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INTRODUCTION |
The transcription factor
STAT11 is activated by
tyrosine phosphorylation mediated by JAK family kinases during cellular
responses to cytokine or growth factor signaling (reviewed in Ref. 1). STAT1 directly regulates expression of key proteins involved in controlling the cellular processes of growth arrest (2, 3) by inducing
expression of p21WAF/CIP (4, 5) and cell death via
expression of caspases (6). Our work (7, 8) and that of others (9-11)
revealed that STAT1 expression is deficient in a number of different
human cancer cell types resistant to type I IFNs in antiproliferative
and antiviral assays. STAT1 has also been shown to be the target of
viral proteins inhibiting IFN signaling responses (12). In addition,
STAT1 expression in tumor cells has been shown to be important for
tumor elimination by immune surveillance mechanisms (13) as well as for
mycobacterial immunity in humans (14). STAT1 is an integral component
in a range of different transcription factor complexes. For example,
the two most commonly identified are GAF and ISGF3, involved in
the signal transduction pathways of type I and II IFNs, respectively
(reviewed in Refs. 1 and 15). The GAF DNA-binding sequence (GAS)
exhibits dyad symmetry across two half-sites, consistent with the
binding of STAT1 proteins as a homodimer. Related GAS elements with
between 2 and 4 intervening nucleotides bind transcription factors that
are activated not only by IFNs, but also by numerous other cytokines
and growth factors. Thus, GAS elements are capable of binding homo- and
heterodimers of the STAT proteins STAT1, STAT3, STAT4, STAT5, and
STAT6. A heterodimer of STAT1 and STAT2 can bind weakly to the GAS
element, resulting in transactivation (16).
ISGF3 is recognized as the major multisubunit transcription factor
activated in response to type I IFNs. ISGF3 comprises two components,
ISGF3
or IRF-9, a single 48-kDa DNA-binding protein, and ISGF3
,
which consists of the STAT1 (91 kDa) or STAT1
(84 kDa) protein in
a heterodimeric complex with STAT2 (113 kDa). Treatment of cells with
IFNs can also result in association of homodimers of STAT1 or STAT2 in
ISGF3. Complexes of STAT2 homodimers with IRF-9 may form, but have only
weak DNA binding and weak transactivating potential. Type II IFN
stimulation can result in formation of ISGF3 as a STAT1
homodimer·IRF-9 complex (17). Thus, a number of different STAT1
complexes have been identified to date.
Once formed, the ISGF3 complexes translocate to the nucleus and bind to
the ISRE to activate ISG expression. The ISRE sequence is
orientation-independent and contains direct repeats of either 5'-TTTC-3' (for example, see Ref. 18) or, more commonly, the complement
5'-GAAA-3', and the repeats are usually separated by a spacing of 1-2
nucleotides (reviewed in Ref. 19). The ISG15 ISRE with
sequence
5'-CTCGGGAAAGGGAAACCGAAACTGAAGCC-3' binds to two different IFN-inducible transcription factors and has been
studied extensively. Not only is the core sequence (the nonamer
underlined in boldface) of the ISRE targeted by the trimeric ISGF3
complexes via IRF-9, but it also binds to IRF-9 alone or to other
members of the IRF family related to IRF-9 such as IRF-1, IRF-2, IRF-3,
IRF-7, and ICSBP. Phosphorylated STAT1-STAT2 heterodimers or
homodimers of either are unable to bind to ISRE sequences, but can bind
to GAS elements. IRF-9 directs binding of ISGF3 to the core ISRE, and
STAT1 stabilizes the interaction with DNA by recognizing and binding to
STAT1 GA(A/T)TC half-sites flanking the core ISRE
sequence (17).
Here we report the mapping of the human STAT1 gene from the
Centre d'Etude du Polymorphisme Humain human YAC library using a PCR-based screening approach, isolation and characterization of the
5'-genomic region, and identification of a 5'-transcriptional start
site. Putative DNA-binding sites for transcription factors were
identified within the intron 1/exon 2 boundary sequence. Transient
expression assays with luciferase reporter constructs and gel mobility
shift assays confirmed the presence of an unusual ISRE
(IRF-E/GAS/IRF-E) binding a high molecular mass IRF-1 complex that
included CBP and was induced by stimulation with either type I or II
IFN. Interestingly, we have not found any binding of ISGF3 components
to this ISRE. We propose that the IRF-1 and STAT1 genes and
their proteins form an intracellular amplifier circuit regulating cellular responsiveness to the IFNs.
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MATERIALS AND METHODS |
Cell Culture and Stimulation--
Melanoma cell line SK-MEL-28
(American type Culture Collection, Manassas, VA) was grown in RPMI 1640 medium supplemented with 10% inactivated fetal calf serum. Human MCF-7
breast cancer and U293T embryonic kidney cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% inactivated
fetal calf serum. In all experiments, cells were stimulated with either
1000 IU/ml IFN-2a or 1000 IU/ml IFN- (Hoffmann-La Roche, Basel,
Switzerland) for the indicated time periods. For IFN- priming, cells
were pretreated with 1000 IU/ml IFN- for 16 h prior to
stimulation with IFN-2.
Antibodies--
Antibodies were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA) and included anti-STAT1 supershift
(sc-464x), anti-p48/ISGF3 supershift (sc-496x), anti-IRF-1 (sc-497),
and anti-IRF-2 (sc-498). Anti-IRF-3 antibody was a gift from Dr. Ricky Johnstone (Austin Research Institute, Heidelberg, Victoria, Australia).
YAC Clones, DNA Purification, and Primers Used for Detecting the
STAT1 Gene--
YAC clones neighboring D2S118 or AFM066xc1
sequence tagged sites (STS), including 737c9, 755b5, 764F5,
805c1, 809e2, 820b12, 821e5, 855g4, 855h9, 984a5, 894a6, and 894a11,
were obtained from Dr. Simon Foote (Walter and Eliza Hall Institute for
Medical Research, Parkville, Victoria). A single colony of each YAC
strain was expanded by growth in 10-ml cultures as described (20), and
the DNA was purified as described, washed with 80% ethanol before it
was dried, and resuspended in 50 µl of MilliQ water. For rapid
preparation of yeast for analysis by PCR, a yeast colony (0.5-2 mm)
taken from a plate was lysed by addition of 2-10 µl of incubation
solution (100 mM sodium phosphate (pH 7.4) and 1.2 M sorbitol) containing zymolyase 20T (2.5 mg/1 ml of
incubation solution) and incubated at 37 °C for 5 min. The digested
product was ready to be used in the PCR or was stored at 
20 °C for
future use. The primers used were as follows: STAT1yac3'a
(18-mer), 5'-CATCTTCTCTGGCGACAG-3', corresponding to nucleotides
2432-2449 of STAT1 cDNA sequence (GenBankTM/EBI accession number M97935); and
STAT1yac3'b (20-mer), 5'-CAGTAAGATGCATGATGCCC-3', corresponding to nucleotides 2599-2618 of STAT1 cDNA sequence.
