From the Department of Cell Biology, University of Massachusetts
Medical School, Worcester, Massachusetts 01655
Received for publication, November 15, 2000, and in revised form, February 2, 2001
Interferon regulatory factors (IRFs) are
transcriptional mediators of interferon-responsive signaling pathways
that are involved in antiviral defense, immune response, and cell
growth regulation. To investigate the role of IRF proteins in the
regulation of histone H4 gene transcription, we compared the
transcriptional contributions of IRF-1, IRF-2, IRF-3, and IRF-7 using
transient transfection assays with H4 promoter/luciferase (Luc)
reporter genes. These IRF proteins up-regulate reporter gene expression
but IRF-1, IRF-3, and IRF-7 are more potent activators of the H4
promoter than IRF-2. Forced expression of different IRF combinations
reveals that IRF-2 reduces IRF-1 or IRF-3 dependent activation, but
does not affect IRF-7 function. Thus, IRF-2 may have a dual function in
histone H4 gene transcription by acting as a weak activator at low
dosage and a competitive inhibitor of other strongly activating IRFs at
high levels. IRF-1/IRF-3 and IRF-1/IRF-7 pairs each mediate the highest
levels of site II-dependent promoter activity and can
up-regulate transcription by 120-150-fold. We also find that interferon
up-regulates IRF-1 and site II-dependent
promoter activity. This up-regulation is not observed when the IRF site is mutated or if cells are preloaded with IRF-1. Our results indicate that IRF-1, IRF-2, IRF-3, and IRF-7 can all regulate histone H4 gene
expression. The pairwise utilization of distinct IRF factors provides a
flexible transcriptional mechanism for integration of diverse
growth-related signaling pathways.
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INTRODUCTION |
Interferon regulatory factors
(IRFs)1 form a large family
of transcription factors involved in antiviral defense, immune
activation, and cell growth regulation. IRFs were initially identified
as regulators of interferon genes in response to viral infection. However, it has subsequently been shown that there are at least nine
cellular IRF proteins (IRF-1, IRF-2, IRF-3, IRF-4/Pip/ICSAT, IRF-5,
IRF-6, IRF-7, ICSBP/IRF8, and ISGF3
/p48/IRF9), as well as virally
encoded forms, with broad biological functions (1, 2). All members of
the IRF family share significant homology in the N-terminal 115 amino
acids, which comprise the DNA-binding domain. For the IRF-3, IRF-4,
IRF-5, IRF-8, and IRF-9 proteins, the homology extends into the
C-terminal region with which these IRFs interact with other proteins or
family members. Current data indicate that IRFs can function as
transcriptional activators (e.g. IRF-1, IRF-3, and IRF-9),
repressors (e.g. IRF-8), or both (e.g. IRF-2,
IRF-4, and IRF-7). Studies with IRF-expressing cell lines and IRF
knockout mice reveal that IRF family members have distinct roles in
various biological processes, including cytokine signaling, responses
to pathogens, cell growth regulation, and hematopoietic development
(1-3).
IRF-1 and IRF-2 are transcription factors that interact with the same
DNA sequence element (designated ISRE/IRF-E) in the promoters of type I
interferon (IFN) and other cytokine inducible genes (4-8). IRF-1 is
up-regulated by type I interferons and the type II interferon, IFN-
(2). IRF-2 is up-regulated by IRF-1 and antagonizes IRF-1 activation by
competing with IRF-1 for its DNA-binding site (4, 9-12). IRF-2 also
functions as a transcriptional activator (13) and has been shown to
activate the genes for histone H4 (14, 15), Epstein-Barr virus nuclear antigen-1 (EBNA-1) (16), and murine muscle vascular cell adhesion molecule-1 (17). In addition, IRF-1 and IRF-2 can co-occupy the
Class II transactivator type IV promoter element IRF-E and synergistically activate this promoter (18).
IRF-1 and IRF-2 are key regulators of cell growth, cell cycle, and
apoptosis, and function as an anti-oncogene and oncogene, respectively
(14, 19-22). Our laboratory has established that IRF-1 and IRF-2 can
each functionally interact with and transcriptionally activate the H4
promoter (14, 15). Furthermore, the gene for p21WAF1/cip1, a member of the family of
cyclin-dependent kinase (CDK) inhibitors, which plays a
primary role in cell cycle control, is regulated in response to DNA
damage by both IRF-1 and p53 (23-25). These observations suggest that
the transcriptional properties of IRF-1 and IRF-2 are linked to their
cell growth regulatory potential.
