Interferon-
-mediated Inhibition of Cyclin A Gene Transcription
Is Independent of Individual cis-Acting Elements in the
Cyclin A Promoter*
Nicholas E. S.
Sibinga
§¶,
Hong
Wang,
Mark A.
Perrella
,
Wilson O.
Endege**,
Cam
Patterson
,
Masao
Yoshizumi
§§,
Edgar
Haber
, and
Mu-En
Lee
§
From the Cardiovascular Biology Laboratory, Harvard School of
Public Health, the
Department of Medicine, Harvard
Medical School, and the § Cardiovascular Division and
Pulmonary and Critical Care Division, Brigham and Women's
Hospital, Boston, Massachusetts 02115
 |
ABSTRACT |
Interferons (IFNs) affect cellular
functions by altering gene expression. The eukaryotic cell cycle is
governed in part by the periodic transcription of cyclin genes, whose
protein products associate with and positively regulate the
cyclin-dependent kinases. To understand better the growth
inhibitory effect of IFN-
on vascular smooth muscle cells (VSMCs),
we compared the expression and activity of G1 and S
phase cyclins in control and IFN-
-treated VSMCs. IFN-
treatment
did not inhibit the G1 cyclins but did decrease cyclin A
protein, mRNA, and associated kinase activity by 85, 90, and 90%,
respectively. Nuclear run-on and mRNA stability determinations
indicated that this decrease was the result of transcriptional
inhibition. To investigate the molecular basis of this inhibition, we
examined protein-DNA interactions involving the cyclin A promoter.
Electromobility shift assays showed little change with IFN-
treatment in the binding of nuclear proteins to isolated ATF, NF-Y, and
CDE elements. In vivo genomic footprinting indicated that
IFN-
treatment changed the occupancy of chromosomal NF-Y and CDE
sites slightly and did not affect occupancy of the ATF site. In a
previous study of transforming growth factor-
1-mediated inhibition
of the cyclin A promoter, we mapped the inhibitory effect to the ATF
site; in the present study of IFN-
treatment, functional analysis by
transient transfection showed that inhibition of the cyclin A promoter
persisted despite mutation of the ATF, NF-Y, or CDE elements. We
hypothesize that IFN-
inhibits cyclin A transcription by modifying
co-activators or general transcription factors within the complex that
drives transcription of the cyclin A gene.
 |
INTRODUCTION |
The pleiotropic cellular effects of the interferons
(IFNs)1 are thought to result
from induction of gene expression. However, because IFNs increase
expression of 50-100 different proteins, many without known function
(1), the changes in cellular activity mediated by IFNs, including
suppression of growth, are at best partially understood. Normal
cellular replication is governed in part by periodic transcription of
the cyclin genes, whose protein products associate with and positively
regulate the cyclin-dependent kinases (cdks). The cdks are
negatively regulated by phosphorylation or by association with a number
of distinct proteins known as cdk inhibitors (reviewed in Refs. 2-5).
Although growth inhibitory properties have been associated with several
of the proteins induced by IFNs (6-8), there is little information
about how genes induced by IFN signaling, particularly those affected
by IFN-
, interact directly with the machinery that regulates
progression of the cell cycle.
IFN-
/
and IFN-
are structurally dissimilar and bind to
distinct cell-surface receptors. The pathways the IFN receptors use to
signal the nucleus to induce gene expression share some components, and
the various IFN types regulate expression of partially overlapping sets
of genes (9). Although induction of gene expression by IFN-
/
is
typically direct, requiring no new protein synthesis, that effected by
IFN-
, which targets a more diverse set of genes, can be direct or
indirect (1). The precise subset of genes affected differs not only
with the type of IFN but also with the type of cell (1).
In neoplastic hematopoietic cell lines, IFN-
treatment suppresses
growth and is linked to decreased phosphorylation of retinoblastoma protein (10, 11) and CDC2 (12); decreased expression of c-Myc (11),
cyclin D3 (13), cyclin A (11, 12), and CDC25A (13); and altered DNA
binding by E2F (14). The independence of some of these effects may
indicate the activation of parallel signaling pathways (11, 13). In
primary bone marrow-derived macrophages, IFN-
treatment inhibits
phosphorylation of retinoblastoma protein (15) and expression of cyclin
D1 coincident with its ability to inhibit DNA synthesis (16). In
irreversibly arrested mammary epithelial cells, IFN-
may mediate
inhibition of cdk2 and cyclin E-associated kinase activity through
p27kip1, which is up-regulated by a post-translational
mechanism (17).
Because overlap among targets of IFN-
and IFN-
/
is only
partial, and because the profile of genes affected by individual IFNs
varies by cell type, extrapolations about mechanisms among IFNs and
cell types are uncertain. For example, cell cycle arrest due to IFN-
treatment in hematopoietic cells corresponds to the G0 (13)
or G0-G1 phase (11), whereas arrest due to
IFN-
treatment corresponds to the mid-G1 phase in
mammary epithelial cells (18) and to the late G1 or early S
phase in primary macrophages (15). These differences in the point of
cell cycle arrest indicate functional differences in the underlying
growth inhibitory mechanisms.
IFN-
inhibits the growth of vascular smooth muscle cells (VSMCs).
Because this effect occurs in vivo (19) as well as in vitro (20), IFN-
may be clinically useful in vascular
pathologies associated with high levels of VSMC proliferation (19). The growth inhibitory effect of IFN-
on VSMCs has been linked with induction of 2'-5'-oligoadenylate synthetase (21), the first enzyme in
the 2'-5'-adenylate IFN-regulated pathway of RNA degradation (22), and
with suppression of c-myc mRNA and protein (23, 24).
Little is known, however, about the effect of these changes in gene
expression on components of the cell cycle machinery in VSMCs.