Bubble PCR and Primers Used to Isolate the STAT1 5'-Genomic
Region--
The procedures for bubble PCR were as described previously
(21) and formed the basis for the methods used here. The
oligonucleotide sequences used for the construction of bubble
anchors/vectorettes were as follows: BubbleVec Top primer
(55-mer),
5'-GCTCTCCCTTCTGCGGCCGCAGTTCGTCAACATAGCATTTCTGTCCTCTCCTTCG-3'; and BubbleVec Bottom primer (60-mer),
5'-AATTCGAAGGAGAGGACGCTGTCTGTCGAAGGTAAACGGACGAGAGAAGGGAGAGCTGCA-3'. Bubble anchors were annealed and ligated to EcoRI- and
PstI-digested DNA from either the 805c1 or 809e2 YAC clone
via the overhanging ends of the anchors/vectorettes. Prior to PCR,
ligated products were extracted from excess bubble anchors/vectorettes
by passage through a QIAQuick PCR purification kit (QIAGEN Inc.) as
outlined by the manufacturer. The final products were used as templates for bubble PCR with the "hot start" procedure and platinum
Taq polymerase (Invitrogen) in a total volume of 50 µl,
consisting of 5 ng of anchored DNA template and 1 µM
primers. One of the primers used was either BubbleprimerIntE or
BubbleprimerIntP (described below), which would prime from the
anchor/vectorette at the 5'-end of the target DNA; the other primer was
based on the nucleotide sequence of the STAT1 cDNA
(GenBankTM/EBI accession number M97935). PCR cycling
parameters were as follows: 94 °C for 3 min, followed by 35 cycles
of 94 °C for 15 s, 63 °C for 10 s, and 72 °C for 3 min. The product was gel-purified before cloning into the pGEMT-Easy
vector used for DNA sequencing reactions (Big Dye sequencing kit,
PerkinElmer Life Sciences). The primers used were as follows:
BubbleprimerIntE (30-mer), 5'-GCGGCCGCAGTTCGTCAACATAGCATTTCT-3'; BubbleprimerIntP (27-mer), 5'-GCTGTCTGTCGAAGGTAAACGGACGAG-3'. In
addition, three primers were prepared as complementary sequences corresponding to the indicated regions from the STAT1 cDNA
sequence (GenBankTM/EBI accession number M97935) as
follows: STAT1 prom primer (30 mer),
5'-GGAATTCCGCTGCTGAAGTTCGTACCACTG-3',
corresponding to nucleotides 203-224; STAT1
upstream primer, 5'-CTTTCCGGCGCAGAGTCTGC-3', corresponding to nucleotides 26-46; and STAT1 upRev
primer, 5'-CTGCGGAGGGGCTCGGCGAG-3', corresponding to nucleotides
10-29.
RNA Ligase-mediated Rapid Amplification of cDNA Ends
(RLM-RACE)--
Messenger RNA was isolated from SK-MEL-28 cells
treated with IFN- (1000 IU/ml, incubated for 16 h) using the
FastTrack 2.0 mRNA isolation kit (Invitrogen). To obtain the
full-length 5'-end of the human STAT1 cDNA, full-length
RLM-RACE was performed using the GeneRacer kit (Invitrogen) according
to the protocols recommended by the manufacturer. The GeneRacer kit
ensures that only full-length transcripts are amplified by prior
elimination of truncated transcripts. The STAT1
gene-specific primer (STAT1prom) was used to synthesize the
first-strand cDNA template. Amplified fragments resulting from
RLM-RACE were cloned into pCR4Blunt-TOPO® (Invitrogen),
and the DNA sequence was determined using the DYEnamic ET terminator
cycle sequencing kit (Amersham Biosciences).
Electrophoretic Mobility Shift Assays--
The
32P-radiolabeled double-stranded oligonucleotides used
included the following: p91-ISREup
(5'-GTACCTGCAGCAAATGAAACTTTCTGCGAAAAGAAGAAAACGA-3') and its complement
p91-ISREdown
(5'-GATCTCGTTTTCTTCTTTTCGCAGAAACTTTCATTTGCTGCAG-3'); p91-ISRE
gas1
(5'-GTACCTGCAGCAAATGAAACTTTGCTAGCGAAAAGAAGAAAACGA-3') and
its complement p91-ISREgas2; IGI mutant 1, p91-ISREmut1
(5'-GTACCTGCAGCAAATCCAACTTTCTGCTAAAAGAATCAAACGA-3') and its complement p91-ISREmut2; IGI mutant 2, p91-ISREmut3
(5'-GTACCTGCAGCAAATGAAACTTTCTGCTAAAAGAATCAAACGA-3') and its complement p91-ISREmut4; ISRE-isg15/sense
(5'-GATCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3') and its complement (a gift
from Dr. Ricky Johnstone); GAS-gbp (5'-AAGTACTTTCAGTTTCATATTACTCTAAATC-3') and its complement; and hSTAT1900prom3'GAS (TGATTCCATGAACAT),
hSTAT1900promIRF-E/GAS/IRF-E (CAG CAA ATG AAA CTT TCT GCG
AAA AGA AGA AAA CGT), hSTAT1900promIGIGAS (GAA ACT TTC
TGC GAA AAG), hSTAT1mut5'IRF-E (CAG CGG ATG GGA CTT TCT GCG AAA AGA AGA AAA CGT),
hSTAT1mut3'IRF-E (CAG CAA ATG AAA CTT TCT GCG AAC
CGA AGC CAA CGT), and IRF-1GAS (CGT CAT TTC CCC GAA ATC
AG) and their complements. Cells (~1 × 107
cells) were induced with either IFN- or IFN-2 as described below
or in the figure legends. During activation, 10 mM NaF was added with the IFNs before lysis of cells to prevent translocation of
ISGF3 to the nucleus (22). Cells were washed with 1×
phosphate-buffered saline and resuspended in 70 µl of HEM buffer (10 mM Hepes (pH 7.9), 1 mM EDTA, 10% glycerol,
0.135 mM MgCl2, 100 mM NaF, 10 mM Na4P2O7, 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml pepstatin, 0.1 mg/ml leupeptin, and 0.1 mg/ml aprotinin). Following lysis on ice for
15 min, cell suspensions were spun at >12,000 × g for 15 min at 4 °C. Supernatants were used as total lysates in gel mobility shift assays using end-labeled oligonucleotide probes. SK-MEL-28 and U293T cell lysates (10-20 µg of protein) were added to
a reaction mixture containing 20 µg of bovine serum albumin, 4 µg
of poly(dI-dC), 5 µl of buffer A (20 mM Hepes (pH 7.9),
100 mM KCl, 0.5 mM EDTA, 0.25% Nonidet P-40, 2 mM dithiothreitol, 20% glycerol, and 1 mM
phenylmethylsulfonyl fluoride), 10 µl of buffer B (20% Ficoll 400, 100 mM Hepes (pH 7.9), 300 mM KCl, 10 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride), and 4 ng of -32P-labeled
oligonucleotide probe in a final volume of 50 µl. The mixture was
incubated for 25 min at room temperature. Recombinant STAT1 was used in
gel shift assays at a concentration of 500 ng. Murine wild-type
IRF-1 protein was synthesized in vitro using a T7-based
coupled transcription/translation kit (Promega, Madison, WI) following
the manufacturer's instructions. For MCF-7 cells, a reaction mixture
consisting of 180 µl of dialysis buffer (20 mM Hepes (pH
7.9), 50 mM KCl, 0.3 mM EDTA, and 20%
glycerol), 45 µl of reaction buffer (10 mM
Na2HPO4 and 1 mM EDTA), 24 µl of
80 mM MgCl2, and 8.8 µl of poly(dI-dC) (3 µg/µl stock) was prepared. For each gel shift reaction, 9 µl of
reaction mixture, ~5 µg of total protein (in 5 µl), and 1 µl of
[-32P]ATP-labeled oligonucleotide probe (~30,000
cpm) were incubated on ice for 20 min and then loaded onto a 6%
nondenaturing polyacrylamide gel. For supershift experiments, either
antiserum was added to the reaction mixture after 10 min and the
incubation was continued for a further 10 min, or extracts were
incubated with 1 µg of each antibody for 20 min before adding the
radioactively labeled probe to the reaction, and samples were
electrophoresed on a 20-cm 6% polyacrylamide gel at 15 mA in 0.25×
Tris borate/EDTA at 4 °C for 10 h.