Cell cycle control of histone gene transcription at the onset of S
phase is required for the functional coupling of histone gene
expression and DNA replication (26, 27). Transcriptional control of the
human histone H4 gene designated FO108 (28) has been extensively
studied. The H4 gene is regulated by two multipartite proximal promoter
elements (sites I and II), which together with two distal auxiliary
domains (sites III and IV) modulate histone H4 promoter activity (27).
Site II mediates cell cycle control of histone H4 transcription by
interacting with three distinct factors, including IRF-2/HiNF-M,
CDP-cut/HiNF-D, and HiNF-P (14, 29-33). The cell cycle element (CCE),
5'-CTTTCGGTTTT-3', which is located in the distal part of site II (34)
and controls transcription at the G1/S phase transition
(29), is known to interact functionally with both IRF-1 and IRF-2 (14,
15).
Recently, other IRF proteins (e.g. IRF-3 and IRF-7) have
been shown to contribute to transcriptional control via IRF-binding sites. For example, the formation of distinct heterodimers between activated IRF-3 and IRF-7 may lead to differential regulation of the
IFN-
and IFN-
genes (2, 35) which were initially characterized as
responsive to IRF-1 and IRF-2. Both IRF-3 and IRF-7 are constitutively
present in several cell types and can be activated in response to
different biological stimuli, including viral infection, type I
interferons, and/or DNA damage (2). These recent findings necessitate
evaluation of the extent to which distinct combinations of IRF proteins
may regulate histone H4 gene expression. To investigate the role of
multiple IRF members in histone H4 gene transcription, we performed
transfection studies with H4 promoter-luciferase reporter genes and a
panel of IRF expression vectors. Our results suggest that IRF-1, IRF-2,
IRF-3, and IRF-7 can all actively regulate histone H4 gene expression and that specific IRF pairs (i.e. IRF-1/IRF-3 and
IRF-1/IRF-7) are strong activators.
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MATERIALS AND METHODS |
Preparation of H4-luciferase Reporter Gene Constructs and IRF
Expression Vectors--
The wild type H4 promoter/luciferase reporter
gene construct wtH4/Luc was derived from pFO108 wt/CAT (30, 31, 36),
which contains the proximal promoter region of the H4 gene (nucleotides
240 to
38 relative to the ATG start codon; mRNA cap site at nucleotide
30) and spans sites I and II. The CAT gene was removed by
PstI and HindIII cleavage and replaced by a
1.65-kilobase PstI/HindIII fragment spanning the
luciferase (Luc) gene. The Luc gene was amplified from pGL3 (Promega,
Madison, WI) by polymerase chain reaction amplification with two
primers: forward PstI primer, 5'-gactgcagGCATTCCGGTACTGTTG-3'; reverse HindIII primer,
5'-gcaagcttACACGGCGATCTTTCC-3'; lowercase nucleotides were
added to create restriction sites. The H4/Luc construct in which the
IRF-binding site is mutated (IRF mutH4/Luc) was prepared from
pMSP16-CAT (33) and the CAT gene was exchanged for the Luc reporter as
described above. The 4X IRF/H4-Site II/Luc plasmid was constructed by
inserting an oligonucleotide cassette containing a tandemly repeated
IRF-binding site
(5'-gatccGCTTTCGGTTTTCAGCTTTCGGTTTTCAGATCCGCTTTCGGTTTTCAGCTTTCGGTTTTCa-3'; 5'-gatctGAAAACCGAAAGCTGAAAACCGAAAGCGGATCTGAAAACCGAAAGCTGAAAACCGAAAGCg-3'; BamHI/BglII overhangs) into the
BamHI site of pFP201CAT (29, 37) and was converted to a
luciferase reporter using the same polymerase chain reaction-derived
fragment described above. The FP201 segment of the H4 promoter spans
nucleotides
97 to
38. All oligonucleotides were synthesized using a
Beckman 1000M DNA synthesizer and all inserts were sequenced (ABI 100 model 377) to verify correct orientation and absence of polymerase
chain reaction or chemical synthesis-related mutations. The 3X H4
distal site II wild type promoter-luciferase reporter gene construct (3X H4 distal Site II/Luc) which contains three copies of an
oligonucleotide spanning the distal segment of H4 site II
(5'-CGCTTTCGGTTTTCAATCTGGTCCGATAC-3') fused to the TATA box of the H2-L
gene was a kind gift from Dr. Keiko Ozato (38). The companion
construct with mutated IRF-binding sites (3X H4 distal site II
IRF-mutant/Luc) was constructed by digesting the wild type plasmid with
XhoI and BglII to remove the multimerized site II
and then inserting the mutant multimer oligonucleotide
(5'-CGCTTCAGGTTTTCAATCTGGTCCGATAC-3'). IRF
expression constructs (pcDNA/IRF-1, pcDNA/IRF-2, and
6X-His-tagged human IRF-3 and IRF-7) were kindly provided by Dr. T. Maniatis (35, 39). The CDK2/Luc construct, containing the 2.4-kilobase
human cdk2 promoter inserted into the pGL2-basic plasmid, was a kind gift of Dr. Dov Shiffman (40).