We evaluated the effect of IFN-
on the expression of cdks and cdk
inhibitors in VSMCs and the expression and activity of cyclins in
VSMCs. IFN-
treatment induced both proliferative (CDK6) and antiproliferative (P15) genes but did not inhibit kinase
activity associated with the G1 cyclins. In contrast,
cyclin A-associated kinase activity decreased markedly after IFN-
treatment, in proportion to decreases in the expression of cyclin A
mRNA and protein. Although this inhibition of cyclin A expression
appeared to be caused by a decrease in transcription, electromobility
shift assays, in vivo genomic footprinting, and functional
analysis by transient transfection indicated that the decrease did not
depend on individual cis-acting elements in the cyclin A promoter.
 |
MATERIALS AND METHODS |
Cell Culture--
VSMCs were harvested from the aortas of
male Sprague-Dawley rats (200-250 g) by enzymatic dissociation
according to the method of Gunther et al. (25). The growth
medium for these cells was Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum
(HyClone), penicillin (100 units/ml), streptomycin (100 µg/ml), and
10 mM HEPES (pH 7.4) (Sigma); cells were made quiescent by
culture for 72 h in medium containing 0.4% calf serum. Rat VSMCs
were used for experimentation within 4-6 passages from primary
culture. Growth medium for human VSMCs (aortic; Clonetics) consisted of
medium 199 (Life Technologies, Inc.) supplemented with 20% fetal
bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml),
and amphotericin B (250 ng/ml). These cells were used within 8 passages
from primary culture. Recombinant rat IFN-
(Life Technologies, Inc.)
and human IFN-
(Genzyme) were reconstituted in sterile water and
stored in aliquots at
80 °C until use. For some experiments, VSMCs
were synchronized by incubation with medium containing 0.4% calf serum
(HyClone) for 72 h before stimulation with growth medium (with or
without IFN-
). For in vivo footprinting experiments,
human VSMCs were synchronized according to a two-step protocol as
follows: cells in growth medium were first blocked in S phase for
20 h with hydroxyurea (1 mM) and then released and
blocked in M phase for 20 h by adding new medium containing nocodazole (500 ng/ml). Synchronized monolayers were washed with phosphate-buffered saline, stimulated with fresh growth medium with or
without IFN-
, and harvested at defined intervals for DNA or RNA extraction.
Protein Immunoblot and Cyclin-associated Kinase
Assays--
Nuclear proteins were extracted as described (26), and
protein concentration was determined by the Lowry method (DC protein assay, Bio-Rad). Total nuclear protein (30 µg) from control and IFN-
-treated cells was fractionated by electrophoresis through 8%
SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride
membrane (Immobilon-P, Millipore). Nonspecific binding sites were
blocked by incubation in 4% skim milk in Tris-buffered saline for 40 min, followed by incubation overnight at 4 °C with primary
antibodies. Anti-cyclin antibodies and working concentrations were
rabbit anti-cyclin D1 (PharMingen 14726E), 1:5000; rabbit anti-cyclin E
(Santa Cruz Biotechnology sc-481), 1:2500; and rabbit anti-human cyclin
A (gift of R. Schlegel, Millennium Pharmaceuticals, Cambridge, MA),
1:2000. Immune complexes were visualized with the ECL detection system
(Amersham Pharmacia Biotech). Cyclin-dependent kinase
assays were performed as described (27), with modifications. In brief,
total cellular protein (50 µg) from control and IFN-
-treated rat
VSMCs was immunoprecipitated with antiserum (2.5 µg) against cyclin
A, D1, or E at 4 °C for 2 h, followed by incubation at 4 °C
for 1 h with protein A-Sepharose beads (16 mg/ml; Amersham Pharmacia Biotech). The beads were incubated for 20 min at 30 °C in
40 µl of kinase buffer containing [
-32P]ATP (6,000 cpm/pmol; 100 mM), with glutathione
S-transferase-retinoblastoma protein (Rb) (40 µg/ml; Rb
amino acids 769-921, Santa Cruz Biotechnology) used as phosphorylation
substrate for anti-cyclin D1 immunoprecipitates, and histone H1 (200 µg/ml; Boehringer Mannheim) used as phosphorylation substrate for
anti-cyclin A or E immunoprecipitates. Reaction products were resolved
on 12% SDS-polyacrylamide gels and detected by autoradiography and
phosphorimaging. Signals were quantified with the ImageQuant version
1.1 software analysis program (Molecular Dynamics).
RNA Half-life Determination--
Quiescent rat VSMCs were
stimulated with growth medium with or without IFN-
for 24 h
before addition of actinomycin D (5 µg/ml, Boehringer Mannheim).
Cells were harvested for RNA extraction at intervals over the ensuing
8 h.
RNA Blot Hybridization--
Total RNA was obtained from cultured
cells by guanidinium isothiocyanate extraction followed by
centrifugation through cesium chloride (28). The RNA was fractionated
on 1.2% formaldehyde-agarose gels and transferred to nitrocellulose
filters, which were then hybridized at 68 °C for 2 h with
random-primed, 32P-labeled cDNA probes (106
cpm/ml) in QuikHyb solution (Stratagene). The filters were washed in 30 mM sodium chloride, 3 mM sodium citrate, and
0.1% SDS at 55 °C (probe and target RNA from same species) or at
25-42 °C (probe and target RNA from different species), stored on
phosphor screens for 12-16 h for phosphorimaging, and autoradiographed with Kodak XAR film for 48 h at
80 °C. Previously described
cDNAs used as probes in Northern analysis included rat cyclin A
(29), human CDK2, CDK4, and CDK6 (30), which were provided by E. Harlow (Massachusetts General Hospital, Boston), and human Stat 1a (31), which
was provided by X. Y. Fu (Yale University, New Haven, CT). Additional rat cDNA probes were generated by reverse
transcription-polymerase chain reaction (PCR) from total rat lung RNA
with primers derived from the corresponding reported rat or mouse
sequences as follows: p15, 5'-ATGATGATGGGCAGCGCCCAGG-3' and
5'-TGTCCAGGAAGCCTTCCG-3' (32); p21, 5'-GTGGACAGTGAGCAGTTGA-3' and
5'-TGGTCTGCCTCCGTTTT-3' (33); and p27, 5'-ATGTCAAACGTGAGAGTGTC-3' and
5'-GAAGGCCGGGCTTCTTGGGC-3' (34). 32P-End-labeled
oligonucleotides complementary to 18 S
(5'-ACGGTATCTGATCGTCTTCGAACC-3') (35) or 28 S
(5'-CGCTCCAGCGCCATCCATTTT-3') (36) ribosomal RNA were hybridized to the
filters to correct for differences in RNA loading. Phosphor screens
were scanned, and radioactive signal intensity was determined with the
ImageQuant version 1.1 software analysis program (Molecular Dynamics).