Luciferase Constructs and Site-directed Mutagenesis--
The
pGL3-Basic luciferase reporter vector (Promega) encoding a
modified firefly luciferase was used to prepare STAT1
intronic enhancer reporter constructs. The primer Rvprimer3
(5'-CTAGCAAAATAGGCTGTCCC-3') was used for DNA sequencing across the
regions cloned into pGL3basic to confirm that the sequences of the
constructs were correct. The constructs prepared included the
following. 1) The full-length (FL) construct contained the full-length
EcoRI fragment of the STAT1900prom region
obtained from pGEMT-Easy. 2) The 3'-luc (where luc is luciferase)
vector contained the SacI/XhoI 3'-half of the STAT1 intronic enhancer region isolated from pGEMT-Easy
(from bp +508 to +1223 relative to the STAT1 transcriptional
start site). 3) The 5'-luc vector in which the 5'-half of the
STAT1 intronic enhancer (from bp +3 to +498) was derived
from the full-length clone by PCR with the p91-ISREup and
STAT1upstream primers, and the product was cloned into
pGEMT-Easy, excised with PstI/NotI, ligated into
pCR-Script SK+ (Stratagene), re-excised with
KpnI/SacI, and cloned into pGL3Luc. 4) The
5'IGI-luc construct was made by inserting a PCR-derived fragment of
the 5'-luc construct between the KpnI and SacI
sites of pGL3basic. The PCR primers used were as follows: forward
primer, GAC GGT ACC GCA CGA CTG GCA AGG ACA ATC; and reverse primer,
GCA CTC GAG GAA TTC GAT TCT TTC CGG CGC. 5) For IGI-luc, p91-ISREup and
its complement p91-ISREdown were annealed at 50 °C and cooled down
slowly to room temperature to allow double-stranded annealing before
cloning into KpnI/BglII-cut pGL3Luc. The sequence
of the IGI-luc construct was confirmed using the forward primer.
Mutation of the GAAANN sites within the IGI element of the pGL3-FL
construct was carried out using the QuikChange site-directed mutagenesis kit (Stratagene) as described by manufacturer. Briefly, the
pGL3-STAT1prom FL construct was subjected to PCR with
primers hSTATmut/sense
(5'-CCATCAGTCACGTTTGATTCTTTTAGCAGAAAGTTGGATTTGCTGTATGCC-3') and hSTATmut/anti
(5'-GGCATACAGCAAATCCAACTTTCTGCTAAAAGAATCAAACGTGACTGATGG-3') for site-directed mutagenesis. After amplification, parental DNA was removed by digestion with DpnI, and the vector DNA
containing the mutant IGI element was transformed into rubidium
chloride-treated, competent Escherichia coli DH5
cells. The DNA sequences of the mutant clones were verified using the
DYEnamic ET terminator cycle sequencing kit.
Cell Transfection and Reporter Assays--
Cells (2 × 105) growing in 35-mm dishes were transfected with 1 µg
of plasmid DNA/dish using purified pSV2-gal (Promega) and/or pGL3basic, 5'-luc, IGI-luc, 3'-luc, FL-luc, 5'IGI-luc, FL-IGImt-luc, or pEF-luc DNA. Either FuGENE 6 (Roche Molecular Biochemicals, Mannheim, Germany) or Transfast reagent (Promega) was used according to
the manufacturers' protocols. At 30-36 h after transfection, cells
were either left untreated or stimulated with 1000 IU/ml IFN- or
IFN-2 and incubated for 8-24 h before harvesting, and extracts were
prepared for luciferase and -galactosidase assays using standard
protocols (Promega). Light units were measured in a Top Count
microplate scintillation counter (Canberra Packard), recording four readings over 2 min and averaging these to measure light
units/min. Each transfection assay was performed in triplicate and
repeated two to three times; the values (light units/min) were
averaged; and S.E. values were calculated.
RNA Preparation and Northern Analysis--
Total RNA was
prepared using Trizol (Invitrogen) according to the manufacturer's
instructions. For Northern blotting, 20 µg of total RNA was separated
on 1.5% formaldehyde gels and transferred to Hybond Plus membranes
(Amersham Biosciences). The STAT1 probe for Northern
blotting was prepared by PCR using the mouse Stat1 cDNA as a template. The primers used to obtain the 177-bp fragment were as follows: forward primer, CCGATG GAG CTT GAC GAC CCT AAG CGA AC;
and reverse primer, CTG TGC TCA TCA TAC TGT CAA ATT CGG CC. The STAT1
probe was labeled with [-32P] dCTP and hybridized to
the membrane-bound RNA overnight at 65 °C in Church's buffer (0.158 M NaH2PO4, 0.342 M
Na2HPO4, 1 mM EDTA, 1% bovine
serum albumin, and 7% SDS). Following stringent washing, membranes
were exposed to film or PhosphorImager screens. Transfers were stripped
and reprobed with the GAPDH probe (pTRI-GAPDH, Ambion Inc.) to assay
for GAPDH expression.
Protein-DNA Cross-linking in Vitro and Western
Blotting--
Streptavidin-agarose beads
(NeutrAvidinTM-agarose, Pierce) were saturated with
5'-biotinylated IGI oligonucleotides at 4 °C for 2 h. The beads
were then washed with HEM buffer and incubated with extracts from
untreated or IFN--treated MCF-7 cells at 4 °C for 1 h. The
beads were washed five times with HEM buffer, resuspended in SDS-PAGE
loading buffer, and loaded onto 10% SDS-polyacrylamide gels. Extracts
from MCF-7 cells were quantitated, and 20 µg of each sample was
separated on 10% SDS-polyacrylamide gels. Proteins were transferred to
polyvinylidene difluoride membranes and analyzed by Western blotting
with either anti-CBP or anti-IRF-1 antibody as indicated.
Construction and Transfection of pEF-STAT1 and pEF-IRF-1
Expression Vectors--
The entire coding region of STAT1
was amplified by PCR using STAT1-pBluescript II
SK
(kindly provided by Dr. Chris Schindler) as a template
plasmid. The primers used were as follows: STAT1 5'-primer,
5'-AAGGAAAAAAGCGGCCGCCACCATGGCTCAGTGGTACGAACTTCAGCAGC-3'; and STAT1 3'-primer,
5'-CGCGGATCCCTATACTGTGTTCATCATACTGTCG-3'. The PCR fragment
was subsequently cloned into the passaging vector pCR-Script.
Subsequently, the STAT1 fragment was excised by digestion with NotI and ClaI and subcloned into the
pEFMCIneopAS6 expression vector (23).
The full-length IRF-1 expression vector was generated by PCR using the
IRF-1 5'-primer
(5'-AAGGAAAAAAGCGGCCGCCACCATGGCCCCATCACTCGGATGCGCATGAGACCC-3') and the IRF-1 3'-primer
(5'-GCTCTAGACTACGGTGCACAGGGAATGGCC-3') from the
IRF-1-pBluescript II SK plasmid as a template (kindly provided by Dr. Richard Pine). The PCR product was cloned into the pEFMCIneopAN9 expression vector (23) via NotI and
XbaI restriction enzyme sites introduced by the above
primers. The resulting constructs, pEF-STAT1 and pEF-IRF-1,
were purified by cesium chloride gradient centrifugation and used to
transfect MM96 cells via electroporation. Stable clones were selected
by culture in the presence of G418.