In Vitro Translation and Electrophoretic Mobility Shift
Assay--
The expression constructs pcDNA IRF-1, pcDNA IRF-3,
and pcDNA IRF-7 were subjected to coupled in vitro
transcription and translation with unlabeled methionine or
[35S]methionine in a rabbit reticulocyte lysate system
according to the manufacturer's instructions (Promega). Aliquots of
35S-labeled IRF-1 (5 µl), IRF-3 (20 µl), and IRF-7 (20 µl) were separated by SDS-PAGE in a 10% gel, which was subsequently
dried and exposed for autoradiography. The bands for IRF-1, IRF-3, and IRF-7 were removed and incorporation of [35S]methionine
for each protein was measured using an LS 6500 multipurpose scintillation counter (Beckman, Fullerton, CA). The measured
radioactivity (in cpm) was used to calculate the molar ratios of IRF-1,
IRF-3, and IRF-7. Electrophoretic mobility shift assays were performed as described previously (14) with unlabeled in vitro
translated IRF proteins. Each reaction contained 10 fmol of
32P-labeled double-stranded CCE oligomer, IRF protein, 2 µg of poly(dG-dC)·(dG-dC), 1 µg of poly (dI-dC)·(dI-dC), and
1 pmol of unlabeled competitor oligomers where indicated. CCE-wt is
5'-GATCCCGGCGCGCTTTCGGTTTTCA and CCE-mut is
5'-GATCCCGGCGCGCTTTCAGGTTTTCA. The binding reactions were separated in
a 4% polyacrylamide gel.
Cell Culture and Transfection Experiments--
Actively
proliferating cultures of NIH3T3 cells were maintained at subconfluency
in Dulbecco's modified Eagle's medium, supplemented with 10% fetal
calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin (Sigma),
and 0.2 µM L-glutamine, at 37 °C in
humidified air containing 5% CO2. Cells were seeded in
6-well culture plates at a density of 1.5 × 105
cells/well, and were transiently transfected 24 h later at ~70% confluency by the Superfect transfection method (Qiagen, Valencia, CA).
We co-transfected 0.8 µg of each H4/Luc reporter gene construct with
different amounts of each IRF expression vector. The amount of DNA in
each well was kept constant by supplementing the transfection mixture
with the empty expression vector. Cells were also transfected with 50 ng/well of the pRL-CMV construct (Promega), which contains a
cytomegalovirus promoter upstream of the Renilla luciferase gene, as an
internal control for transfection efficiency (41). Cell lysates were
prepared for luciferase assay or for Western blot analysis 24 h
after transfection. To monitor the effect of IFN-
, cells transfected
with reporter gene constructs at 0.8 µg/well were incubated with
0-1.0 ng/ml IFN-
at 24 h post-transfection, and analyzed
12 h after treatment. Each transfection was performed in
triplicate and repeated at least three times.
Measurement of Reporter Gene Activity by Dual-luciferase
Assay--
Cells were washed twice with 1 × PBS buffer 24 h
after transfection and lysed with 1 × lysis buffer (Promega).
Luciferase assays were carried out according to the manufacturer's
specifications using a dual-luciferase reporter assay system (Promega)
and a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). The activity of Renilla luciferase was used to normalize for variation in transfection efficiency by calculating the
ratio of firefly and Renilla luciferase activities.
Western Blot Analysis and Densitometry--
Cell lysates were
centrifuged at 14,000 × g (4 °C for 30 min), and
protein concentrations were determined using the Coomassie protein
assay reagent (Pierce Chemical Co., Rockford, IL) according to the
manufacturer's instructions. Equal amounts of total cellular protein
were mixed with loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 2%
-mercaptoethanol, and bromphenol blue), boiled for 5 min, and subjected to 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp., Bedford, MA). The membranes were saturated with phosphate-buffered saline containing 0.05% Tween 20 (1 × PBS-T buffer) and 5% fat-free dry milk (42) for 1 h at room temperature and incubated overnight with primary IRF antibodies at 1:1,000 dilution
(
-IRF-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); 1:3,000
dilution (
-IRF-2 (14)), or
-His antibodies at 1:1,500 dilution
(for detection of IRF-3 or IRF-7; Qiagen) in 1% fat-free dry milk in
1 × PBS-T buffer. After washing with 1 × PBS-T buffer
containing 1% milk, blots were further incubated for 1 h at room
temperature with horseradish peroxidase-conjugated secondary antibody
(Santa Cruz Biotechnology, Inc.) diluted 1:10,000 in milk/PBS-T buffer.