Nuclear Run-on Analysis--
Quiescent rat VSMCs were stimulated
for 24 h with growth medium with or without IFN-
. The cells
were lysed and nuclei were isolated as described (26). Nuclear
suspension samples (200 µl) were incubated with 0.5 mM
each of CTP, ATP, and GTP and 250 µCi of 32P-labeled UTP
(3000 Ci/mmol, NEN Life Science Products) for 30 min. The samples were
extracted with phenol/chloroform, precipitated, and resuspended in
water for scintillation counting; equivalent amounts of radioactive
probe were added to nitrocellulose filters bearing target cyclin A and
control
-actin cDNAs. Filters were hybridized for 72 h at
40 °C in the presence of formamide, washed, and subjected to
autoradiography. Radioactivity hybridizing to cyclin A target cDNA
was normalized to the activity of the corresponding
-actin control.
In Vivo DNA Footprinting by Ligation-mediated (LM)
PCR--
In vivo dimethyl sulfate (DMS) treatment was
performed as described by Mueller and Wold (37) with some modification
(38). In brief, for in vivo DNA methylation, intact human
VSMC monolayers were exposed to 0.1% DMS at room temperature for 2.5 min. Cells were washed and lysed, and methylated DNA was recovered and
deproteinized overnight at 37 °C. For in vitro
methylation, naked genomic DNA was treated with 1% DMS for 4 or 6 min
at room temperature. Methylated DNA was cleaved by treatment with 1 M piperidine. Human VSMCs were treated in vivo
with DNase I according to the method of Pfeifer and Riggs (39) with
some modification. Cells in a monolayer were permeabilized with 0.05%
lysolecithin (Sigma) and then exposed to DNase I (Boehringer Mannheim,
150 Kunitz units/ml) for 4 min at room temperature. Genomic DNA was
treated in vitro with DNase I (0.03 Kunitz units/ml) for 8 min at room temperature.
LM-PCR was performed according to the method of Garrity and Wold as
described by Patterson et al. (38). The 5' set of primers was derived from the published human cyclin A promoter sequence (40)
and designed according to the criteria of Mueller et al. as
described by Ausubel et al. (28). Primers 5'-1, 5'-2, and 5'-3, with their respective positions (40) and calculated
Tms were as follows: 5'-CCCTAAATCCTACCTCTCCC-3'
(
214 to
195) (Tm 62.9 °C),
5'-TCCCGCCCCAGCCAGTTTGTTTCTC-3' (
174 to
150) (Tm 64.9 °C), and 5'-CCCAGCCAGTTTGTTTCTCCCTCCTGCCC-3' (
168 to
140) (Tm 68.5 °C).
The 3' set of primers was described by Zwicker et al. (41).
For first-strand synthesis, DNA was denatured at 95.5 °C for 5 min,
annealed at 55 °C for 30 min, and extended at 76 °C for 10 min.
Ligation to asymmetric linkers was performed for 6 h at 17 °C.
PCR cycling conditions for the 5' primer set were 95.5 °C for 1 min,
65 °C for 2 min, and 76 °C for 3 min, for a total of 21 cycles.
PCR cycling conditions for the 3' primer set were 95.5 °C for 1 min,
68 °C for 2 min, and 76 °C for 3 min, for a total of 21 cycles.
Two cycles of primer extension were performed. Primers were annealed at
68.5 °C with 32P-end-labeled primer 5'-3 and at 72 °C
with 32P-end-labeled primer 3 derived from Zwicker et
al. (41). Primer extension products were separated on 6%
denaturing polyacrylamide gels and visualized by autoradiography.
Electromobility Shift Assay--
Electromobility shift assays
were performed as described (42), with modifications. Sense strand
sequences of DNA probes with respective positions in the cyclin A
promoter were NF-Y, 5'-GAGCGCTTTCATTGGTCCATTTCAATAGTC-3'
(
65 to
36), and CDE-CHR, 5'-CAATAGTCGCGGGATACTTGAACTGCAA-3' (
43 to
16).
(Underline indicates consensus protein-binding sites.) The ATF 22-mer
probe was described elsewhere (42). Typical binding reactions contained
double-stranded DNA probe (20,000 cpm), 1 µg of
poly(dI-dC)·poly(dI-dC), 50 mM KCl, 20 mM
HEPES (pH 7.5), 10 mM MgCl2, 10% glycerol, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, and 10 µg of
nuclear extract in a final volume of 25 µl. The specificity of
binding interactions was assessed by competition with a 50-fold excess
of unlabeled double-stranded oligonucleotide of identical sequence.
Transient Transfection Analysis of Promoter
Activity--
Reporter constructs were made by ligating wild-type (42)
or mutated cyclin A promoter fragments into the promoterless pCAT3 basic vector (Promega). Deletion fragments and mutants were generated by PCR with Pfu polymerase (Stratagene) by using standard
cycling conditions. Primers for the
83 to +5 fragment were forward
5'-GAATGCACGTCAAGGCCGCA-3' and reverse 5'-CCGCTGGAGCGCGGCTGTTC-3'.
Site-directed mutagenesis of the reverse NF-Y site (ATTGG)
was performed by PCR from a wild-type template with the forward primer
5'-GAGCGCTTTCcggttTCCATTTCAATAG-3'. The CDE-CHR sequence
(CGCGGGATACTTGAA) was mutated by PCR with the
primer 5'-TCCATTTCAATAGTatattGATcaggtAACTGCAAGAACAGCC-3'. (Lowercase
denotes mutant bases.) The ATF mutant was described elsewhere (42).