Antiviral Assays--
Antiviral assays were performed as
described previously (7, 8).
| |
RESULTS |
Mapping of the STAT1 Gene from the CEPH Human YAC
Library--
When this work commenced, the DNA sequence of human
chromosome 2 was incomplete, and the 5'-genomic sequence of the human STAT1 gene was unknown. The STAT4 and
STAT1 genes had been located between q32.2 to q32.3 on human
chromosome 2 (24, 25). The CEPH-Genethon
Database2 revealed
STAT4 located at marker D2S118 or STS AFM066xc1. YACs from
the CEPH human contig library (26) and overlapping or neighboring D2S118 and STS AFM066xc1 were selected and cultured, and DNA was obtained from each of clones 737c4, 755b5, 764f5, 805c1, 809e2, 820b12,
821e5, 855g4, 855h9, 894a5, 894a6, and 894a11. PCR was carried out on
YAC DNA samples using primers STAT1yac3'a and
STAT1yac3'b. DNA products predicted from the
STAT1 cDNA sequence (27) of 187 bp were obtained from
clone 805c1 and 809e2 YAC DNAs as templates (Fig.
1A). DNA from clones 805c1 and
809e2 was purified, digested with either PstI or
EcoRI (selected because these are infrequent sites present
every 4-6 kb), and analyzed by Southern blotting with an
-32P-labeled STAT1 cDNA fragment as a
probe. Positive bands were identified, confirming the presence of the
STAT1 gene (data not shown).
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Fig. 1.
A, YACs containing the STAT1
gene in regions of human chromosome 2 identified by PCR. DNA was
obtained from each of the YAC clones 737c4, 755b5, 764f5, 805c1, 809e2,
820b12, 821e5, 855g4, 855h9, 894a5, 894a6, and 894a11 using the
zymolyase 20T rapid lysis method. The STAT1yac3'a
and STAT1yac3'b primers (corresponding to nucleotides
2432-2449 and 2599-2618, respectively, of STAT1 cDNA
sequence (GenBankTM/EBI accession number M97935)) were used
for PCR, and the products were analyzed on a 1.5% agarose gel.
M, pUC19/HpaI markers. B, use of
bubble PCR to isolate the human STAT1 intron 2/exon 3 boundary. EcoRI- and PstI-digested 805c1 and
809e2 YAC DNAs were ligated to bubble anchors as indicated before PCR.
Products were analyzed on a 1% agarose gel. M,  DNA
EcoRI/HindIII markers. Products were designated
PromIcl2 and PromIIcl3 as indicated by the arrows.
C, isolation of the human STAT1 5'-genomic region
by bubble PCR. PstI- and EcoRI-digested 805c1 YAC
DNA was ligated to bubble anchors before PCR with the
STAT1upstream primer, and the products were analyzed on a
1% agarose gel. M, DNA
EcoRI/HindIII markers.
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Use of Bubble PCR to Clone the Human STAT1 5'-Genomic
Structure--
We used bubble PCR (21) of PstI- and
EcoRI-digested YAC DNA to obtain specific 5'-fragments of
the STAT1 gene, and GenBankTM/EBI accession
number M97935 provided the initial 5'-cDNA sequence information
used as the basis for primer design. Initially, we selected the
nucleotide STAT1prom (Fig. 2)
adjacent to the ATG initiation codon of STAT1 to use with either
BubbleprimerIntE or BubbleprimerIntP as the primer set. Specific PCR
products were obtained whether template 805c1 or 809e2 YAC DNA digested
with either EcoRI or PstI was used (Fig.
1B). The bubble PCR fragments cloned were called PromIcl2
(2.2 kb, obtained with EcoRI) and PromIIcl3 (1.5 kb,
obtained with PstI). The resulting DNA sequences determined
from both fragments were aligned and overlapped at the 3'-end such that
the larger fragment (PromIcl2) extended ~700 bases more 5' than
PromIIcl3 (Fig. 2). Analysis of the sequence adjacent to the
STAT1prom primer confirmed the intron/exon boundary previously identified (GenBankTM/EBI accession number
U18666) (Fig. 2B). Attempts to use primers designed to be
near the 5'-end of the published STAT1 cDNA nucleotide sequence (accession number M97935) (27) for bubble PCR were unsuccessful, and no specific products resulted from either the PstI- or EcoRI-digested 809e2 DNA used as a
template (data not shown). However, using a primer farther in from the
5'-end (STAT1upstream, nucleotides 26-45 of the
STAT1 cDNA sequence) together with the BubbleprimerIntE
primer for bubble PCR produced a specific PCR product of ~900 bp,
STAT1900prom (Fig. 1C). No specific PCR product could be detected when the PstI-digested 805c1 anchor was
used as the target template. DNA sequencing of STAT1900prom
revealed that it was 1223 bases long and that the 3'-end overlapped
with the 5'-end of the published cDNA sequence (accession number
M97935) (see Fig. 4), but contained marked differences to the M97935 cDNA sequence. Thus, the 12 nucleotides at the 5'-end of the
published cDNA sequence (27) are not present in the genomic
sequence. In addition, our sequencing also revealed that the next 29 nucleotides in the published cDNA sequence are inverted in the
reverse direction.
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Fig. 2.
A, partial map of the human
STAT1 5'-genomic region. The relationship of PromIcl2 and
PromIIcl3 to the human STAT1 cDNA
(GenBankTM/EBI accession number M97935) and genomic
sequences (accession number U18666) is shown. E,
EcoRI; P, PstI. Exons 1-3
(e1-3) are boxed. B, DNA sequence of
the human STAT1 intron 1/exon 2/intron 2 region indicating
putative enhancer elements. The sequence was compiled from that of
STAT1900prom and the cloned 5'-RLM-RACE human
STAT1 product. Regions containing exons are shown in
boldface uppercase letters; introns are in lowercase
letters. MatInspector Version 2.2 (see Footnote 2) analysis of the
sequence revealed the presence of TATA boxes, GC-rich regions, Sp1
sites, and IRF-E as well as possible GAS and retinoic acid response
element (RARE) elements as indicated.
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Characterization of the 5'-Transcriptional Start Site in the STAT1
Gene--
To resolve the problems identified above with the published
cDNA sequence as well as to determine the 5'-transcriptional start site of the human STAT1 cDNA, RLM-RACE was undertaken. A
product of 225 bp was obtained (data not shown), which was cloned and sequenced. The start site was identified as shown in Fig. 2, and the
region of 5'-cDNA sequence overlapping with the 5'-DNA sequence from STAT1900prom was found to be identical (Fig. 2).
Comparative analysis of the resulting cDNA and genomic sequences
indicated the presence of two exons in the STAT1900prom DNA
fragment (exons 1 and exon 2) (Fig. 2).
Human STAT1 Genomic Intron 1 Contains an IFN-regulated Enhancer
Element--
MatInspector Version
2.2 3 analysis of the
STAT1900prom sequence revealed the presence of IRF-E
elements, two GAS elements, and a retinoic acid response element
(RARE, highlighted in Fig. 2B). One
unusual feature of the sequence was the presence of one of the GAS
sites in between two IRF-Es that each contained GAAANN repeats
(boxed sequence in Fig. 2B). Previous studies
have identified the presence of intronic enhancers within several
IFN-regulated genes (28, 29). Given these observations, vectors were
constructed using different regions of the STAT1900prom
fragment subcloned into the basal luciferase reporter plasmid,
pGL3basic (Fig. 3A). The
constructs were transiently transfected into cells that were then
induced with either IFN- or IFN- and analyzed for the levels of
induced luciferase activity (Fig. 3, B-D). Constructs
containing either the whole region or either halves of the human
STAT1900prom 5'-genomic region were examined in three
different human cell lines, MCF-7, U293T, and SK-MEL-28. The results
were very consistent (Fig. 3, B-D). In two of the cell
lines, the 3'-portion (3'-luc) produced a dramatic reduction in the
level of constitutive luciferase activity, ~10-fold lower than the
control levels resulting from the basal pGL3basic vector alone, and
showed no effect of IFN treatment (Fig. 3, B and
C). This result indicates the presence of a repressor
element in the 5'-end of the STAT1 genomic intron 2 region.