Blots were then washed five times with the same buffer before
visualization of immunoreactive protein bands by enhanced
chemiluminescence detection (ECL kit; Amersham Pharmacia Biotech Inc.,
Piscataway, NJ). Densitometry was performed by using the Alpha Imager
2000 densitometer (Alpha Innotech Corp., San Leandro, CA) according to
the manufacturer's instructions. The protein level of each IRF member
in untransfected cells was set as control and the relative protein
level of each IRF member in transfected cells was determined by
dividing the densitometry measurements of IRF transfected cells by the
densitometry measurements of the control.
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RESULTS |
IRF-3 and IRF-7 Bind to the Cell Cycle Element (CCE) of Histone H4
Site II--
The CCE within site II of the histone H4 promoter has
previously been shown to interact with IRF-1 and IRF-2 (14) and has high similarity to the IRF-E and ISRE consensus elements. To determine whether more recently identified IRF family members (e.g.
IRF-3 and IRF-7) are also capable of binding to the histone H4
promoter, we performed protein-DNA interaction studies with IRF
proteins produced by coupled in vitro transcription and
translation. The IRF-1, IRF-3, and IRF-7 proteins were analyzed by
SDS-PAGE in a 10% gel for radiometric quantitation (Fig.
1, A and B).
Approximately equimolar amounts of these IRF proteins were evaluated by
electrophoretic mobility shift assay for binding to an oligonucleotide
spanning the CCE in the distal segment of histone H4 site II (Fig.
1C). All three proteins (Fig. 1), as well as IRF-2 (Ref. 14
and data not shown), form complexes with the CCE and these complexes
are competed specifically by the unlabeled wild type but not the mutant CCE oligonucleotides. The relative intensities of the signals of
protein-DNA complexes suggest that IRF proteins have different affinities for the same site (IRF-1 = IRF-2 > IRF-3 = IRF-7) (Fig. 1 and data not shown). Our results indicate that in
addition to IRF-2 (14), IRF-1, IRF-3, and IRF-7 are also capable of
binding to the CCE in the histone H4 promoter.

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Fig. 1.
IRF-3 and IRF-7 interact with distal site II
of the histone H4 gene promoter. A,
35S-labeled IRF-1, IRF-3, and IRF-7 proteins (see
"Materials and Methods") were separated by SDS-PAGE in a 10% gel.
Mr standards are shown on the left.
B, equimolar amounts of IRF-1, IRF-3, and IRF-7 were used in
electrophoretic mobility shift assays and shown in the gel. Molar ratio
was calculated by measuring the [35S]methionine
radioactivity and the number of methionines in each IRF protein.
C, electrophoretic mobility shift assays with in
vitro transcribed and translated IRF-1, IRF-3, and IRF-7 (as
indicated above each panel) show sequence specific binding
to the CCE/site II of histone H4 promoter. Competition analysis of the
IRF·CCE complexes was carried out with 50-fold excess unlabeled
wild-type (CCE-wt) and mutant (CCE-mut) oligonucleotides.
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Multiple IRF Proteins (IRF-1, IRF-2, IRF-3, and IRF-7) Regulate
Site II-dependent H4 Promoter Activity--
We have
previously shown that the transcription factor IRF-2 can activate
histone H4 gene expression (14) and is involved in cell cycle
regulation of histone H4 gene transcription (15). To determine whether
IRF-1, IRF-3, and IRF-7 function as activators or repressors of histone
H4 gene expression, we performed co-transfection assays with IRF
expression vectors and histone H4 gene promoter/luciferase reporter
gene constructs (Figs. 2 and
3). We tested IRF-dependent activation in the context of the wild type histone H4 promoter spanning
sites I and II, as well as with a mutant H4 promoter in which the IRF
binding element in site II was altered by a two-nucleotide substitution
that prevents IRF binding (33). The results show that IRF-3 and IRF-7
are each capable of activating transcription by 5-6-fold (Fig.