Mutations were confirmed by dideoxy-sequencing with Thermosequenase
(Amersham Pharmacia Biotech). Rat VSMCs in 6-well plates were
transfected with reporter plasmid (2 µg/well) by the DEAE-dextran
method (43). After 4 h, cells were treated with growth medium with
vehicle (control) or rat IFN-
for 24 h until harvest.
Chloramphenicol acetyltransferase activity (CAT) was assayed as
described (44). To correct for variability in transfection efficiency,
we co-transfected cells with 0.5 µg/well of pCMV-
-gal
(CLONTECH) and determined
-galactosidase
activity by solution assay. Transfections were performed three times,
each time in triplicate. Corrected CAT activities (mean ± S.E.)
were normalized in relation to a parallel transfection with pCAT3
control (Promega), the activity of which was set as 100%.
Data Analysis--
Comparisons among groups were made by
factorial analysis of variance followed by a Bonferroni/Dunn post
hoc analysis. Statistical significance was accepted for a
p value < 0.05.
 |
RESULTS |
IFN-
Has Varied Effects on Expression of Cdks and Their
Inhibitors--
After confirming the growth inhibitory effect of
IFN-
treatment (19-21) on VSMC DNA synthesis and cell replication,
we determined how IFN-
affected the expression of genes involved in
progression and arrest of the cell cycle. Northern analysis was
performed with RNA derived from rat VSMCs synchronized in a quiescent
state and then stimulated to enter the cell cycle by the addition of 10% fetal bovine serum with or without IFN-
. A 4-5.5-fold
induction of Stat 1a mRNA in IFN-
-treated cells (Fig.
1) confirmed their responsiveness to
IFN-
(45). Although we postulated that this growth inhibition
mediated by IFN-
might be reflected in a decrease in the expression
of cdks or an increase in that of cdk inhibitors, we found little
change in the level of CDK2, CDK4, p21, or p27 mRNA during the
48 h after stimulation. Note that CDK6 and p15, which contribute
to and inhibit, respectively, activity associated with D-type cyclins
(46), were both induced by approximately 2-fold in cells treated with
IFN-
in comparison with time-matched controls.

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Fig. 1.
Northern analysis of cdks and cdk inhibitors
in control and IFN- -treated VSMCs. Rat
VSMCs were incubated for 72 h under low serum conditions and then
stimulated with growth medium with or without IFN- (300 units/ml).
Total cellular RNA was extracted at the indicated time points after
stimulation. Samples (10 µg) were resolved by electrophoresis through
denaturing agarose gels and transferred to nitrocellulose filters,
which were hybridized with 32P-labeled cDNA probes as
indicated. Filters were washed as described under "Materials and
Methods" and analyzed by exposure to radiography film and phosphor
screens.
|
|
IFN-
Inhibits Expression and Activity of Cyclin A but Not Those
of Cyclin D1 or E--
IFN-
treatment is associated with arrest of
the cell cycle in the G1 phase in fibroblasts (47, 48) and
mammary epithelial cells (18). In myeloid cells, growth arrest
associated with IFN-
is linked to a decrease in expression of a
principal G1 cyclin, cyclin D1, even when the cytokine is
added late in the G1 phase (16). In light of these
observations and the induction of CDK6 and p15 mRNAs in VSMCs that
we observed (Fig. 1), we next assessed the effect of IFN-
on
G1 and G1-S phase cyclin expression and activity.
Cyclin D1 expression in rat VSMCs was also induced by IFN-
at both
the mRNA (data not shown) and protein levels, with immunoblot analysis showing a 3.8-fold increase in the amount of cyclin D1 (Fig.
2A). IFN-
treatment had no
significant effect on cyclin E protein levels, whereas it decreased
cyclin A expression by 85% (Fig. 2A); this decrease was
present also at the mRNA level (data not shown).

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Fig. 2.
Effect of IFN-
on cyclin expression and activity in VSMCs. A,
rat VSMCs in growth medium were treated with vehicle (control) or
IFN- (300 units/ml). After 24 h, nuclear protein was extracted,
fractionated through 8% SDS-polyacrylamide gels, and transferred to
membranes for immunoblotting. Primary antibody hybridization was
performed at 4 °C overnight, and immune complexes were visualized by
chemiluminescence autoradiography. Bands correspond to cyclin D1 (34 kDa), cyclin E (51 kDa), and cyclin A (58 kDa). B,
cyclin-associated kinase activity was assayed in total cellular protein
(50 µg) from control and IFN- -treated rat VSMCs, with antibodies
directed against cyclins D1, E, and A used for immunoprecipitation.
Glutathione S-transferase-Rb, containing Rb amino acids
769-921, was used as kinase substrate for cyclin D1
immunoprecipitates, and histone H1 was used as kinase substrate for
cyclin E and A immunoprecipitates. 32P-Labeled reaction
products were resolved on 12% SDS-polyacrylamide gels and detected by
autoradiography and phosphorimaging.
|
|
We then assayed cyclin-associated kinase activity to determine how
these changes affected net cell cycle kinase activity. Despite the
IFN-
-mediated induction of p15, we found that cyclin D1-associated
kinase activity increased 1.7-fold and that cyclin E-associated kinase
activity changed little. Cyclin A-associated kinase activity, on the
other hand, was inhibited by 90% (Fig. 2B).
These results are consistent with an IFN-
-mediated arrest of the
VSMC cycle that involves late G1 or G1-S phase
regulatory mechanisms. Because cyclin A-associated kinase activity was
inhibited in proportion to the decrease in cyclin A expression, and
cyclins D1 and E were not inhibited, we investigated the means by which IFN-
decreased cyclin A expression.
