(Received for publication, August 28, 1996, and in revised form, December 19, 1996)
From the Divisions of Molecular Virology and Hematology-Oncology, Departments of Internal Medicine and Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8594
A positive regulatory element in the interleukin-2 (IL-2) promoter, designated the antigen receptor response element-2, is essential for the induction of IL-2 gene expression upon the binding of an inducible multiprotein complex of proteins known as nuclear factor of activated T cells. In the current study, we demonstrated that the winged-helix transcription factor IL-2 enhancer binding factor (ILF) is constitutively expressed in both resting and activated Jurkat cells and binds to two adjacent sequence motifs immediately downstream of the binding site for NFAT. One of these elements has a high degree of homology with consensus binding sites for a variety of winged-helix DNA binding proteins, and the second site functions to modulate ILF binding. Mutagenesis of each of the two sequence elements required for ILF binding decreased IL-2 promoter activity when assayed in transfection assays. Although ILF bound constitutively to the IL-2 promoter, it was not detected as a component of the NFAT complex. These results suggest that important regulatory sequences in the IL-2 promoter are bound by ILF and that this binding may be involved in the control of IL-2 gene expression.
Induction of IL-21 gene expression is
a pivotal event in early T-lymphocyte activation. Binding of the T cell
receptor complex to its cognate antigen results in protein kinase C
activation and elevated intracellular calcium levels with subsequent
activation of IL-2 mRNA synthesis (1). Studies of the IL-2 promoter
(2, 3) have revealed a positive regulatory element situated between 288 and
255 relative to the transcription start site that is essential for increased IL-2 expression upon the activation of T-lymphocytes (2). This purine-rich site, designated the antigen receptor response element 2 (ARRE-2), is bound by an inducible multi-protein complex known as the nuclear factor of activated cells
(NFAT) following the activation of T-lymphocytes (4, 5).
T cell-specific activation of the IL-2 gene expression via the ARRE-2
site is a complex phenomenon regulated by a variety of proteins.
Several members of the AP-1 and NFAT families of transcription factors
participate in NFAT complex formation (6-10). NFATp and NFATc
represent the first identified members of a family of transcription
factors closely related to Rel proteins (9-14). Integration of these
proteins into the NFAT complex is achieved by two diverse mechanisms.
NFATp is present in the cytoplasm in resting T cells and translocates
to the nucleus upon immunologic activation in a manner reminiscent of
the transcription factor NF-B (11). In contrast, NFATc is newly
synthesized upon T cell activation (10). The two pathways are
intertwined because both activation events are blocked by the
immunosuppressive agent cyclosporin A (10, 11). Identification of a
functional AP-1 site directly adjacent to the binding site for the NFAT
proteins demonstrated additional transcription factors are able to
regulate the activity of the NFAT enhancer element (7). Jun, JunB, Fos,
and Fra-1 have all been demonstrated to be present in NFAT complexes
isolated from activated T cells (6-8). The involvement of multiple
NFAT and AP-1 family members in the NFAT complex suggests that this complex may in fact represent a closely related collection of multi-protein transcriptional activators.
Constitutively expressed transcription factors have also been proposed
to play a role in NFAT complex formation (15-19). Two such
constitutive factors NF90 and NF45 have been purified and cloned (20,
21) and found to bind to the same purine sequences as the NFAT
proteins. While these factors appear to represent novel genes, sequence
analysis of NF45 reveals homology to the prokaryotic -54
transcription factor. Other proteins including E1f-1, a member of the
Ets family of transcription factors (22), and ILF, a member of the
winged helix family of DNA binding proteins (23, 24), have also been
demonstrated to bind to the IL-2 ARRE-2 site. Winged-helix or forkhead
proteins share a conserved 100-amino acid DNA binding domain and
represent a family of transcription factors that participate in a
number of processes that regulate cellular gene expression (25-30).
Constitutively expressed factors such as ILF that bind to the ARRE-2
region may help to modulate the rapidity and tissue specificity of IL-2
mRNA induction.
In this study, we present evidence that ILF binds constitutively to the
ARRE-2 element in the IL-2 promoter. The optimal in vitro
binding site for ILF closely resembles the non-consensus AP-1 site in
ARRE-2 indicating that sequences recognized by ILF and the
multi-protein NFAT complex overlap. The 3 portion of the ILF binding
site comprised of purine-rich sequences is not required for NFAT
binding but is important for maximal IL-2 promoter activity. These
results suggest that ILF may have a positive role in regulating IL-2
gene expression, although ILF is not a component of the multi-protein
NFAT complex. Thus, ILF may be important both to prevent constitutive
expression from the IL-2 promoter and to maintain the IL-2 promoter in
a configuration that allows NFAT and AP-1 to activate IL-2 gene
expression.