3A). For comparison, IRF-1 and IRF-2 increase transcription
by ~11- and 2-fold, respectively. When the IRF-binding site was
mutated, activation of the histone H4 promoter by IRF factors was
completely abrogated (Fig. 3B). These results show that
IRF-3 and IRF-7 can activate the histone H4 promoter via the IRF
recognition motif in site II.

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Fig. 2.
Organization of the H4 gene proximal promoter
and five H4 gene promoter-luciferase constructs. A,
promoter organization of the human histone H4 gene designated FO108
(28). B, schematic diagram of the H4 promoter-luciferase
reporter constructs used in this study: 1) wtH4/Luc,
2) IRF mutH4/Luc, 3) 4X IRF/H4-site II/Luc, 4) 3X
H4 distal site II/Luc, 5) 3X H4 distal site II IRF
mutant/Luc.
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Fig. 3.
The effects of four IRFs on histone H4
transcription assayed with different promoter constructs. NIH3T3
cells were transfected with 0.8 µg/well of different luciferase
promoter constructs: A, wtH4/Luc; B, IRF
mutH4/Luc; C, 4X IRF/H4-site II/Luc; D, 3X H4
distal site II/Luc. The cells were co-transfected with 0.4 µg/well of pcDNA/IRF-1, pcDNA/IRF-2, pcDNA/IRF-3,
pcDNA/IRF-7, and pcDNA as control. Renilla luciferase construct
(50 ng/well) was used as an internal control for each sample. Samples
were analyzed by the dual-luciferase assay 24 h after transfection
as described under "Materials and Methods." The graphs
were based on at least three independent experiments with triplicate
samples.
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To assess further the role of H4 site II promoter elements in mediating
activation by IRF factors, we prepared two promoter constructs in which
either the IRF element or the entire distal site II segment was
tandemly repeated upstream of distinct minimal promoters,
i.e. human histone H4 or the mouse MHC class I H2-L TATA box
regions, respectively (Fig. 3, C and D). Our
results indicate that IRF-1 can synergistically activate H4-related
transcription in the presence of multimerized IRF elements (compare
36-60-fold activation in Fig. 3, C and D, with
11-fold activation in Fig. 3A). In contrast, IRF-2, IRF-3,
and IRF-7 show approximately the same levels of activity for both the
wild type histone H4 promoter and the multimerized IRF promoter
constructs. Thus, it appears that IRF-1, but not other IRFs, can
synergize with itself to up-regulate H4 site II-related transcription.
Dose-dependent Activation of H4 Site
II-dependent Transcription by IRF Proteins--
The
differences in site II-dependent transcriptional activation
observed for the four IRF proteins may be influenced by the relative
levels of these factors. Therefore, we analyzed reporter gene activity
(3X H4 distal site II/Luc) at different levels of each IRF factor by
monitoring transcription as a function of time after transfection (Fig.
4) or concentration of expression vector (Fig. 5). The results show that
IRF-related transcriptional activation by IRF-1, IRF-3, and IRF-7, but
not IRF-2, is dramatically increased as these proteins accumulate at
later times (e.g. 10-24 h) after transfection (Fig. 4). To
relate IRF-dependent activation directly to protein levels,
we performed Western blot analysis of cells transfected with different
amounts of expression constructs and also monitored IRF activity by
measuring reporter gene expression in parallel. Western blot analysis
demonstrates that increasing the amount of IRF expression vector
results in the expected elevation of cellular IRF proteins (Fig. 5,
right panels). Reporter gene assays reveal that
IRF-1-dependent activation through site II reaches
saturation at a relatively modest level (0.4 µg) of IRF-1 expression
vector, whereas with increasing IRF-3 and IRF-7 levels, transcriptional
activity continues to increase (Fig. 5, left panels). These
data are consistent with the apparent affinities of the IRF factors for
site II (see Fig. 1) in that the protein with the highest affinity
(i.e. IRF-1) may reach binding site saturation inside the
cell at lower protein concentrations than IRF-3 and IRF-7.

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Fig. 4.
Time course effects of IRF proteins on H4
site II promoter activity. NIH3T3 cells were transfected with 3X
H4 distal site II/Luc construct (0.8 µg/well) together with pcDNA
expression vectors containing IRF-1, -2, -3, -7, or pcDNA alone
(0.4 µg/well). Renilla luciferase construct (50 ng/well) was used as
the internal control. Samples were taken at time points 0, 4, 6, 8, 10, 12, and 24 h after transfection and analyzed by the
dual-luciferase assay. Results shown were based on three independent
experiments, each comprised of triplicate samples.
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Fig. 5.