IFN-
Inhibits Transcription of the Cyclin A Gene Without
Affecting mRNA Stability--
IFNs can inhibit gene expression by
induction and activation of RNA degradation pathways (49) and by
post-transcriptional mechanisms (50). To determine the underlying
mechanism of the decrease in cyclin A mRNA mediated by IFN-
, we
directly assessed the mRNA half-life and transcriptional rate. The
stability of cyclin A mRNA in the presence or absence of IFN-
was evaluated by inhibiting transcription in rat VSMCs with actinomycin
D. The calculated cyclin A mRNA half-life of 2.7 h was not
affected by IFN-
treatment (Fig.
3A). In nuclear run-on
experiments, IFN-
did not change transcription of the control
-actin gene, but it inhibited radioactive signal hybridizing to
cyclin A sequences by 67% (Fig. 3B). These studies indicate
that the decrease in expression of cyclin A resulting from IFN-
treatment is due to inhibition of transcription.

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Fig. 3.
Effect of IFN- on
cyclin A mRNA stability and rate of transcription in rat
VSMCs. A, 24 h after quiescent rat VSMCs had been
stimulated with growth medium with or without IFN- (300 units/ml),
the cells were treated with actinomycin D (5 µg/ml). Total RNA was
harvested over the next 8 h and used in Northern analysis for
cyclin A mRNA. Cyclin A signals were corrected for differences in
loading against the activity of a radiolabeled oligonucleotide probe
for the 18 S ribosome. Corrected values are plotted in log scale as a
percentage of the 0-h value. B, the rate of cyclin A
mRNA transcription was evaluated by nuclear run-on analysis.
Subconfluent rat VSMCs were incubated in medium containing 0.4% calf
serum for 72 h and stimulated by addition of growth medium with or
without IFN- (300 units/ml). Nuclei were isolated after 28 h of
stimulation, and in vitro transcription was allowed to
resume in the presence of [ -32P]UTP. Equivalent
amounts of 32P-labeled, in vitro transcribed RNA
probes from control and treated cells were hybridized to 1 µg of
denatured cyclin A cDNA and -actin cDNA on nitrocellulose
filters. The filters were washed, and signal activities were assessed
by autoradiography.
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|
Transcriptional Activity of the Proximal Cyclin A Promoter Is
Inhibited by IFN-
--
Our finding that IFN-
treatment decreased
cyclin A transcription in VSMCs suggested that we could map the
inhibitory effect to cis-acting elements in the cyclin A
promoter and so identify downstream signaling pathways affected by the
cytokine. In growing rat VSMCs transiently transfected with cyclin A
promoter constructs driving expression of a CAT reporter gene (Fig.
4), the activity of the
406/+205
fragment was similar to that of the pCAT3 control plasmid. Deletion of
5' sequences to yield the fragment
266/+205 had relatively little
effect on overall activity, despite the removal of a consensus
AP1-binding site. The further removal of 5' sequences including three
consensus Sp1 sites and 3' sequences including two consensus E2F sites,
one consensus p53 site, and a potential composite Yi/Sp1 element (40),
to yield the
83/+5 fragment, decreased activity to approximately 30%
of the activity of the reference plasmid. Inhibition of cyclin A
promoter activity by IFN-
was retained in all constructs tested. The
degree of inhibition by IFN-
, ranging from 55 to 66%, was similar
to that found in the nuclear run-on analysis (Fig. 3B). On
the basis of these findings, we narrowed our search for an
IFN-
-responsive element to the
83/+5 fragment; further deletions
within this fragment severely impaired promoter activity (data not
shown).

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Fig. 4.
Transient transfection analysis of cyclin A
promoter activity in rat VSMCs. Constructs bearing fragments of
the human cyclin A promoter and driving expression of a CAT reporter
gene were transiently transfected into rat VSMCs. Transfected cells
were treated with growth medium with or without IFN- (300 units/ml).
After 24 h, cellular lysates were harvested, and CAT activity was
determined in a solution assay. CAT activities were divided by
corresponding (co-transfected) -galactosidase activities to correct
for variation in transfection efficiency and normalized against the
activity of the pCAT3 control plasmid, which was given the arbitrary
value of 100. Open bars, control; filled bars,
IFN- . *, p < 0.05.
|
|
Protein Occupancy of Proximal Cyclin A Promoter Elements Shows
Little Change with IFN-
Treatment--
To determine whether IFN-
treatment changed protein binding to elements in the proximal cyclin A
promoter, we performed in vivo footprinting with DNase I
(Fig. 5), electromobility shift assays
(Fig. 5), and in vivo footprinting with dimethyl sulfate (Fig. 6). In comparison with DNA treated
in vitro, in vivo DNase I footprinting in control
(growing) VSMC cultures showed protection of the native cyclin A
promoter in three distinct regions between
83 and +5, and at a fourth
site near
150 (Fig. 5A, heavy bars). Each
protected region encompassed an identified consensus binding site (ATF,
NF-Y, CDE-CHR, and Sp1) (Fig. 5A, thin bars). The
footprinted area containing the ATF site extended in the 5' direction
more than 20 bases beyond the consensus ATF sequence itself. The
protected regions were demarcated by hypersensitive sites (Fig.
5A, asterisks). The hypersensitive site at
60,
between the ATF and NF-Y core sites, maps to the area between the NF-Y
core and putative flanking regions of the cyclin A promoter described
by Zwicker et al. (41, 51). Although there were no obvious
differences between the footprints derived from control or
IFN-
-treated VSMCs, our finding (Fig. 5A) confirmed that
the ATF, NF-Y, and CDE-CHR consensus binding sites were actually
occupied by protein in the native chromosomal context. We did not find
additional protected elements between bases
83 and +5.

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Fig. 5.
Protein interactions with the proximal cyclin
A promoter defined by in vivo DNase I footprinting and
electromobility shift assay. Quiescent human VSMCs were stimulated
with growth medium with vehicle or human IFN- at 10 ng/ml for
24 h before harvesting. A, cellular monolayers were
permeabilized with lysolecithin and treated with DNase I in
vivo. Purified human VSMC DNA that had been treated in
vitro was used as a control. Protection of the coding strand from
the activity of DNase I was analyzed by LM-PCR with the 3' set of
primers described under "Materials and Methods." The
scale shows nucleotide positions within the human cyclin A
promoter. Protected regions are indicated by heavy bars and
consensus binding sites by thin bars. Asterisks
correspond to hypersensitive residues. B, nuclear proteins
were extracted from human VSMCs and used for electromobility shift
analysis of protein binding to the ATF, NF-Y, and CDE-CHR elements in
the proximal human cyclin A promoter. Competition assays with 50-fold
excess of unlabeled probe were performed to verify specificity.