Jurkat cells were
maintained in RPMI media with 10% fetal bovine serum, 100 units/ml
penicillin, 100 µg/ml streptomycin sulfate, and 2 mM
glutamine. T cells were activated by stimulation with 25 ng/ml phorbol
myristate acetate (PMA) and 2 µM ionomycin for 30-120
min. When used, cyclosporin A was added to a concentration of 100 ng/ml. Total RNA was prepared from cells with the RNAZol reagent
(Biotecx Laboratories) using the manufacturer's protocol. 10 µg of
RNA was used as a template for cDNA synthesis primed by 1 µg of
random hexamer oligonucleotides (Pharmacia Biotech Inc.) and catalyzed
by avian myeloblastosis virus-reverse transcriptase (Promega) in the
presence of 4 mM dNTPs. The 4 µl of the cDNA synthesis reaction was used as a template in a 100-µl PCR reaction containing 50 mM KCl, 10 mM Tris-HCl, pH 8.9, 0.1% Triton X-100, 1.5 mM MgCl2, 0.8 mM dNTPs, and 50 pmol of each specific primer. Primers
specific for ILF, IL-2, TATA-box binding protein (TBP), and NFATc human
cDNAs contained the following sequences and nucleotide numbers. ILF
sense (917-941): 5-GGAAGCTTCAGGTGGAGACAGCCCG-3
, ILF antisense
(2206-2183): 5
-GGCA-CCACAGAGTTGATATCGTT-3
, IL-2 sense (198-222):
5
-TGGAATTAATAATTACAAGATCCC-3
, IL-2 antisense (423-400):
5
-TGTTTCAGA-TCCCTTTAGTTCCAG-3
, TBP sense (220-244): 5
-TGCCAAGAAGAAAGTG-AACATCATG-3
, TBP antisense (565-541):
5
-AGGCAAGGGTACATGAGAGCCA-TTA-3
, NFATc sense (239-268):
5
-ATGCCAAGCACCAGCTTTCCA-GTCCCTT-3
, NFATc antisense (613-591):
5
-GTCTTGGGAGACACGCAGGGAGACT-3
.
Predicted PCR product sizes using these primers are 240 bp (IL-2), 334 bp (TBP), and 600 bp (NFATc). ILF primers amplify PCR products of different sizes for each transcript (ILF-1, 1289 bp; ILF-2, 1711 bp; ILF-3, 411 bp). PCR reactions were generally denatured for 5 min at 95 °C followed by 25-40 cycles of 55 °C annealing, 72 °C extension, and 95 °C denaturation (1 min each step). The 20 µl of each PCR reaction was analyzed by agarose gel electrophoresis followed by ethidium bromide staining. PCR reactions were allowed to progress for the lowest number of cycles at which ethidium bromide-stained PCR product could be visualized by UV light.
Preparation of ILF and Other ProteinsILF was expressed in
bacteria as a fusion protein with glutathione S-transferase
(23). GST-ILF constructs contained either 148 amino acids including the
ILF (amino acids 210-357) fused to GST which included the forkhead
domain (amino acids 251-348) or 445 amino acids (amino acids 210-655)
(23). The fusion proteins were expressed in Escherichia coli
and induced by the addition of 0.2 mM
isopropyl-1-thio--D-galactopyranoside. Proteins were purified by glutathione-agarose chromatography (31). Factor Xa
(Promega) was used to cleave the GST moiety and inactivated using 1 mM Pefablock (Boehringer Mannheim).
Full-length ILF and NFATc cDNAs used for CASTing experiments (32, 33) were transcribed and translated in vitro using the TNT T7 polymerase system (Promega). In vitro translation of CREB, Jun, or NFATc cloned into pGEM3 or -4 was also performed in a coupled transcription and translation system in either the presence or absence of [35S]methionine. The 12-amino acid influenza hemagglutinin sequences were inserted on the carboxyl terminus of ILF and Jun by PCR, and these proteins were translated in the absence of [35S]methionine. Immunoprecipitation of unlabeled Jun and ILF proteins was performed with a 1:100 dilution of 12CA5 antibody that recognizes the influenza hemagglutinin sequences.
Nuclear extracts were prepared by a needle lysis method (34). Nuclear extracts were prepared in the presence of a protease inhibitor mixture including dithiothreitol (1 mM), phenylmethylsulfonyl fluoride (1 mM), aprotinin (0.015 mg/ml), benzamidine (0.2 mM), antipain (1 µg/ml), pepstatin (1 µg/ml), leupeptin (0.5 µg/ml), amd chymostatin (0.1 µg/ml).
Western Blot Analysis of ILF ExpressionNuclear extracts from resting and activated Jurkat cells containing the same amount of soluble proteins were resolved on 8% SDS-polyacrylamide gels along with ILF-1 in vitro translated using the TNT rabbit reticulocyte lysate system (Promega). Proteins were transferred from the gel to Hybond C nitrocellulose (Amersham Corp.) by electroblotting for 8 h at 35 mA constant current. The filter was blocked for 1 h in 1 × TBS, 0.2% Tween (TBS-T), 5% nonfat milk. Incubation with the primary antibody was performed in TBS-T, 0.5% milk with ILF (1:2000 dilution), or c-Jun (1:500 dilution) rabbit polyclonal antisera. An anti-rabbit IgG conjugated to horseradish peroxidase was subsequently incubated with the blot at a 1:5000 dilution. All incubations were performed at room temperature. Immunoreactive proteins were visualized using enhanced chemiluminescence detection reagents (Amersham Corp.). Blots were generally exposed to x-ray film for 10-60 s.
Gel Retardation AnalysisOligonucleotides corresponding to
the IL-2 ARRE-2 (3) were annealed and radiolabeled with
[-32P]ATP and polynucleotide kinase. Conditions for
binding of bacterially expressed ILF to DNA were as follows: 50 mM NaCl, 10 mM Tris, pH 7.5, 1 mM
dithiothreitol, 1 mM EDTA, 5% glycerol, 5 mM
MgCl2, 1 µg of poly(dG-dC), 5 µg of GST-ILF, 0.1-0.5
ng of radiolabeled oligonucleotides (20,000 cpm) in a 20-µl reaction.