By contrast, in the absence of added IFNs, the 5'-portion of
STAT1900prom (5'-luc) showed constitutive luciferase
activity, ~5-10-fold above the pGL3basic background levels,
and a further 2-3-fold increase in activity when induced by either
IFN- or IFN- (Fig. 3, B-D). However, the full-length
STAT1900prom construct, in the absence of IFNs, produced a
similar low level of activity compared with the basal pGL3basic vector,
but was induced 5-10-fold after treating cells with either IFN- or
IFN- for 16 h. The kinetics of induction were similar when type
I or II IFN was used to induce the full-length or 3'-luc constructs
such that the luciferase activity peaked between 16 and 24 h after
IFN induction.
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Fig. 3.
Functional analysis of constitutive and
IFN-induced human STAT1 intronic enhancer
activity. A, schematic diagram depicting the
STAT1 promoter fragments cloned into the pGL3basic
luciferase vector. The human STAT1 intron 1 region contains
IFN-inducible enhancer activity. The human STAT1 5'-genomic
constructs analyzed included the full-length construct (exon 1 (e1)/intron 1/exon 2 (e2)/intron 2 5'-region),
the 5'-end and 3'-end regions, the IGI element alone, the 5'-end with
the IGI element deleted, and the full-length construct containing a
mutant IGI element. Each DNA fragment was isolated by PCR, cloned into
pGL3basic, and confirmed by DNA sequencing. B-C, mean total
values (±S.E.) of luciferase activity (light units/min) normalized
from sets of results from two to three repeated experiments. The cells
indicated were transfected with reporter constructs as indicated and
were treated or not with IFNs. Analyses were carried out with U293T
(B) or SK-MEL-28 cells (C) transfected under
standardized conditions using 1 µg of purified plasmid DNA/reaction.
In D, MCF-7 cells were transfected with the pSV2-gal
construct and the empty vector (pGL3basic), pEF-luc (positive control),
or the 5'-luc, 3'-luc, or IGI-luc construct. Transfected cells were
split 36 h post-transfection and either left untreated or treated
with IFN- for 8 h. Extracts from untreated and IFN--treated
and transfected cells were then analyzed for -galactosidase and
luciferase activities according to standard protocols. Luciferase
activity units in D were normalized against
-galactosidase activity for each sample. RARE, retinoic
acid response element.
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Site-directed mutagenesis of the full-length STAT1900prom
construct was used to specifically modify the GAAANN repeat sites contained within the IGI element. When the full-length reporter gene
construct with altered GAAANN sites was assayed, the results (Fig.
3B) revealed a complete loss of the responsiveness of the region to IFN- and IFN-.
The IGI Element within the Human STAT1 Intronic Enhancer Binds a
Novel IFN-inducible Complex That Contains the IRF-1
Protein--
The above results indicated the presence of an
IFN-responsive element contained within the
STAT1900prom region, and the IGI element was
implicated as the most likely site of IFN regulation. Therefore, EMSA
was carried out using double-stranded oligonucleotide sequences
encompassing the IGI element as probes. Extracts prepared from the
three different human cell lines (MCF-7, U293T, and SK-MEL-28) were
used in EMSA reactions, the latter cell line having been extensively
characterized in previous studies as highly sensitive to both type I
and II IFNs (7, 8). In control EMSA reactions (data not shown), the use
of the ISG15 ISRE and guanylate-binding protein GAS
oligonucleotides revealed potent induction of ISGF3 and GAF activities
in extracts from SK-MEL-28 cells treated with IFN- and IFN-,
respectively. In addition, ISGF3 was successfully supershifted by
addition of anti-IRF-9 antibody, and anti-IRF-1 antibody supershifted
the IRF-1 bands obtained with extracts from cells treated with either
IFN- or IFN- (data not shown), confirming the specificity of
these antibodies for supershift experiments.
With samples from all three cell lines, when EMSA was carried out using
the -32P-radiolabeled IGI DNA element as the probe (Fig.
4), a single prominent IFN-induced band
was observed, particularly after incubation with extracts from IFN--
or IFN--treated cells. Low levels of the IGI complex were detected
in some preparations of untreated cells (Fig. 4). However, the amount
of IGI complex detected was greatly increased after IFN treatment and
was especially noticeable in EMSA reactions of cell samples that had
been previously primed with IFN- for 16 h before IFN-
activation for 30 min (data not shown). The levels of the novel
IGI-binding complex obtained from IFN--treated MCF-7 cells were
higher in the 30-min than in the 60-min treated samples (Fig.
4A). The IGI complex exhibited slower mobility in EMSA than
the STAT1 homodimeric complex bound to the GAS element from the human
IRF-1 gene (Fig. 4A). A smaller oligonucleotide probe
containing the GAS element within the IGI sequence motif of
STAT1900prom (GAA ACT TTC TGC GAA AAG) was unable
to bind the IFN--induced STAT1 homodimer (Fig. 4A). In
addition, recombinant STAT1 protein was assessed for its ability to
bind to this IGI GAS element. Binding of the recombinant STAT1 dimer to
the IGI GAS sequence was detected (Fig.
5A), albeit at low levels
compared with levels of recombinant STAT1 protein binding to the GAS
site from the human IRF-1 gene promoter (cf. Fig.
4A).
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Fig. 4.
A, the IGI motif of the human
STAT1 intronic enhancer binds a novel IFN-inducible complex.
MCF-7 cells growing in Dulbecco's modified Eagle's medium + 10%
fetal calf serum were treated with IFN- for 30 or 60 min before
preparing whole cell extracts for EMSA. Oligonucleotide probes
corresponding to the 3'-GAS site from the STAT1900prom
sequence (TGATTCCATGAACAT and its complement), the IGI motif from the
human STAT1 (hSTAT1) intron 1/exon 2 boundary
(CAG CAA ATG AAA CTT TCT GCG AAA AGA AGA AAA CGT and its complement),
and the GAS element from the human IRF-1 (hIRF-1) gene
promoter were analyzed. Binding of recombinant STAT1
(rSTAT1) was also examined by adding 500 ng to the EMSA
reaction. DNA-bound complexes were resolved on 6% polyacrylamide gels
and detected by autoradiography. B-D, the novel
IGI-binding complex contains IRF-1. Cell extracts from MCF-7
cells (B) as described for A or from
SK-MEL-28 (C) or U293T cells (D) treated with
IFN- or IFN-2a for 30 min were used in EMSA reactions. Whereas
the shorter IGI element of STAT1900prom was used in
A and B, the slightly longer IGI element of
STAT1900prom (p91-ISREup/down annealed together) was used in
C and D. Supershift analysis of the novel
IGI-binding complex was carried out by adding 1 µg of the indicated
antibodies (Ab) to the EMSA reaction before addition of the
radiolabeled IGI probe (p91-ISREup/down oligonucleotides).
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Fig. 5.