Arrows indicate specific protein-DNA complexes.
|
|

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Fig. 6.
In vivo DMS footprinting analysis
of protein interactions with the proximal cyclin A promoter in human
VSMCs, with and without inhibition by
IFN- . Human VSMCs blocked in
G2 were released into growth medium containing vehicle
(control) or human IFN- at 10 ng/ml and harvested at the
indicated time points. A, Northern analysis was performed on
RNAs from cells treated in parallel. Two cyclin A mRNA isoforms,
2.7 kilobases and 1.8 kilobases, are indicated. B and
C, DNA in cellular monolayers was methylated with DMS
in vivo for 2.5 min; purified DNA was methylated with DMS
in vitro for 4 or 6 min (control). Methylated DNA
was cleaved and used in LM-PCR. The coding strand (B) was
analyzed with the 3' primer set, and the noncoding strand
(C) was analyzed with the 5' primer set. In the vicinity of
the ATF-binding site, diamond a indicates a
hypersensitive guanine residue at 70, and asterisk b
indicates a methylated adenine at 75 seen only in vivo. In
the vicinity of the NF-Y site, diamond c and
asterisk c indicate a protected guanine at 56 and a
hypersensitive adenine at 58, respectively, and diamond
d indicates a protected guanine at 60. In the region of
the CDE, diamond e denotes guanines at 32 and
33 that show some variation in protection through the time
course.
|
|
To obtain an independent means of evaluating DNA-protein binding, we
also performed electromobility shift assays with nuclear proteins
extracted from control and IFN-
-treated VSMCs. Consistent with the
absence of change in protein binding to these sequences revealed by
in vivo DNase I footprinting, treatment with IFN-
did not
affect the intensity of specific binding of nuclear proteins to
oligonucleotides bearing ATF, NF-Y, or CDE-CHR sites (Fig. 5B).
To look for IFN-
-mediated changes in protein interactions with the
proximal cyclin A promoter not detectable by DNase I footprinting or
electromobility shift assay, we performed in vivo
footprinting with DMS. Synchronized VSMCs were released into growth
medium with or without IFN-
, and DNA was harvested after 18, 24, and 30 h. These time points corresponded to early, mid, and late S phase on the basis of [3H]thymidine incorporation studies
(data not shown). To confirm the effect of IFN-
on cyclin A
expression, we harvested RNA from cells cultured and treated with
IFN-
in parallel. Northern analysis showed that in comparison with
control samples, IFN-
treatment decreased cyclin A mRNA by 95%
at 18 h after release, by 90% at 24 h, and by 69% at
30 h (Fig. 6A).
The results of DMS footprinting in vivo are shown in Fig. 6,
B (coding strand) and C (noncoding strand). DNA
ladders from both the coding and the noncoding strands indicated
protein interactions with the ATF, NF-Y, and CDE sites, consistent with
previous reports (41, 52). The ATF site was protected in all in
vivo samples and showed little change with IFN-
treatment. On
the coding strand (Fig. 6B), guanines
76 and
79 were
visible in samples treated in vitro but not in
vivo. The same was true for noncoding strand guanines
74 and
77 (Fig. 6C). Protein binding to the ATF site of the
proximal cyclin A promoter was also indicated by a hypersensitive coding strand guanine at
70 (Fig. 6B, diamond
a), whereas on the noncoding strand an adenine at
75 was
faintly visible in all in vivo samples (Fig. 6C,
asterisk b). Like guanine, adenine can be methylated by DMS
but is less susceptible to cleavage than is methylated guanine; its
hypersensitivity may reflect torsional strain on the DNA (37).
In comparison with the in vitro samples, the CDE site was
partially protected at all in vivo time points. However,
there was some variability in the extent of protection as follows: on
the coding strand, guanines
32 and
33 (Fig. 6B,
diamond e) were more protected at time 0 and in
the IFN-
-treated samples and less protected in the control samples.
Thus, maximal protection of the CDE site corresponded to a decrease in
transcription of cyclin A, as reflected in the Northern analysis (Fig.
6A). The decrease in transcription is consistent with the
model proposed by Zwicker et al. (41) in which occupancy of
this site mediates repression of positive upstream elements. In the CHR
site, guanine
24 (Fig. 6B, coding strand) showed a slight
but consistent protection at all in vivo time points.
In the NF-Y core site (41) (Fig. 6B,
NF-YC), guanines
51 and
52 on the coding
strand were relatively protected throughout the time course, but
protection appeared greatest in control samples. In the NF-Y flanking
region (41) (Fig. 6B, NF-YF), there was
some variability of the footprint corresponding to six guanines located
between
71 and
61 (between the ATF and NF-Y sites), but there was
no consistent pattern that correlated with IFN-
treatment. On the
noncoding strand (Fig. 6C), guanine
56 was always
protected in vivo (diamond c). A
hypersensitive residue 5' to the core NF-Y site (corresponding to
adenine
58) appeared in all in vivo samples; this residue
was most prominent in the G2-M (time 0) sample
(asterisk c). Signal from the guanine at
60 on the
noncoding strand (diamond d) was prominent in the
G2-M (time 0) sample but was largely protected in both the
growing and the IFN-
-treated samples.
Inhibition of Cyclin A Promoter Activity by IFN-
Is Not Affected
by Mutation of Protein-bound Elements--
The electromobility shift
assays and in vivo footprinting with DNase I indicated
little apparent change with IFN-
treatment in protein binding to
cis-acting elements in the proximal cyclin A promoter.