Binding conditions and protein amounts for gel retardation with nuclear
extracts were identical using 1 µg of poly(dI-dC) or 1 µg of
poly(dA-dT) used instead of poly(dG-dC). Use of poly(dI-dC) proved
optimal for generating NFAT binding activity, whereas the presence of poly(dA-dT) enhanced ILF binding activity. Binding reactions were performed at room temperature and were loaded and run on 4.5% polyacrylamide gels (0.5 × TBE). Antibodies used in
electrophoretic mobility shift assays were purified using protein
A-Sepharose chromatography, with 2 ml of rabbit serum loaded on a 1-ml
protein A column, washed with 20 ml of 1.5 M glycine, 3 M NaCl, pH 8.9, eluted with 0.1 M citric acid,
pH 3.0, into 50 µl of 1 M Tris, pH 8.9, and dialyzed into
phosphate-buffered saline.
Oligonucleotide sequences (sense strand)/IL-2 ARRE-2 oligonucleotides
with (mutated sequences underlined) are as follows. Wild type
(286/
257), 5
-AATTGGAGGAAAAACTGTTTCATACAGAAGGCGT-3
; 5
purine
mutant, 5
-AATTGGA
ACTGTTTCATACAGAAGGCGT-3
; core mutant, 5
-AATTGGAGGAAAAAC
CATACAGAAGGCGT-3
; 3
purine
mutant, 5
-AATTGGAGGAAAAACTGTTTCATAC
CGT-3
; double
core/3
purine mutant,
5
-AATTGGAGGAAAAAC
CATAC
CGT-3
.
Gel retardation analysis using Jurkat nuclear extract was performed as described above. Reactions were scaled up 5-fold to a 100-µl size. Gels were exposed to x-ray film for 2 h and the shifted complexes subsequently purified. Gel slices containing either NFAT complex or ILF were placed in the wells of an SDS-polyacrylamide protein gel with 50 µl of 5 × protein loading dye layered on top. Gels were subject to electrophoresis and proteins transferred to nitrocellulose with immunoblotting performed as described previously.
Cyclic Amplification and Selection of Targets (CASTING)Determination of the optimal binding sites of DNA
binding proteins using degenerate oligonucleotides using the so-called
CASTING procedure was performed as described previously (32). ILF and NFATc proteins containing a hemagglutinin epitope (amino acid sequence
YPYDVPDYA) at their amino termini were in vitro translated using the TNT system (Promega). Proteins were allowed to bind double-stranded 75-bp fragments containing a degenerate 35-bp core for
20 min at room temperature. Magnetic beads (Dynabeads) coated with an
anti-hemagglutinin monoclonal antibody were used to isolate proteins
and bound DNA fragments. PCR primers 5-GCGTCGACAAGCTTTCTAGA-3
(forward), 5
-CGCTCGAGGGATCCG-AATTC-3
(reverse) flanking the degenerate core were used to amplify the selected fragments, and products were visualized on agarose gels. Aliquots of each PCR reaction
were collected after 10, 14, 18, and 22 cycles. The lowest cycle number
PCR aliquot that contained visible product was identified and saved,
completing the first round of CASTING. Five additional rounds of
CASTING were performed using this and subsequent PCR products. Products
were cloned into the pCRII TA vector (Invitrogen) and analyzed by DNA
sequencing.
The IL-2 promoter from 340 to +47 was cloned
upstream of the chloramphenicol acetyltransferase (CAT) gene (2, 3).
Mutant IL-2 promoter plasmids were constructed using the Sculptor
in vitro mutagenesis system (Amersham Corp.) according to
the manufacturer's protocol. Mutant IL-2 constructs extending from
286 to
257 had the following underlined alterations in the ARRE-2:
wild type, 5
-GGAGGAAAAACTGTTTCATACAGAAGGCGT-3
; 5
purine mutant,
5
-GGA
AAAAACTGTTTCATACAG-AAGGCGT-3
; AP-1/ILF mutant,
5
-GGAGGAAAAAC
TTCATACAGAAGGC-GT-3
; 3
purine mutant,
5
-GGAGGAAAAACTGTTTCATAC
GCGT-3
.
IL-2 promoter activity was assayed by transient transfection of IL-2
CAT plasmids into H9 or Jurkat T cells stimulated with PMA and
ionomycin with subsequent determination of CAT activity from
transfected cell extracts. 107 cells were used per
transfection using 200 µg of DEAE-dextran and 10-20 µg of IL-2 CAT
plasmid DNA. Cells were split into two flasks 24 h after
transfection with one set of cells stimulated with 25 ng/ml phorbol
myristate acetate (PMA) and 2 µM ionomycin for 8 h.
Cells were harvested, washed with phosphate-buffered saline lysed by
multiple freeze-thaw cycles, and used in assays of CAT activity as
described previously (36). Approximately 2 µg of an
RSV-galactosidase plasmid was cotransfected with the CAT
reporter to standardize for transfection efficiency.
-Galactosidase activity was measured by incubation of 10 µl of whole cell extract with 5 mM CPRG (70 mM sodium phosphate, 8 mM KCl, 0.8 mM
-mercaptoethanol) at 37 °C
followed by determination of absorbance at 574 nm (37).
Several cDNA
libraries and were screened to obtain additional ILF clones containing
full-length coding sequences. 1.2 × 106 phage from a
ZAP Sup T3 T cell leukemia library and 500,000 plaques from a
EXlox HeLa cDNA library (Novagen) were probed with a
32P-radiolabeled fragment spanning nucleotides 527-876 of
the originally described ILF-1 and ILF-2 cDNAs. Several positive
phage were isolated and sequenced, three of which contained new ILF
coding sequence. The new ILF-1, ILF-2, and ILF-3 composite cDNA
clones were assembled using these new sequences and compared for ILF-1,
ILF-2, and ILF-3 to previously isolated cDNAs (24). The sequences
were placed into GenBank and have the accession numbers U58196[GenBank], U58197[GenBank], and U58198[GenBank] respectively.