IRF proteins up-regulate H4 promoter activity
in a dose-dependent manner. NIH3T3 cells were
co-transfected with 3X H4 distal site II promoter/Luc plasmid (0.8 µg/well) and increasing amounts of IRF-1, IRF-2, IRF-3, and IRF-7
expression constructs. Dual luciferase assays performed 24 h after
transfection show promoter activity in response to increasing amounts
of IRF expression (left panels). The same samples were
analyzed by Western blot assays with specific IRF antibodies for IRF-1
and IRF-2 (right panel, the upper part of
A and B) or with antibody to the His epitope tag
for IRF-3 and IRF-7 (right panel, C and
D). The graphs on the right show IRF expression
levels relative to the levels found for untransfected cells, and are
based on quantitation of Western blot data (see "Materials and
Methods"). The reported values represent an average of two (for
Western blots) or three (for luciferase assays) independent experiments
of triplicate samples.
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Interestingly, increasing concentrations of IRF-2 result in enhancement
of H4-site II driven promoter activity at low dosage, but not at higher
levels (Fig. 5B). At the highest concentrations tested,
IRF-2 does not display transactivation potential. These data indicate
that IRF-2 is only transcriptionally active in a very narrow
concentration range. Taken together, our IRF titration results indicate
that these proteins have highly distinct
concentration-dependent activity profiles. These distinct
profiles suggest that differences in transcription observed on the wild
type H4 promoter and site II related test promoters (see Fig. 3) can be
accounted for by both IRF protein concentration and intrinsic
transactivation potential.
IRF-1/IRF-3 and IRF-1/IRF-7 Pairs Are Strong Activators of H4 Site
II-dependent Transcription--
Recently it has been shown
that IRF-3 and IRF-7 can function together in the regulation of the
IFN
promoter (35). To assess whether IRF-3 and IRF-7 are capable of
co-regulating histone H4 gene transcription together with IRF-1 or
IRF-2, we performed co-transfection assays with pairwise combinations
of IRF proteins (Fig. 6). The experiments
show that co-expression of increasing amounts of IRF-2 in the presence
of a fixed amount of IRF-1, IRF-3, or IRF-7 results in either a minor
decrease or no effect on promoter activity (Fig. 6, A, C,
and D). When IRF-2 concentrations are maintained at a
constant level in the presence of increasing amounts of IRF-1, IRF-3,
or IRF-7, enhancement of promoter activity is observed (Fig.
6B). These findings are consistent with limited competition
of the IRF-2 protein with IRF-1, IRF-3, or IRF-7, each of which has
higher activation potential at H4 site II than IRF-2. Strikingly, all
pairwise combinations of IRF-1 with either IRF-3 or IRF-7 yield
significantly stronger activation of reporter gene expression than any
one of these three factors by itself (Fig. 6). For comparison, the
combination of IRF-3 and IRF-7 (Fig. 6, C and D)
results in quantitatively modest levels of reporter gene transcription.
We conclude from these data (Fig. 6) that IRF-1/IRF-3 and IRF-1/IRF-7
pairs are the strongest activators of H4 site II-dependent
transcription.

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Fig. 6.
Combinatorial effects of IRF protein
expression on the up-regulation of the H4 site II promoter. NIH3T3
cells were transfected with 3X H4 distal site II/Luc, a fixed amount
(0.4 µg/well) of IRF-1 (panel A), IRF-2 (panel
B), IRF-3 (panel C), or IRF-7 (panel D), and
increasing amounts (0, 0.1, 0.2, and 0.4 µg/well) of the remaining
three IRF factors, except for IRF-2 in panel A, where the
amounts were 0, 0.05, 0.1, 0.2, 0.3, and 0.4 µg/well. At least four
independent experiments with triplicates were performed and normalized
as described in Fig. 3.
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Interferon-
Selectively Up-regulates Histone H4 Site
II-dependent Transcription--
Upon establishing that
IRF-1 in combination with IRF-3 and IRF-7 is a strong enhancer of cell
cycle controlled histone H4 gene transcription, we addressed the
possibility that H4 gene promoter activity may be responsive to
signaling mechanisms that up-regulate IRF-1. To address whether H4 site
II is capable of integrating IRF-related cellular responses, we
transfected cells with H4/Luc constructs and treated cells with
interferon-
(Fig. 7). When transfected
mouse NIH3T3 cells were treated with mouse interferon-
(0.5 ng/ml),
we observed a strong 4-5-fold up-regulation of H4 site II-driven
luciferase activity (Fig. 7, A and B) which did
not occur when the IRF element within Site II was mutated (Fig.