In vivo footprinting with DMS showed complex and relatively subtle changes in binding through a more extended time course. Whereas
protection of the ATF site was relatively invariant, the changes in
footprint involving the CDE and NF-Y elements suggested that they might
be involved in mediating the inhibitory effect of IFN-
. To determine
the functional importance of these sites for promoter inhibition by
IFN-
, we performed further transient transfection experiments with
mutated cyclin A promoter constructs. In transiently transfected
growing cells, mutation of the ATF, NF-Y, and CDE-CHR sequences led in
each case to a decrease in cyclin A promoter activity (Fig.
7) in comparison with the activity of the
wild-type (parent) construct (
266/+205, Fig. 4). The greatest reduction in activity occurred with the ATF site mutant, and the activity of the NF-Y mutant was closest to that of the wild type. Nevertheless, in transiently transfected cells treated with IFN-
, inhibition of promoter activity was not affected significantly by any
of these mutations; with all three constructs, activity fell by
55-61% with IFN-
treatment.

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|
Fig. 7.
Transient transfection analysis of mutated
cyclin A promoter activity in rat VSMCs. Cyclin A promoter
fragments ( 266/+205) with mutations of proximal cis-acting
elements ATF, NF-Y, and CDE-CHR were transiently transfected into rat
VSMCs. Transfected cells were treated with growth medium supplemented
with vehicle (control) or rat IFN- (300 units/ml). After 24 h,
cellular lysates were harvested, and CAT activity was determined by a
solution assay, as described in the legend to Fig. 4. Open
bars, control; filled bars, IFN- . *,
p < 0.05.
|
|
 |
DISCUSSION |
We have investigated the molecular basis of VSMC growth inhibition
by IFN-
. First we found that whereas most of the cdks and cdk
inhibitors were not affected by exposure to IFN-
, CDK6 and p15 were
both induced at the mRNA level (Fig. 1). Despite these changes,
however, cyclins D1 and E and the activities of their associated
kinases were not inhibited (Fig. 2, A and B), although cyclin A protein and cyclin A-associated kinase activity decreased substantially. These findings suggest that IFN-
treatment of VSMCs results in a relatively late G1 or
G1-S junction cell cycle block. The simplest explanation
for these findings is that the decrease in cyclin A protein leads to
the decrease in cyclin A-associated kinase activity; this observation
suggested that regulation of cyclin A expression might be a target of
IFN-
-mediated signaling in VSMCs. Cyclin A mRNA stability and
nuclear run-on studies indicated in turn that the decrease in cyclin A
expression resulting from IFN-
treatment was due to inhibition of
cyclin A gene transcription.
Cyclin A promotes cell cycle progression by associating with and
stimulating cyclin-dependent kinases CDC2 and CDK2 and is a
critical regulator of the cell cycle at both the G1-S
junction (53, 54) and during the G2-M transition (53). Like
that of other cell cycle molecules, the level of cyclin A protein is controlled primarily by cyclical changes in mRNA expression. The human cyclin A promoter has been cloned (40) and characterized in a
number of growth-related contexts such as contact inhibition (42),
adhesion dependence (55), and TGF-
1 treatment (56, 57). Inhibition
of cyclin A promoter activity by TGF-
1 requires an intact ATF site,
as we (57) and others (56) have reported previously; this effect
involves decreased phosphorylation of CREB and ATF-1 (57). We
hypothesized that it would be possible to identify the
cis-acting elements in the cyclin A promoter required for
its inhibition by IFN-
. In turn, this identification would point to
downstream targets of IFN-
signaling that are critical to regulation
of cell cycle progression, as our analogous identification had pointed
to downstream targets of TGF-
1 signaling.
Our initial transient transfections involved cyclin A promoter
constructs driving expression of a luciferase reporter gene. With this
system, however, we encountered a problematic nonspecific effect of
IFN-
on luciferase activity that extended even to control promoters.
Others have also reported difficulties with using luciferase as a
reporter for analysis of IFN-
-mediated effects (58). The CAT system,
in contrast, did not show nonspecific effects with IFN-
, and we used
it for all subsequent transfections.
Transient transfections with a CAT reporter showed that the inhibitory
effect of IFN-
was retained in an 88-base pair fragment of the
proximal promoter. This fragment contained consensus binding sites for
ATF and NF-Y transcription factors, as well as the CDE and CHR elements
(40, 41, 52). One model of regulation of cyclin A promoter activity
during the cell cycle involves phase-specific protein binding to the
CDE and CHR elements to periodically repress transcriptional activation
mediated through the upstream positive regulatory elements NF-Y, ATF,
and Sp1 (41, 52). We anticipated that the inhibitory effect of IFN-
would be mediated through one of the elements between bases
83 and
+5.
However, complementary lines of evidence from in vivo DNase
I footprinting (Fig. 5A) and electromobility shift assays
(Fig. 5B) argued against a substantial change with IFN-
treatment in the level of protein binding to cis-acting
elements in the
85/+5 cyclin A fragment. Further investigation by
in vivo DMS footprinting (Fig. 6, B and
C) showed only limited changes in the protection pattern
with IFN-
treatment, despite confirmation of inhibition of cyclin A
mRNA expression by as much as 95% (Fig. 6A). We then evaluated the functional significance of protein binding to these sites
by transiently transfecting mutated promoter constructs. These
experiments indicated the functional importance of the ATF, NF-Y, and
CDE-CHR binding sites in the overall activity of the cyclin A promoter
but showed further that inhibition by IFN-
occurs regardless of
mutations at these specific sites. The mechanism of down-regulation of
cyclin A promoter activity by IFN-
is thus not a simple extension of
the repressive mechanism that normally operates in G1 (41,
52) nor does it involve the ATF site in a manner analogous to
down-regulation by TGF-
1 (56, 57).