The
ARRE-2 site in the IL-2 promoter is extremely well conserved between
species as depicted in Fig. 1. Comparison of the human,
bovine, and murine ARRE-2 elements in the IL-2 promoter reveal three
absolutely conserved regions each of which are indicated by brackets
(Fig. 1). Two of these elements are remarkably purine-rich and flank a
third conserved region that we designated the core element. The 5
purine and core regions are bound in vivo by NFAT and AP-1
transcription factors, respectively, and both of these elements have
been demonstrated to be essential for maximal ARRE-2 transcriptional
activity (6, 7, 11, 22). The functional importance of the 3
purine
element in IL-2 gene expression has not been previously determined.
Gel retardation analysis was performed with an IL-2 ARRE-2 probe to
characterize the binding of constitutive and inducible factors that
were present in nuclear extract prepared from nonactivated and PMA and
ionomycin-activated Jurkat cells. A single gel-retarded species was
present in nuclear extract prepared from nonactivated Jurkat cells
(Fig. 2A, lane 1). When the IL-2 probe was
incubated with extract prepared from PMA and ionomycin-treated Jurkat
cells, the faster mobility gel-retarded species as well as a slower
migrating inducible NFAT complex were present (Fig. 2A, lane
2). Probes containing mutations in each of the three conserved
regions in the IL-2 ARRE-2 were then used to determine the binding
specificity of constitutive and inducible binding activities (Fig.
2A, lanes 3-5). The inducible NFAT complex present in
extracts prepared from activated Jurkat nuclear extract was markedly
decreased when the 5 purine or the core regions were mutated (Fig.
2A, lanes 3 and 4). Mutation of the 3
purine
region did not prevent NFAT binding in nuclear extract prepared
from activated Jurkat cells but decreased the constitutive
binding activity in the nuclear extract (Fig. 2A, lane 5).
These results were consistent using different preparations of Jurkat
nuclear extract. Thus, a factor present in both resting and activated
Jurkat nuclear extract bound to the IL-2 ARRE-2 promoter, and this
binding was dependent on the core and 3
purine sequences.
Previously we have demonstrated that the transcription factor ILF when
produced as a fusion with glutathione S-transferase was
capable of binding to the IL-2 ARRE site (23). To further characterize
the constitutive binding activity, nuclear extract prepared from
nonactivated Jurkat cells was incubated with the radiolabeled wild-type
IL-2 ARRE-2 probe (Fig. 2B). In addition to the predominant
constitutive binding activity, a nonspecific faster mobility
gel-retarded complex that was present in some Jurkat nuclear extract
preparations was also detected (Fig. 2B). This latter
complex bound to the wild-type IL-2 ARRE-2 probe in addition to mutant
probes in the 5 purine, core, or 3
purine regions (data not shown).
Incubation of Jurkat nuclear extract with affinity purified rabbit
polyclonal antisera to ILF resulted in a supershift of the majority of
the slower mobility binding activity with no change in the amount of
the nonspecific binding activity (Fig. 2B, lane 2). A
similar supershifted band was not seen when preimmune antisera were
incubated with the extract (Fig. 2B, lane 3) or when ILF
antisera were incubated with the probe alone (Fig. 2B, lane
4). Mutations in the 5
purine region should preclude the binding
of several ARRE-2 binding proteins present in resting T cells including
NFATp (9), in addition to the NF45 and NF90 proteins (20, 21). ILF
antisera also supershifted the majority of the slower mobility complex
bound to an IL-2 ARRE-2 probe mutated in the 5
purine region when used
in gel retardation analysis with nuclear extract prepared from
nonactivated Jurkat cells (Fig. 2C, lane 2). Again the
ILF antibody did not alter the intensity of the nonspecific binding
activity (Fig. 2C, lane 2). Therefore, the constitutive
binding activity that is reactive with ILF antisera is distinct from
previously described proteins present in nonactivated
T-lymphocytes.
Next it was important to determine whether
bacterially produced ILF had the same binding specificity for the IL-2
ARRE-2 as the endogenous cellular protein. Gel retardation analysis
with IL-2 probes containing mutations in the 5 purine, core, and 3
purine regions in the IL-2 ARRE-2 were assayed using recombinant ILF
(Fig. 3A, lanes 1-4). A probe containing a
mutation of the 5
purine (Fig. 3A, lane 6) did not alter
ILF binding as compared with the wild-type probe (Fig. 3A, lane
5), whereas probes containing mutations of either the core
sequences (Fig. 3A, lane 7) or the 3
purine sequences (Fig.
3A, lane 8) markedly decreased ILF binding. The amounts of
wild-type and mutant IL-2 probes that were bound by ILF were
quantitated using PhosphorImager scanning in three separate
experiments, and the results are expressed graphically (Fig.
3B). Although mutation of the 5
purine region had only slight effects on ILF binding to the IL-2 probe with a reduction to
84% of wild type, mutation of the core and 3
purine regions resulted
in marked diminution of ILF binding giving values of 17 and 23% of the
wild type levels, respectively (Fig. 3B). A probe containing
mutations in both the core and 3
purine regions reduced ILF binding to
2.9% of wild type (data not shown). These results indicate that both
endogenous and recombinant ILF protein require the core and 3
purine
element for its binding.