A, the GAS site within the IGI element
exhibits low level binding to the STAT1 dimer. MCF-7 cells untreated or
treated with IFN- for either 30 or 60 min were used to prepare
extracts that were combined with radiolabeled probe in the EMSA
reaction. The probe used (GAA ACT TTC TGC GAA AAG and its
complement) contained the GAS site in a truncated IGI element. Binding
of recombinant STAT1 (rSTAT1) was also examined by adding
500 ng of protein o the EMSA reaction. B, mutations in the
IRF-E sequences in the IGI element reduce gel shift complex formation.
Both the 5'- and 3'-IRF-Es within the IGI element are important for
formation of the novel IGI-binding complex. Extracts for EMSA were
prepared from MCF-7 cells that were treated with IFN- for 30 min.
Oligonucleotides used as probes in EMSA reactions included the
wild-type IGI element (hSTAT1900promIRF-E/GAS/IRF-E)
and the IGI element containing mutations within the 5'-IRF-E
(hSTAT1mut5'IRF-E) and the 3'-IRF-E
(hSTAT1mut3'IRF-E). C, mutations in the
IGI GAS sequence do not affect complex formation, unlike mutations in
the IRF-E sequences, which can eliminate it. SK-MEL-28 cells were
treated with IFN- or IFN- for 30 min, and extracts were added to
EMSA reactions with radiolabeled probes as follows: the wild-type IGI
sequence; the IGI sequence with a mutant GAS element (TTCTGCGAA  TGCTAGCGAA, using the p91-ISREgas1/2 oligonucleotides); or the IGI
sequence with mutant GAAANN regions, IGI mutant 1 (p91-ISREmut1/2) with
all three GAAANN sequences mutated and IGI mutant 2 (p91-ISREmut3/4)
with the two 3'-GAAANN sequences mutated. Anti-IRF-1 antibody
(Ab) supershifted the IGI complex.
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A significant fraction of the IGI complex was supershifted upon
addition of anti-IRF-1 antibody (Fig. 4, B-D). However,
addition of antibodies against STAT1, STAT2, STAT3, IRF-2, or IRF-9
(Fig. 4, B-D) or IRF-3 (data not shown) did not
significantly affect the resulting levels of IGI complex formation.
These results indicate that the IGI complex contains IRF-1 protein, but
does not contain significant levels of any of the other IFN-activated
transcription factors.
Mutations in the Human STAT1 Intronic Enhancer IGI Element Affect
Its Ability to Form Protein Complexes--
To determine which of the
sites in the IGI element are important for binding to protein
complexes, EMSA was carried out with -32P-radiolabeled
oligonucleotides containing different mutations in the DNA sequences of
the IGI enhancer elements. When mutations were made individually to
either the 5'- or 3'-IRF-E GAAANN repeat elements in the IGI sequence,
they resulted in a reduced level of IGI complex formation (Fig.
5B). Mutation of the IGI GAS domain (TTCTGCGAA) to
TTGCTAGCGAA (p91-ISREgas1) (Fig. 5C) did not
affect the formation of the IFN-activated IRF-1-containing IGI complex.
However, mutations to the GAAAN repeats that included a mutation of the
GAAANN site overlapping the IGI GAS 3'-end (TTCTGCGAAAGT to
TTCTGCTAAGT) resulted in significant reductions in IGI complex formation. Thus, IGI mutant 1 (p91-ISREmut1 and its complement p91-ISREmut2) contained mutations in three GAAAN repeats, whereas IGI
mutant 2 (p91-ISREmut3 and its complement p91-ISREmut4) contained mutations in only the two 3'-GAAAN repeat sequences. The 5'-GAAAN sequence was mutated to CCAAN, the middle sequence to
TAAAN, and the 3'-sequence to TCAAN. With either
the IGI mutant 1 or 2 oligonucleotides, the EMSA binding of the
IRF-1-containing IGI complex was no longer detected (Fig.
5C), indicating that the IGI 3'-GAAAN sequences are critical
for IRF-1 binding. No gel shift bands with comparable mobility to ISGF3
or STAT1-3 dimeric complexes were detected in any of the assays with
any of the IGI probes examined.
Induction of IRF-1 Expression Alone Does Not Result in DNA Binding
of the IGI Complex or Transcription of the Human STAT1 Gene--
IRF-1
gene expression is regulated by activation of STAT1 (30), and the
TNF- and IFN- signaling pathways converge to synergistically increase expression of IRF-1 via a composite GAS/NF
B promoter element (31, 32). Thus, we compared the IFNs and TNF- for their
ability to induce increased levels of IRF-1 expression and whether the
increased expression was associated with the increased formation of the
IGI DNA-binding complex by these cytokines. MCF-7 cells were stimulated
with IFN-, IFN-, or TNF- either alone or in different
combinations for 8 h to compare their effects on IRF-1 and STAT1
expression as well as activation of IGI complex binding. IFN- and
TNF- both increased expression of the IRF-1 protein (Fig.
6). However, only IFN-- or
IFN--treated MCF-7 cells showed increased expression of
STAT1 mRNA after 8 h, particularly in the
IFN--treated samples. In addition, activation of the IGI-binding complex was only detected in MCF-7 cells treated with IFN-. Thus, from these cellular studies, activation of the IGI DNA-binding complex
was associated with increases in STAT1 mRNA expression, supporting a role for the IGI complex in activating cellular
STAT1 gene expression. However, the presence of high IRF-1
protein levels by itself was not sufficient to induce STAT1
gene expression in these cells, but required activation by IFN-, as
TNF- did not activate the formation of the IGI DNA-binding
complex.
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Fig. 6.
Overexpression of IRF-1 alone does not induce
DNA binding of the novel complex or transcription of the human
STAT1 gene. Samples were prepared from MCF-7
cells untreated or treated for 8 h with IFN-, IFN-, TNF-,
or a combination of either IFN- and TNF- or IFN- and TNF-
as indicated. IRF-1 expression was analyzed by Western blotting,
whereas GAPDH and STAT1 gene transcription levels were
detected by Northern blotting. In the EMSA, cell samples were treated
with the cytokines indicated for 30 min.
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The Novel IRF-1-containing Complex Bound to the IGI Element Has a
Reduced Mobility Compared with IRF-1--
IRF-1 has previously been
shown to form a range of different associations, e.g.
forming complexes with itself and other members of the IRF family
(33-35), STAT1 (36), and CBP (34, 35, 37). Given this ability to
associate with other proteins and the size of the IGI complex, a study
was carried out to further define its components. The mobility in gel
shifts of the IGI complex was compared with that produced by
preparations of in vitro translated murine IRF-1 protein
added to the IGI DNA element as a probe (Fig. 7A). A single dominant band
was visible in gel shifts with the in vitro translated IRF-1
protein (indicated as IRF-1 in Fig. 7A), and a
band of mobility equivalent to that produced by the in vitro
translated recombinant IRF-1 protein was also obtained using the
IFN--treated MCF-7 cell samples. Another band of much slower
mobility (indicated as IGI complex in Fig. 7A)
was also detected in the IFN--treated MCF-7 cell samples.
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Fig. 7.
A, the novel IRF-1-containing complex
bound to the IGI enhancer exhibits reduced mobility compared with the
complex formed by addition of recombinant IRF-1 alone. EMSA was
performed using the IGI probe with either extracts from MCF-7 cells
treated with IFN- for 30 min or with in vitro translated
(IVT) murine IRF-1 protein. Complexes were resolved on 6%
polyacrylamide gels and detected by autoradiography. *, nonspecific
DNA-binding protein complex. B, CBP binds to the IGI element
in an IFN- inducible manner. Neutravidin-agarose saturated with
biotinylated IGI oligonucleotides were used to pull-down DNA-bound
proteins from extracts of MCF-7 cells untreated or treated with IFN-
for 30 or 60 min. Beads were then washed extensively to remove
nonspecifically bound proteins before resuspension in SDS-PAGE loading
buffer. IGI-binding proteins were resolved by 10% SDS-PAGE and
identified by Western blotting using specific antibodies as
indicated. I.P., immunoprecipitated.