Change in protein-DNA binding is by no means required for altered
transcriptional activity. For example, in vivo footprinting indicated no significant difference in protein interaction with the
essential serum response element and flanking c-fos promoter sequences after A431 cells had been stimulated with epidermal growth
factor, a treatment that resulted in dramatic induction and then
repression of c-fos mRNA as determined by Northern
analysis (59). In the case of the cyclin A promoter, we previously
found that TGF-
1 treatment led to changes in transcription factor
activity through modifications not reflected in protein abundance or
DNA binding per se (57). These findings suggested that for
transcription of certain genes, specific protein-DNA interactions are
maintained and do not have to be disrupted to effect either activation
or repression of transcriptional activity. In our analysis of TGF-
1 signaling, however, we identified a functional role for the ATF site in
the cyclin A promoter, because its mutation eliminated the inhibitory
effect of TGF-
1. With IFN-
, we found that change in protein-DNA
binding involving the proximal cyclin A promoter was quite limited and
that mutation of the protein-bound DNA elements in the proximal
promoter did not reduce the inhibitory effect of IFN-
.
Indeed, although IFN-
inhibits transcription of many genes (60-66),
no IFN-
-specific inhibitory elements have been identified. One
proposed mechanism for inhibition of gene expression by IFN-
, derived from studies of the macrophage scavenger receptor gene promoter, involves competition between positive regulatory factors (AP-1, ETS) and a direct target of IFN-
signaling (activated Stat
1a). AP-1, ETS, and Stat 1a all depend on the essential co-activators CBP and p300 for full transactivation, so when cellular levels of these
co-activators are limiting, the increase in activated Stat 1a due to
IFN-
treatment limits AP-1-and ETS-mediated transactivation by
titrating away the co-activators (67). Such a mechanism, however,
requires DNA binding by individual positive regulators, and thus
inhibition should be susceptible to mutation of these individual
cis-acting elements; our transfections with site-directed mutations of these elements showed that cyclin A promoter activity was
still fully inhibited by IFN-
. Moreover, co-transfection of a p300
expression plasmid in transient transfection analysis did not relieve
IFN-
-mediated inhibition of cyclin A promoter activity (data not shown).
In contrast to our limited understanding of how IFN-
inhibits gene
expression, functionally significant cis-acting elements mediating transactivation in response to IFN-
signaling have been
precisely identified in a number of genes (68, 69). Nevertheless, despite the directness of signaling through the JAK-Stat pathway, alternative mechanisms for gene induction by IFN-
that do not require changes in cis element binding do exist. Class II
MHC genes are strongly induced by IFN-
, but expression of the
transcription factors that bind directly to class II MHC promoters is
not affected by IFN-
(70). In vivo footprinting of class
II MHC gene promoters showed that promoter occupancy in the class II
MHC-negative mutant cell line RJ2.2.5 was no different from that in the
class II MHC-positive Raji line, implicating a defect in a co-activator
protein (71). This co-activator was subsequently identified as class II
transactivator (CIITA) (72), an IFN-
-inducible protein whose
expression is strongly correlated with that of the class II MHC genes
(73). CIITA has separable functional domains: the N-terminal domain provides transcriptional activation and the C-terminal domain confers
promoter specificity (74). Mutation of any one of five distinct
elements in the proximal promoter of the class II MHC DR
gene resulted in impaired
CIITA-dependent transactivation (74), consistent with a
model of CIITA as a second tier co-activator that does not bind DNA
directly but activates transcription of specific promoters by a
mechanism that involves multiple cis-acting elements.
IFN-
can induce gene expression by direct and indirect mechanisms
and can also inhibit gene expression, presumably through indirect
pathways. Despite the opposite natures of these effects, induction of
class II MHC transcription and inhibition of cyclin A transcription
resulting from IFN-
treatment both share a similar dissociation of
promoter activity from individual protein-DNA interactions in the
proximal promoter region. RNA polymerase II-mediated transcription
involves a complex of factors that probably varies in composition from
one promoter to the next (75). Evidence from yeast indicates that some
cell cycle-regulated genes have unique requirements for particular
components of the general transcription factor TFIID complex (76).
Indeed, the complex associated with cyclin A expression is known to be
functionally distinctive, as a specific mutation in TAF(II)250, the
largest subunit of TFIID, disrupts transcription from the cyclin A but
not the c-fos or c-myc promoter (77). This
suggests that transcription complexes assembling on growth-related
genes may be selectively modified in response to changes in cellular
growth status. Our findings about IFN-
support the existence of a
means of regulating transcriptional activity of the cyclin A promoter
that does not involve direct DNA-protein interactions. More likely it
involves alteration of co-activators or other components of the
transcription complex that assembles on the cyclin A gene. Given the
array of cellular targets affected by IFN-
, and its known growth
inhibitory effects on a number of distinct cell types, we speculate
that its regulatory capacity may extend to components of the
transcriptional apparatus that drives expression of the cyclin A gene.
 |
ACKNOWLEDGEMENTS |
We thank Thomas McVarish for editing the
manuscript; Bonna Ith for technical assistance; Robert Schlegel for the
anti-cyclin A antibody; Ed Harlow for the CDK2, CDK4, and CDK6
plasmids; and Xin-Yuan Fu for the Stat 1a plasmid.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants HL03274 (to N. E. S. S.), HL03194 (to M. A. P.), and GM53249 (to M.-E. L.), a National Research Service Award (to C. P.),
and by a grant from Bristol-Myers Squibb Co..The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Deceased.
¶
To whom correspondence should be addressed: Cardiovascular
Biology Laboratory, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-3586; Fax: 617-432-2980; E-mail:
sibinga{at}cvlab.harvard.edu.
**
Present address: Millennium Pharmaceuticals, Cambridge, MA 02139.

Present address: Sealy Center for Molecular Cardiology,
University of Texas Medical Branch, Galveston, TX 77555.
§§
Present address: University of Tokyo Hospital, Tokyo 113-8655, Japan.
 |
ABBREVIATIONS |
The abbreviations used are:
IFN, interferon;
cdk, cyclin-dependent kinase;
VSMC, vascular smooth muscle
cell;
LM, ligation-mediated;
PCR, polymerase chain reaction;
DMS, dimethyl sulfate;
CAT, chloramphenicol acetyltransferase;
Rb, retinoblastoma protein;
TGF, transforming growth factor;
MHC, major
histocompatibility complex.
 |
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