ILF and NFATc Have Optimal DNA Binding Sites Closely Resembling Their Target Sequences in the IL-2 ARRE-2
It was important to
determine whether the sequences in the IL-2 promoter were bound by ILF
were similar to the optimal ILF binding sequences. For that reason, we
used degenerate oligonucleotides to select for sequences optimally
bound by ILF (32, 33). As a control, we also used degenerate
oligonucleotides to determine the optimal binding site for NFATc. These
proteins that contained both 3 influenza hemagglutinin sequences were
in vitro translated in rabbit reticulocyte lysate, bound to
degenerate oligonucleotides, and immunoprecipitated with 12CA5 antibody
that can recognize the hemagglutinin-tagged proteins. This was followed
by repeated cycles of PCR and protein binding prior to the isolation of
these DNAs and the DNA sequence analysis.
Using this analysis, the consensus ILF binding site determined from the
alignment of 19 independent sequences was TGTTTAC. As depicted in Fig.
4A, each of the first five nucleotides
(TGTTT) derived by site selection was conserved in 79-89% of the
aligned sequences. This sequence was present in the core region of the IL-2 ARRE-2 and mutations of the sequence reduced ILF binding greater
than 5-fold. The ILF consensus sequence was also highly related to
binding sites seen in promoters bound by other winged-helix proteins
(30). The consensus NFATc sequence determined by site selection was an
octamer with the sequence TGGAAAAT (Fig. 4B). This consensus
sequence was even more highly conserved than that obtained for ILF with
the GGAAA motif present in 100% of sequences. The most striking aspect
of the NFATc sequence determined in this analysis was its strong
homology to the IL-2 NFAT binding site. Thus the use of site selection
defined optimal ILF and NFAT binding sites and demonstrated that these
sequences were present in the IL-2 ARRE-2 region.
Gel retardation analysis using both Jurkat nuclear extract (Fig. 2) and
recombinant ILF (Fig. 3) indicated that the core element in the IL-2
ARRE-2 was critical for ILF binding and the 3 purine sequence also
influenced the degree of ILF binding. Next, the role of the 3
purine
sequence on regulating ILF binding was analyzed when the core sequence
was mutated to the optimal ILF binding sequence as determined by
CASTING. Gel retardation was performed with recombinant ILF protein
using oligonucleotides containing either the wild-type ARRE-2, this
oligonucleotide with a mutation of the 3
purine sequences, the ARRE-2
sequence with the ILF CASTING consensus in place of the wild-type core,
or this oligonucleotide in the presence of a mutated 3
purine
sequence. Altering the IL-2 wild-type core sequences to the ILF CASTING
consensus increased ILF binding approximately 3-fold (Fig.
5, lanes 2 and 4). Mutation of the
3
purine sequence reduced ILF binding to both the wild-type core
sequence and the ILF CASTING consensus sequence (Fig. 5, lanes
3 and 5). These results in conjunction with those of
Figs. 2 and 3 indicate that the core sequence is critical for ILF
binding and that the 3
purine sequences modulate this binding.
The Core and 3
To assess the role of the ILF binding site on IL-2
gene expression, we performed site-specific mutagenesis on the elements in the ARRE-2 that were required for ILF and NFAT binding. The sequence
of this region and the base pair changes introduced are indicated (Fig.
6A). These individual mutations were each
inserted into an IL-2 promoter construct fused to CAT, and the gene
expression of these constructs was analyzed following transfection into
PMA and ionomycin-treated Jurkat cells. Mutation of either the 5 purine or the core region sequences reduced IL-2 promoter activity dramatically to 9.1 and 17.4% of wild type levels, respectively (Fig.
6B). The deleterious effect of the core site mutation on IL-2 promoter activity could be explained by changes in the AP-1 binding site resulting in the lack of formation of a multimeric NFAT
complex (6-10). Due to the overlapping AP-1 site, it is not possible
to assess the importance of ILF binding to the core region by
mutagenesis. The role of ILF binding on IL-2 gene expression could best
be tested by analyzing mutations in the 3
purine region since this
region does not alter the binding of the NFAT complex comprised of both
NFAT and AP-1 factors. Mutation of the 3
purine site decreased IL-2
promoter expression to 10.5% of wild type (Fig. 6B)
indicating that this conserved element that is required for ILF binding
is essential for maximal IL-2 gene expression. Mutations of the IL-2
promoter upstream of the NFAT binding site extending between
344 and
300 did not alter IL-2 gene expression (data not shown). Finally, we
determined whether transfection of an expression vector containing ILF
into Jurkat cells was able to activate IL-2 CAT gene expression.
Transfection of increasing amounts of the ILF expression vector
resulted in activation of IL-2 gene expression (Fig. 6C, lanes
4-6) as compared with increasing amounts of the expression vector
alone (Fig. 6C, lanes 1-3). The level of activation by ILF
ranged from 3- to 6-fold in three independent experiments and was
dependent on the ARRE-2 site (data not shown). These data suggest that
ILF plays a positive role on activating IL-2 gene expression.
Analysis of ILF mRNA Levels in Jurkat T Cells
Three
composite ILF cDNAs corresponding to alternatively spliced
transcripts have been assembled based on PCR analysis of Jurkat RNA and
submitted to GenBank. ILF-1 and ILF-2 represent updated versions of the
originally published clones (24) and contain additional 5 sequences.