7B). Consistent with the species specificity of
interferon-
signaling, the same concentration of human
interferon-
did not influence reporter gene activity in mouse NIH3T3
cells (Fig. 7A). We also did not observe effects of mouse
interferon-
on a promoter-less construct (pGL2) or an unrelated
promoter (CDK2/Luc) (Fig. 7B). Interferon-
did not
further enhance site II-dependent transcription when cells
were preloaded with IRF-1 or IRF-7 (Fig. 7C). This desensitization of interferon-
is consistent with IRF-1 being a
downstream mediator of interferon-
effects on site II. Consistent with this concept, we find that interferon-
significantly increases IRF-1 protein levels in NIH3T3 cells (Fig. 7D). Taken
together, interactions of IRF-1 and other IRF factors with the cell
cycle regulatory element in site II of the histone H4 gene may support interferon-
-dependent cell signaling mechanisms.

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Fig. 7.
Activation of histone H4 gene promoter and
IRF-1 expression in response to IFN- .
NIH3T3 cells were transfected with 3X H4 distal site II/Luc wild type
or IRF-mutant constructs and treated with interferon- . A,
dose-dependent activation of the wild type promoter in
response to mouse (mIFN ( ) or human (hIFN ( ) interferon- .
B, effect of mouse IFN- treatment (0.5 ng/ml)
on activity of vector control (pGL2), histone H4 wild type, histone H4
IRF-mutant, and Cdk2 gene promoters. C, effects
of IRF-1, IRF-2, IRF-3, and IRF-7 or control pcDNA vector in
combination with mouse IFN- (0.5 ng/ml) on H4 promoter activity. The
reported values are an average of three independent experiments of
triplicate samples. D, Western blot analysis of
IRF-1 levels in the experiment shown in B; actin protein
levels are shown for comparison.
|
|
 |
DISCUSSION |
Cell cycle control of histone H4 gene transcription involves a
3-fold enhancement of promoter activity at the G1/S phase
transition and is mediated by the CCE. The results of this study show
that the CCE represents a universal IRF recognition motif capable of interacting with all IRF members analyzed to date (i.e.
IRF-1, -2, -3, and -7). We find that these IRF factors can each
up-regulate histone H4 gene transcription and that IRF-1 is a stronger
activator than IRF-2, IRF-3, or IRF-7. More interestingly, pairwise
combinations of IRF-1 with either IRF-3 or IRF-7 mediate the strongest
level of activation. Thus, dimeric IRF interactions with the CCE may function to integrate signals of two distinct classes of IRF factors. The multitude of IRF factors involved in diverse immunological, genotoxic, and cell growth regulatory functions may provide a broad
spectrum of gene regulatory options to control H4 gene transcription during the cell cycle.
We have previously shown that IRF-2 is identical to HiNF-M, the major
DNA-binding protein that interacts with the CCE within H4 site II in
HeLa S3 cells. Cell cycle analyses using mouse fibroblasts in which the
genes for IRF-1 and/or IRF-2 have been ablated show that IRF-2 is
important for cell cycle regulation of histone H4 gene expression (15).
The deregulation of histone gene expression in these cells may be due
to direct effects on the promoter or to a general effect of the IRF-2
oncoprotein on cell growth. Studies using human HeLa cervical carcinoma
cells have revealed that abolishing the IRF/site II interaction by
mutating the CCE has a major effect on basal level transcription (29,
33), but does not affect the timing of transcription (43). These data
suggest that the role of IRF-2 in histone gene expression may be
related to the growth regulatory phenotype of the cell and is possibly
subject to compensatory mechanisms. The main finding in this study is that different IRF members can substitute for IRF-2 in modulating transcription, which may reflect the in vivo complexity of
cell cycle regulatory mechanisms at the site II element.
IRF-2 is a weak activator of histone H4 gene transcription (14) and is
also known to activate other genes, including EBNA/Qp and vascular cell
adhesion molecule-1 (16, 17, 44). For some genes, IRF-2 has been shown
to inhibit IRF-1 activation (4, 13), ostensibly by competing with IRF-1
for IRF-binding sites. The data presented here indicate that IRF-2 has
a biphasic transcriptional activity curve in response to elevations in
IRF-2 levels. Activation of the H4 gene is only observed in cells
expressing low levels of IRF-2, and IRF-2 becomes transcriptionally
neutral at high dosage. Thus, the dual role of IRF-2 in both
transcriptional activation and inhibition of different genes may in
part be attributable to cellular IRF-2 levels. The biphasic response of
IRF-2 in modulating H4 gene transcription is consistent with data on
IRF-2 dependent activation of the EBNA/Qp promoter in different cell
types, which displays a similar dependence of promoter activity on
IRF-2 concentrations (45).
