase of the cell cycle. We have established in the
present communication that a p92 recognition site conferred activation
of the SV40 minimal promoter, suggesting that the proteins that bind to
the 5`-AATTGCTTGCATAA motif act as transcription factors. We think,
however, that p92 might be the responsible factor-mediating
transcriptional activation because p92 is the major retarded complex
detectable by EMSA in 444 cells(4) . This issue will have to be
analyzed after regulated in vivo expression of p92 cDNAs. We
are currently engaged in sequencing p92 protein for cDNA cloning.
Diverse Phase-specific Nuclear Inhibitors Link Cell Cyle
Regulation with Transcription Factor Regulation
The major
finding of this report is the identification of a novel mechanism of
phase-specific gene regulation due to the activity of phase-specific
nuclear inhibitors, termed I-92s. The consequence of this regulation is
that the DNA binding activity of the transcription factor p92 is
restricted to S phase of the cell cycle. In nuclear extracts of 444
cells, we have identified two G
phase inhibitors (G
I-92a, and G
I-92b), one G
phase
inhibitor (G
I-92) and a G
phase inhibitor
(G
I-92). In S phase of the cell cycle I-92 was
undetectable, demonstrating that the different I-92s regulate the DNA
binding activity of the transcription factor p92 in a phase-specific
manner, thereby linking cell cycle regulation with transcription factor
regulation.The existence of G
I-92a, G
I-92b, G
I-92, and G
I-92 was revealed by
biochemical cell synchronization experiments of 444 cells and
fractionation of crude I-92 preparations by Mono Q chromatography. The
efficiency of cell synchronizations was assessed by measurements of the
DNA content (FACS). Western blot analysis controlled for G
and the G
phase populations using the Ki67 antibody
(G
) and a cyclin B antibody (G
). The latter was
used to determine empirically the onset of G
phase after an
aphidicolin release. Chromatography of crude I-92 on Sephacryl S300 HR
columns revealed I-92 activities in a molecular mass range from 15 kDa
to 50 kDa. These results were confirmed by preparative, reducing
SDS-polyacrylamide gel electrophoresis of crude I-92, subsequent
elution of proteins from individual gel slices and renaturation of
eluted proteins. I-92 was monitored functionally by an EMSIA. The
identification of multiple I-92 activities with different molecular
weights in this experiment supports the notion that diverse I-92s
exist. A molecular weight determination of the G
I-92a,
G
I-92b, G
I-92, and G
I-92
inhibitors from Mono Q fractions will be part of future experiments.
We have characterized I-92 functionally and determined that the
inhibition of p92 DNA binding by the two G
phase
inhibitors, the G
phase inhibitor, and the G
phase inhibitor was reversible, because p92 could be released
from inactive I-92-p92 complexes by treatment with the detergent
deoxycholate. The G
phase inhibitor G
I-92 is
present in the 200 mM NaCl Mono Q fraction of exponentially
growing 444 cells and in G
phase-synchronized 444 cells (Fig. 4C, lane 4 and Fig. 5D, lane 4), and low activities of this inhibitor could be
detected in M phase and G
cells (Fig. 5, panel
E, lane 4, and panel F, lane 4). We
noted that there were additional faster moving DNA-protein complexes
present in the 200 mM NaCl Mono Q fractions that might have
been generated during I-92 assays (Fig. 5D, lane
4), because they were undetectable in electrophoretic mobility
shift inhibition assays without added p92. In contrast, fast moving
complexes could not be detected in Sephacryl S300 fractions and in
SDS-PAGE eluates (see Fig. 4, A and B). In
addition, Sephacryl S300 fractions from normal human diploid
fibroblasts likewise did not contain any detectable faster moving
complexes.
Furthermore, we have identified a cancer-derived
cell line that contains only G
I-92.
G
I-92 from this cell line was present in fraction 46 of a
Sephacryl S300 column and in the 200 mM NaCl fraction of a
Mono Q column. G
I-92 assays from these cells revealed that
this Mono Q fraction generated similar faster moving complexes as the
one from 444 cells, whereas G
I-92 from Sephacryl S300
fraction 46 did not contain any faster moving complexes
.
It is therefore possible that additional factors are present in the 200
mM NaCl Mono Q fraction that lead to a partial degradation of
p92 in EMSIA. Partial degradation of p92 could also explain why in DOC
release experiments lower amounts of p92 were recovered when compared
with input levels (Fig. 6, lanes 3 and 8).
Future experiments will be required to resolve this issue.
We have
analyzed whether I-92 is selective in its ability to form complexes
with p92. For this reason we selected three other transcription factors
and asked whether they are targets for I-92. We could show that the DNA
binding activities of AP-1 (Fig. 3), E2F, and NF-
B were not
affected by one or two units of crude I-92, which completely
inactivated a given amount of p92. These data demonstrate that I-92 is
selective, and this result is further supported by the fact that the
DNA binding activity of Oct-1 was not regulated by crude
I-92(3) . A number of transcription factors are regulated by
specific inhibitors, which modulate their DNA binding activities. AP-1
is regulated by IP-1(27) , the POU transcription factor Cf1-a
is regulated by I-POU(30) , and the helix-loop-helix protein
MyoD is regulated by Id (31) , and in these cases nonfunctional
heterodimers are formed. I-92s negatively regulate p92 DNA-binding in
G
, G
, and G
phase by physical
association with p92. During the process of p92 purification we have
observed that other proteins exist that specifically bind to the p92
recognition site present in RP3 and have shown that the DNA binding
activities of most of these were regulated by crude I-92(3) .
These results show that I-92s selectively regulate, besides p92, the
DNA binding activities of other related proteins, and this suggests
that I-92s could link the activities of multiple factors with the cell
cycle. It is conceivable that this regulatory principle could be a more
general mechanism whereby multiple specific inhibitors for a variety of
different transcription factors could link the regulation of many genes
with the cell cycle.