ILF-3 represents a newly identified alternatively spliced transcript
that lacks the coding sequence for a carboxyl-terminal portion of the
winged-helix DNA binding domain and is unable to bind to DNA. All three
cDNAs have different 5
splice donor sites fused to a common 3
splice acceptor. A schematic of the cDNAs encoding proteins of 655, 609, and 323 amino acids, respectively, and designated ILF-1, ILF-2,
and ILF-3 are indicated (Fig. 7).
To determine whether ILF cDNAs were expressed in resting or PMA and
ionomycin-treated Jurkat cells, mRNA from these cells was extracted
and amplified with two PCR primers flanking the common 3 splice
acceptor site for the three alternatively spliced ILF transcripts. The
ILF primers were capable of amplifying PCR products of different sizes
for each transcript (ILF-1, 1289 bp; ILF-2, 1711 bp; ILF-3, 411 bp).
Primers corresponding to the constitutively expressed TATA-binding
protein (TBP) were used as controls in this PCR analysis. The PCR
products were analyzed following agarose gel electrophoresis and
ethidium staining. In Fig. 8A, ILF expression was analyzed in both resting and PMA and ionomycin-stimulated Jurkat
cells as well as in stimulated Jurkat cells incubated with cyclosporin
A. No differences were observed in PCR-amplified mRNA from these
cells corresponding to ILF-1 and ILF-3 (Fig. 8A, lanes 1-3). ILF-2 mRNA that is present in extremely low levels in
Jurkat cells relative to ILF-1 and ILF-3 was not detected using the
number of cycles in this PCR analysis (data not shown). Consistent with many published reports, IL-2 mRNA levels (Fig. 8A, lanes
5-7) and NFATc (Fig. 8B, lanes 1-3) mRNA levels
were increased upon T cell activation, whereas IL-2 induction was
blocked by cyclosporin A treatment (Fig. 8A, lanes 6 and
7). TBP mRNA was expressed equally in all samples tested
(Fig. 8B, lanes 5 and 6). Similar levels of ILF
expression were also present in resting and stimulated PBMCs (data not
shown).
Immunoblot analysis of ILF was next used to characterize the endogenous ILF protein present in Jurkat nuclear extracts and to further determine the specificity of the ILF antibody (Fig. 8C, lanes 1-5). Equal quantities of Jurkat nuclear proteins from nonactivated and PMA and ionomycin-treated Jurkat cells were analyzed by Western blot analysis. ILF-1 that represents the cDNA clone with the longest open reading frame was the most abundantly expressed ILF cDNA in Jurkat cells (Fig. 8C, lanes 1-3). In vitro translated ILF-1 comigrated with the predominant endogenous Jurkat ILF protein and was detected at an approximate molecular weight of 75 kDa (Fig. 8C, lanes 1-4). ILF protein levels remained unchanged 60 min after Jurkat stimulation with PMA and ionomycin (Fig. 8C, lanes 1-3) consistent with the PCR data of ILF mRNA. While the predominant protein detected in Jurkat cells corresponded to ILF-1, two smaller, less abundant proteins of approximately 65 and 30 kDa were also detected that may represent ILF-2 and ILF-3 or other closely related factors. As the ILF antisera were raised against a fusion protein containing the intact winged-helix domain, ILF-3 would likely be less immunoreactive since it lacks a large portion of this domain, and thus, our immunoblot analysis may not accurately reflect ILF-3 levels (Fig. 6). These results indicate ILF is constitutively expressed in Jurkat cells, and its levels do not change upon T cell activation.
ILF Is Not a Component of the NFAT ComplexFinally, we wished
to determine whether ILF was a component of the NFAT complex. This
would distinguish between two alternative models for ILF action. ILF
could bind to the IL-2 ARRE-2 in the absence of the NFAT complex and
precede NFAT binding or alternatively ILF could be the first protein
binding to the ARRE-2 followed by the formation of a multi-protein NFAT
complex containing ILF as a member. To determine if ILF was a component
of the NFAT complex, we assayed the immunoreactivity of the NFAT
complex with ILF antisera in gel retardation experiments (Fig.
9A). Nuclear extract was prepared from both
nonactivated and PMA and ionomycin-treated Jurkat cells and used in gel
retardation analysis with the IL-2 ARRE-2 probe. In contrast to an
earlier extraction procedure used in this study, this extract was
prepared according to a protocol to optimize the extraction of NFAT
proteins (38) and resulted in a variety of proteins that bound
nonspecifically to the ARRE-2 probe. ILF formed a major gel-retarded
species that bound to the ARRE-2 probe with Jurkat nuclear extract
prepared from nonactivated cells (Fig. 9A, lane 1). The NFAT
complex was the predominant species in nuclear extract prepared from
PMA and ionomycin-treated Jurkat cells (Fig. 9A, lane 4).
The addition of ILF antibody supershifted the constitutive ILF
gel-retarded complex (Fig. 9A, lane 2) but did not alter the
mobility of the NFAT complex (Fig. 9A, lane 5). In contrast,
the addition of Jun antibody inhibited the binding of the NFAT complex
(Fig. 9A, lane 6). The addition of preimmune rabbit sera did
not alter the mobility of either the ILF or NFAT complexes (Fig.
9A, lanes 3 and 7). These results indicate that ILF antibody did not alter the mobility of the NFAT complex bound to
the IL-2 promoter.