In this study, we have shown that IRF-1 by itself is the strongest
transactivator of histone H4-related transcription when compared with
IRF-2, IRF-3, and IRF-7. IRF-1 has been shown to regulate the genes for
IL-4, IL-5, IL-7 receptor, IL-12, guanylate-binding protein, EBNA/Qp,
cyclin D1, ICAM 1, p53, ICE, 2-5A synthetase, p21, c-Myb,
IRF-2, as well as H4 (10, 13-16, 19, 21, 22, 25, 46-48). The strength
by which IRF-1 can activate transcription via the CCE suggests that the
H4 gene is a physiological target of IRF-1 at least in some biological
circumstances. IRF-1 dependent control of H4 gene expression would be
consistent with the cell growth and/or apoptotic properties of this
protein (1, 2). One novel finding of our study is that IRF-3 and IRF-7
are also capable of mediating CCE-related control of H4 gene
transcription and that these proteins, when paired with IRF-1, are the
strongest modulators of promoter activity. Hence, the H4 gene appears
to be targeted by heterodimers comprised of IRF-1/IRF-3 and
IRF-1/IRF-7.
IRF-3 and IRF-7 are phosphorylated in virus-infected cells.
Phosphorylation is required for nuclear-cytoplasmic transport of IRF-3
and IRF-7, transcriptional activation, and association of IRF-3 with
p300 (35, 49-54). IRF-7 was first described to bind and repress the Qp
promoter region of the Epstein-Barr virus encoded gene EBNA-1 which
contains an ISRE-like element (44, 55). Recent results indicate that
IRF-3 and IRF-7 can be detected in both uninfected and virus-infected
cells and that IRF-7 expression is up-regulated by type I IFNs,
lipopolysaccharide, and virus infection (35, 56). Our results clearly
indicate that IRF-3 and IRF-7 together with IRF-1 activate the histone
H4 gene promoter in the absence of viral infection.
Recent studies have indicated that IRF family members form homo- and/or
heterodimers and upon binding to DNA can regulate the same gene. For
example, IRF-8 forms multiple protein complexes with both IRF-1 and
IRF-2 (57, 58), while IRF-3 and IRF-7 form dimers upon binding to the
IFN
promoter (52, 56, 59, 60). Similar complexes were reported for
IRF-1 and/or IRF-2 (16, 38, 58, 61, 62). Our protein/DNA interaction
data, as well as results from transient co-expression experiments,
suggest that IRF family members may also form dimers at the CCE to
regulate histone H4 gene expression. These combined observations
reinforce the idea that protein-protein complexes play central roles in the ability of IRFs to bind their cognate target genes and mediate biological functions. We conclude that the multiplicity of IRF family
members apart from roles in virus-mediated signaling perform essential
functions in the regulation of cell growth control in normal and tumor cells.
IFN-
is a multifunctional cytokine with a highly cell-type dependent
activity that plays an important role in immunity and cell growth
control (63-67). For example, IFN-
mediates activation of an
antiviral state and regulates cell growth (64, 68). Furthermore,
IFN-
is capable of inducing cell cycle arrest and apoptosis in
primary hepatocytes, but not in hepatoma cell lines (HepG2, H4IIE, and
Hepa1-6) (63). Interferon-
can stimulate cell growth depending on
serum levels in malignant human T cells (68). Our data demonstrate that
CCE dependent activity of the histone H4 gene promoter but not CDK2
promoter activity is selectively elevated in response to IFN-
through IRF-1. IRF-1 dependent activation of interferon-
responsive
genes has also been observed for other genes (1-5). The biological
effects of IFN-
are mediated through a heterodimeric transmembrane
receptor which is capable of stimulating the JAK-STAT pathway (69) and
result in activation of IRF-1. Not withstanding the known role of
interferon-
in cell growth suppression of different cell types (69),
these data suggest that IRFs, IFN-
, and the CCE of the histone H4
gene are components of a cell signaling mechanism that may contribute
to regulation of histone H4 gene transcription during the cell cycle in
a cell-type or serum-dependent manner.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010391200
The abbreviations used are:
IRF, interferon regulatory factor;
Luc, luciferase;
IFN, interferon, EBNA-1,
Epstein-Barr virus nuclear antigen-1;
CDK, cyclin-dependent
kinase;
CCE, cell cycle element;
CAT, chloramphenicol
acetyltransferase;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline.
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