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Agarose beads coupled to the IGI DNA element were used to pull-down
specific IGI-binding complexes from extracts of MCF-7 cells treated
with IFN-. The proteins in the complex were resolved by SDS-PAGE and
identified by Western blotting (Fig. 7B). IRF-1 was detected
in the IGI-binding complex. The complex also contained CBP, but not
STAT1. CBP was detected in the IGI-binding complex only after IFN- treatment.
IFN-resistant Human Melanoma Cell Line MM96 Transfected to
Constitutively Express IRF-1 Exhibits Increased Expression of STAT1 and
Greater Sensitivity in Biological Assays to IFN---
We had
previously shown that the human melanoma cell line MM96 is resistant to
the effects of IFN- in biological assays (7, 8) as a result of a
deficiency in STAT1 protein expression and reduced ISGF3 activity. In
addition, cellular resistance to IFN is partly restored by transfecting
MM96 cells to constitutively express higher levels of STAT1 protein
(7). Analysis of the wild-type MM96 cell line and the STAT1-expressing
MM96 subline (7) in the present study by Western blotting revealed that they contained very low levels of IRF-1 protein (Fig
8A). Therefore, the wild-type
MM96 cell line was transfected to constitutively express greater levels
of IRF-1 protein. Cell extracts from transfected clones were examined
for IRF-1 protein expression levels, and a highly expressing clone
(c55) was analyzed for cellular levels of STAT1, STAT2, and p48 protein
(Fig. 8A). From the results, it can be seen that the MM96
pEF-IRF-1c55 line expressed significantly increased levels of IRF-1
protein, and this was associated with an increased expression of STAT1
to levels similar to those observed in the STAT1-transfected MM96
pEF-STAT1c7 line. In addition, the levels of STAT2 and
p48/IRF-9 protein expression were increased to a similar extent in both
MM96 sublines transfected to express STAT1 or IRF-1 protein. The
response of the MM96 pEF-IRF-1 line to IFN- was examined by
antiviral assay (Fig. 8B) and compared with that of the
wild-type cell line. The MM96 pEF-IRF-1 line exhibited increased
sensitivity to IFN- (CPE50, 50% inhibition of
cytopathic effect of 5 IU/ml compared with 280 IU/ml for the wild-type
cell line). Together, the results from Fig. 8 are consistent with a
role for IRF-1 acting in vivo as a key regulator of the human STAT1 gene.
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Fig. 8.
A, increased human STAT1 expression in
MM96 human melanoma cells transfected to express high levels of IRF-1.
The wild-type MM96 human melanoma cell line (7, 8) expressing low
levels of STAT1 and IRF-1 was transfected with the pEF-IRF-1 vector,
and clones were selected for growth in G418. The highly
IRF-1-expressing clone (c55) was further analyzed by immunoblotting and
compared with wild-type and pEF-hSTAT1-transfected MM96 cell
lines. Equal amounts of total cellular protein from lysates of 1 × 105 cells prepared in SDS-PAGE sample buffer were
examined for the presence of ISGF3 components. The blots were stripped
and reprobed with anti-GAPDH antibodies to confirm that sample loadings
in each lane were equivalent. B, antiviral assay of IFN-
in MM96 and MM96 pEF-IRF-1c55 cell lines. Shown are the dose-response
curves for protection against Semliki Forest virus challenge afforded
by treating wild-type ( ) compared with pEF-IRF-1-transfected MM96
cells ( ) as indicated over a range of IFN- concentrations.
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DISCUSSION |
The STAT1 gene was mapped from the CEPH human YAC
library, and clones containing the 5'-genomic region were isolated by a PCR-based screening approach. Two positive YAC clones (809e2 and 805c1)
containing the STAT1 gene were identified (Fig. 1). Using bubble PCR, the 5'-genomic region of the STAT1 gene has been
characterized and extends previous observations of a closely related
organization in the human STAT1 and STAT2
5'-genomic regions (38). Thus, both genes have an intron adjoining near
the exon containing the ATG initiation codon and an additional 5'-exon
containing 50 bases (for STAT2). However, the
STAT1 gene is distinct in that it contains two additional
5'-exons upstream of the exon containing the start codon (Fig.
2A). We mapped and sequenced the 5'-region including exon
1/intron 1/exon 2/intron 2/exon 3. The second intron of the human
STAT1 gene is ~2.8 kb in size. In the STAT2
gene, the first intron is ~3 kb in size. Thus, STAT1 and
STAT2 genes show close similarities in genomic organization
up to this point. However, the human STAT1 gene contains an
additional 5'-noncoding exon.
We isolated a 5'-genomic region of 1223 bp from the STAT1
gene, STAT1900prom (Figs. 3 and 4), amplified using
EcoRI-digested 805c1 YAC DNA as a template for bubble PCR
with the STAT1upstream primer. The sequence obtained from
this DNA fragment overlapped with the reported 5'-end of the
STAT1 cDNA (GenBankTM/EBI accession number
M97935). The discrepancy between our sequence and 12 nucleotides at the
5'-end of the published cDNA sequence is most probably the result
of artifacts generated while preparing the cDNA library reported
previously (27). This would also explain why the STAT1upRev
primer, mismatched at the four 3'-bases, failed to produce a bubble PCR
product, whereas use of the STAT1upstream primer gave rise
to the correct product. Further support that accession number M97935
has an incorrect 5'-end comes from the finding that the next 29 nucleotides in this cDNA sequence (27) are inverted in the reverse
direction compared with our DNA sequence (Fig. 2). Analysis of the
transcriptional start site using RLM-RACE identified the 5'-end
indicated as +1 in Fig. 2B. However, it is unlikely that the
human STAT1 gene has a unique start site. An examination of
the 5'-dbEST Database of Expressed Sequence Tags in the
GenBankTM/EBI Data Bank revealed the presence of 21 human
STAT1 5'-dbESTs, and with the inclusion of our 5'-end, 18 of
the 22 start at different sites, mostly within +20 bp of the site we
have identified. Thus, it is highly probable that the human
STAT1 gene uses multiple transcriptional start sites.
Analysis of the human STAT1 intron 1 sequence revealed
putative regulatory elements, and this was confirmed when transient transfection assays and EMSA studies were undertaken. All three cell
lines (MCF-7, U293T, and SK-MEL-28) transfected with pGL3basic constructs containing the 5'-half of STAT1900prom including
intron 1 (5'-luc) displayed IFN-induced increases in expression of the luciferase reporter gene (Fig. 3), indicating the presence of an
IFN-regulated enhancer. Intriguingly, this region also resulted in
significant constitutive luciferase activity in the absence of IFN
treatment, ~10-fold above the basal level produced in cells transfected with the pGL3basic control vector alone. The luciferase activity resulting from the 5'-luc construct could be further elevated
2-3-fold by treating transfected cells with either type of IFN. It is
likely that this constitutive gene transcription results from Sp1
binding to several of the numerous GC box elements located in the
5'-end, as Sp1 sites are commonly found in the regulatory regions of ISGs.