ILF is not associated with the NFAT complex. A, gel retardation analysis was performed with nuclear extract prepared from nonactivated (lanes 1-3) or PMA and ionomycin-treated Jurkat cells (lanes 4-7) using a wild-type IL-2 ARRE-2 probe in the absence of antibody (lanes 1 and 4), in the presence of ILF polyclonal antibody (lanes 2 and 5), Jun antibody (lane 6), or with preimmune rabbit sera (lanes 3 and 7). B, NFAT and ILF gel-retarded complexes were isolated following electrophoresis and autoradiography from both activated and nonactivated Jurkat nuclear extract. Each of the gel-retarded complexes was subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis with NFAT, Jun, ILF, or TBP antibody. The antibodies used are indicated at the top of each panel, and the Jurkat nuclear extract input used in the gel retardation analysis and the gel-retarded complexes (ILF or NFAT) used in the Western blot analysis are indicated at the bottom of each panel. The NFAT gel-retarded complex was analyzed with NFAT antibody (panel 1), Jun antibody (panel 2), ILF antibody (panel 3), or TBP antibody (panel 4). The ILF complex was analyzed with ILF antibody (panel 3) or TBP antibody (panel 4). C, gel retardation with an IL-2 ARRE-2 probe was performed with either 3.0 µg of GST (lane 1) and ILF (lane 2) or nuclear extract (5.0 µg) prepared from PMA and ionomycin-treated Jurkat cells (lane 3). In addition, 1 or 3 µg of ILF (lanes 5 and 6) or GST (lanes 7 and 8) were added to the nuclear extract prior to gel electrophoresis. D, coupled in vitro transcription and translation was performed in rabbit reticulocyte lysate in the presence of [35S]methionine for complete cDNAs coding for Jun (lanes 1, 4, and 7), CREB (lanes 2, 4, and 6), and NFATc (lanes 3, 6, and 9). Both ILF (lanes 4-6) and Jun (lanes 7-9) cDNAs that contained carboxyl-terminal influenza hemagglutinin sequences were in vitro translated in rabbit reticulocyte lysate in the absence of [35S]methionine. Both the [35S]methionine-labeled and unlabeled proteins (10 µl) were incubated prior to immunoprecipitation with the 12CA5 monoclonal antibody that recognizes the influenza hemagglutinin sequences followed by SDS-polyacrylamide gel electrophoresis and autoradiography.
An additional approach was also used to analyze whether ILF was a component of the NFAT complex (Fig. 9B). Gel retardation analysis was performed using the IL-2 ARRE-2 probe with nuclear extract prepared from PMA and ionomycin-treated Jurkat cells, and the NFAT complex was isolated from the polyacrylamide gel following electrophoresis and autoradiography. Gel slices containing the NFAT activity were then subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting with either ILF, NFAT, TBP or Jun antibodies (Fig. 9B). In addition, nuclear extract prepared from nonactivated Jurkat cells was used in gel retardation analysis with the IL-2 ARRE-2 probe to isolate the ILF gel-retarded species. This complex was immunoblotted with either ILF or TBP antisera as a control for the sensitivity of the Western blot analysis. The TATA-box binding protein (TBP) antibody was used to rule out nonspecific association of other nuclear proteins with the NFAT and ILF gel-retarded complexes. The same quantity of Jurkat nuclear extract used in the gel retardation assays was also analyzed by Western blot analysis and was designated as the input fraction (Fig. 9B). Using this procedure, we could analyze by immunologic means for the presence of ILF in the NFAT complex. Immunoblotting of the NFAT complex revealed the presence of NFATc (Fig. 9B, panel 1) and Jun (Fig. 9B, panel 2) but not ILF (Fig. 9B, panel 3) or TBP (Fig. 9B, panel 4). In contrast, the ILF antibody detected ILF in a gel-retarded complex using nonactivated Jurkat extracts (Fig. 9B, panel 3), and this complex was not reactive with TBP antibody (Fig. 9B, panel 4). These results indicate that ILF bound to the IL-2 ARRE-2 in nuclear extract prepared from nonactivated Jurkat cells but was not a component of the NFAT complex.
Finally, we determined whether the addition of recombinant ILF produced as a GST fusion and cleaved with factor X resulted in alterations of NFAT binding to the IL-2 ARRE-2 oligonucleotide. A partially truncated ILF protein was used in these studies to differentiate the bacterially produced ILF from endogenous ILF present in Jurkat extract. Gel retardation analysis with the recombinant ILF demonstrated that it bound to the IL-2 oligonucleotide and generated a species corresponding to intact ILF protein in addition to several degradation products (Fig. 9C, lane 2). Extracts prepared from Jurkat cells treated with PMA and ionomycin resulted in the generation of the inducible NFAT complex in gel retardation analysis (Fig. 9C, lane 3). The addition of increasing amounts of recombinant ILF to the gel retardation assays with extract prepared from activated Jurkat cells resulted in the loss of binding of the NFAT complex and appearance of a gel-retarded species indicative of recombinant ILF (Fig. 9C, lanes 4 and 5). In contrast there was no change in the appearance of NFAT complex following the addition of increasing amounts of GST protein (Fig. 9C, lanes 6 and 7). These results further demonstrate that ILF and NFAT were not able to form a complex on the IL-2 ARRE-2 site.