By contrast, pGL3 containing the 3'-half of STAT1900prom
(3'-luc) resulted in a 10-fold reduction in the levels of luciferase activity, below those produced by the pGL3basic vector in two of the
three cell lines (U293T and SK-MEL-28). These data indicate the
presence of a strong repressor or negative regulatory sequence within
this 3'-half of the STAT1900prom region active in some cell
types. Further investigation is required to define the nature of this
negative regulatory mechanism and to explain why no suppression was
detected in the MCF-7 cell line with 3'-luc. Placing the two halves of
the STAT1 intronic enhancer region together in the
full-length construct restored control to the 5'-half because the
full-length construct exhibited tight regulation with similar levels of
luciferase activity compared with the pGL3basic vector in the absence
of IFNs as well as a 5-10-fold induction after treatment with IFN- or IFN- (Fig. 3). These results suggest that the 3'-repressor site
is important for maintaining tight IFN-inducible regulation from the
5'-half of the STAT1 intronic enhancer region. The poor IFN
response obtained with either the IGI-luc construct (containing the IGI
element alone) or the 5'IGI-luc construct (in which only the IGI
element was deleted) suggests that the presence of additional sequences
in intron 1 act together with the IGI element and that both are
required before IFN-activated transcription can occur. The identity of
these other sites remains to be determined.
Nevertheless, the IGI DNA element containing IRF-E GAAANN repeats
separated by an intervening GAS site in intron 1 was found in gel
mobility shift assays to bind a novel complex induced by IFN- and
IFN- in all three cell lines examined. In addition, this IGI-binding
complex migrated relative to the adjacent reference lane containing the
ISG15 ISRE with a slower mobility than that of the
prototypic IRF-1 complex (39), but not as slow as the ISGF3-bound
complex (data not shown). The IGI-binding complex was also slower than
the STAT1 homodimeric complex bound to the human IRF-1 GAS element
(Fig. 4A). The supershift of the IGI complex observed with
anti-human IRF-1 antibodies, but not with any of the other anti-IRF and
anti-STAT antibodies tested (Fig. 5, B and C),
indicates that the IGI complex is unusual in that it contains IRF-1,
but does not appear to contain any of the other common IFN-activated
transcription factors. No ISGF3 or other complexes of molecules
containing p48 or STAT1, STAT2, or STAT3 were visible or could be
detected by EMSA or supershift experiments. Although the GAS site
within the IGI element was capable of low level binding to the STAT1
dimer in in vitro EMSA reactions, this complex was not
detected using extracts from IFN--treated cells (Fig.
5A), and no STAT1 homodimer binding to the 3'-GAS site was
detected (Fig. 4A). The inability to detect STAT1 or ISGF3
binding to the ISRE/GAS-like STAT1 sequence was surprising
given that two GAS half-sites exist in the sequence
TTCTGCGAA (15). In addition, mutations introduced
to eliminate the IGI GAS site did not affect the DNA binding of the IGI
complex induced by IFN- and IFN- (Fig. 5C).
The consensus sequence for IRF-1 binding to DNA has been characterized
as a repeat of the GAAANN hexameric oligonucleotide, common to the
promoters of the type I IFN genes, the IFN- gene, and other ISGs,
usually present as a direct repeat (reviewed in Ref. 41).
Crystallography studies revealed that IRF-1 binds as a protein dimer to
the GAAA repeat (41-43), and IRF-1 has been shown to form dimers
in vivo (44). In support of this proposal, purified IRF-1 at
high concentrations was shown by EMSA to form a novel complex called
ISGF2 that bound to the ISRE and that migrated with a slower mobility
than the prototypic IRF-1 band (39). The ISGF2 band migrates with
similar mobility compared with the larger IRF-1-containing complex
(IGI) identified here. Whether these two bands are related remains to
be clarified.
With few exceptions, changes to the GAAANN (NNTTTC) repeat motif have
been shown to result in a loss of IRF-1 binding and IFN-induced
activation (45-53). Our results were consistent with these
observations. In the context of the full-length
STAT1900prom region of DNA, mutation of all three
GAAANN repeats within the IGI element resulted in the complete loss of
IFN responsiveness (Fig. 3B). All the GAAANN site mutations
assayed by EMSA for their effect on IGI complex formation resulted in
reduction or loss of IGI binding. It was of interest that mutation of
either the 5'- or 3'-IRF-E resulted in only a partial reduction in IGI
binding, whereas mutations of both IRF-Es caused complete loss of
binding (Fig. 5, B and C). Thus, the GAAANN
repeat sequences are essential for IGI DNA binding of IRF-1 protein
complexes. Many studies have demonstrated the existence of functional
"imperfect" ISREs, and some also adjoin GAS elements in a
arrangement similar to that of the IGI element. In relation to the
present study, one of the most interesting is the Sp100 gene
promoter, which is also induced by both IFN types and contains two GAS
sites and an unusual ISRE (GGAAAAGAGAAGAGAAAGT) that binds
preferentially to IRF-1 and only weakly to ISGF3 (54). Thus, the
Sp100 promoter resembles the human STAT1 intronic
enhancer IGI element with its related sequence, GAAAAGAAGAAAAC, which
we have shown binds IRF-1 preferentially. These two promoters must be
distinguished from the more common ISGs such as the guanylate-binding
protein gene, in which an ISRE and an overlapping GAS element
are both necessary for cooperative transcriptional induction by type I
IFN (55).
The observed increase in IRF-1 protein levels in U293T and SK-MEL-28
cells (but not MCF-7 cells) (cf. Figs. 4 and 5 with Fig. 6)
was interesting given that IFN- treatment induced increased STAT1 mRNA expression in all three cell lines. It
follows that an IGI-independent signaling pathway exists in MCF-7 cells
by which IFN- can up-regulate STAT1 expression. Although TNF-
treatment of MCF-7 cells raised their IRF-1 protein levels, no
associated increase in STAT1 mRNA expression or IGI
complex formation occurred. Thus, the TNF- signaling pathway is not
sufficient to activate the IGI complex even though it does increase
IRF-1 expression levels. By contrast, IFN- was the strongest inducer
of IRF-1 protein expression as well as in promoting the formation of
the IGI complex in MCF-7 cells. IFN- treatment of these cells was also associated with much greater increases in STAT1
transcription levels. It can be concluded from these results and from
EMSA studies with the IGI element that IRF-1-containing IGI complex
formation and activation play a key role in the increased human
STAT1 mRNA expression in cells treated with IFN-.
The histone acetylase proteins CBP/p300 have recently been shown to be
important in binding to IRFs and in recruiting them to chromatin to
activate transcription (34, 35). Our analysis using IGI-conjugated
beads in pull-down experiments with cell extracts revealed that the IGI
complex also contained CBP after treating cells with IFN- (Fig.
7B). This observation is consistent with the proposal that
IFN- signaling activates IRF-1 to bind the IGI element within the
STAT1 intronic enhancer in complex with CBP, thereby turning
on RNA transcription from the STAT1 gene. Additional
evidence that IRF-1 regulates the STAT1 gene in
vivo was obtained with the transfected MM96 melanoma cell line expressing IRF-1 protein, as these cells showed elevated expression of
STAT1 and other ISGF3 proteins as well as an accompanying enhanced responsiveness to IFN-. Thus, our evidence in toto
supports a key role for IRF-1 in regulating human STAT1
expression (40). Given that STAT1 regulates the IRF-1 gene promoter
(30), we propose that these two genes and their encoded proteins form
an intracellular amplifier circuit or feedback loop that acts to regulate cellular responsiveness to the IFNs. The elucidation of
reciprocating regulation involving the IRF-1 and STAT1 genes and encoded proteins, two pivotal transcription factors in IFN signaling, provides novel insight into the mechanisms regulating cellular responsiveness to the IFNs. This understanding will also help
in further identifying the basis of the changes that take place in
cancer cells as they become resistant to IFNs and whereby abnormalities
in the expression of key IFN signaling molecules have been elucidated
(7-11).
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ABSTRACT
INTRODUCTION