The addition of the Jun antibody to the gel retardation in Fig. 9A completely disrupted the binding of the NFAT complex and also slightly decreased ILF binding. To address whether ILF might interact directly with either Jun or NFATc, cDNAs encoding these proteins were in vitro translated in rabbit reticulocyte lysate in the presence of [35S]methionine (Fig. 9D, lanes 1-3). CREB was also in vitro translated for use as a negative control. In addition, ILF and Jun cDNAs that contained carboxyl-terminal influenza hemagglutinin sequences and could be recognized by the 12CA5 monoclonal antibody were also in vitro translated in rabbit reticulocyte lysate in the absence of [35S]methionine. The presence of these unlabeled proteins was confirmed by Western blot analysis (data not shown). The unlabeled Jun and ILF proteins were incubated with each of the [35S]methionine-labeled proteins followed by immunoprecipitation with 12CA5 antibody and autoradiography. This analysis indicated that ILF was not able to co-immunoprecipitate with Jun or NFATc (Fig. 9D, lanes 4-6), whereas Jun was able to co-immunoprecipitate with Jun as these proteins can form homodimers (Fig. 9D, lane 8). These results support the fact that ILF cannot directly interact with components of the NFAT complex.
The regulation of IL-2 gene expression is dependent on multiple
cis-acting sequences in its promoter element that bind both constitutive and inducible cellular factors (1-4, 6, 7, 9-11, 19, 20,
22, 39, 40). A critical region required for induction of IL-2 gene
expression in response to T cell activation is an element located
between 288 and
255 relative to the transcription start site known
as ARRE-2 (2). This site is bound by an inducible protein complex known
as NFAT that is present in activated T cell nuclear extract and is
required for IL-2 mRNA synthesis (1, 4, 6, 8-11, 29). The NFAT
complex is comprised of a group of proteins known as NFAT in addition
to the Jun and Fos proteins (6-11). The NFAT proteins are comprised of
four highly related members, and various spliced forms include NFATc,
NFATp/NFAT1, NFAT3, and NFATx/NFAT4 (9-14) that have homology in their
300-amino acid Rel homology domain (41). In contrast to other Rel
proteins that form homodimers on heterodimers on their target
sequences, NFAT proteins bind as monomers to their recognition
sequences (41). The stability of NFAT protein binding to the ARRE-2
site is dependent on a non-consensus AP-1 site with the sequence
TGTTTCA immediately downstream of the NFAT binding site (6-9, 22). A
variety of members of the Jun/Fos family have been demonstrated to be
present in the NFAT protein complex including Jun, Fos, JunB, JunD, and
Fra-1, and their binding to the non-consensus AP-1 site is essential
for the activation of IL-2 gene expression (6-9, 22).
Activation of IL-2 gene expression is dependent on NFAT translocation from the cytoplasm to the nucleus in response to an increase in intracellular calcium and activation of the Jun family through a protein kinase C-dependent pathway (1, 4, 5, 10, 11). The translocation of the NFAT proteins occurs following dephosphorylation of these proteins by the calcium/calmodulindependent serine/threonine phosphatase calcineurin (42). Although the regulation of IL-2 gene expression in activated T-lymphocytes has been studied in detail, much less is known about the cellular factors that bind to the IL-2 promoter in resting T-lymphocytes. Two polypeptides designated NF45 and NF90 have similar DNA binding specificity to NFAT and bind to the ARRE-2 site in resting T-lymphocytes (20, 21). DNase I footprinting and gel retardation assays have demonstrated changes from constitutive to inducible factor binding to ARRE-2 in the IL-2 promoter upon T cell activation (15-18). These results suggest that constitutive factors that are likely different from NFAT and Jun/Fos bind to the IL-2 promoter and may be important for the subsequent induction of IL-2 gene expression.
To identify constitutive factors that are bound to the IL-2 ARRE-2
site, gel retardation analysis was performed using an IL-2 ARRE-2 probe
in the presence of nuclear extract prepared from nonactivated Jurkat
cells. This analysis indicated that ILF was able to bind specifically
to the IL-2 promoter and that the binding specificity of ILF in Jurkat
nuclear extract correlated with the binding properties seen with
recombinant ILF. Site selection and mutagenesis were performed to
define the sequences in the IL-2 promoter responsible for ILF binding.
ILF binding is dependent on sequences that correspond to a degenerate
AP-1 binding site in the IL-2 promoter and that have a high degree of
homology with a consensus binding site for other members of the
winged-helix family of DNA binding proteins. For example, the sequences
in the IL-2 promoter TGTTTCA that are critical for ILF binding are similar to the optimal binding site sequences TGTTTAC for ILF and other
winged-helix family members (30). The two 3 nucleotides in this
consensus binding sequence vary considerably between different winged-helix binding proteins. In addition, 3
purine-rich sequences flanking the ILF binding site in the IL-2 promoter are also critical for ILF binding.
Mutations in either the core or 3 purine regions of the IL-2 ARRE-2
site that are required for ILF binding reduce IL-2 gene expression to
approximately 10% of wild-type levels. Mutations in the core region
have previously been shown to decrease IL-2 gene expression by
disrupting Jun and Fos assembly into the NFAT complex (6, 7). However,
other DNA binding proteins have not been demonstrated to require the 3
purine region for binding to the IL-2 promoter, and the detrimental
effects of mutations of this region on IL-2 gene expression are likely
due to decreased ILF binding to the promoter. This suggests that ILF
may be a positively acting transcription factor involved in regulating
IL-2 gene expression. However, our inability to demonstrate that ILF
was a component of the NFAT complex suggests that its mechanism of
action was not likely due to association with either NFAT or Jun or
Fos. The predominant ARRE-2 binding activity detected in gel
retardation assays with nuclear extract prepared from resting T cells
was immunoreactive with ILF antibody and had similar DNA binding
specificity to bacterially produced ILF. Previous studies describing
ARRE-2 binding activity in nuclear extract prepared from resting T
cells have predominantly detected nonspecific DNA binding. We were able to detect specific ILF-immunoreactive activity using the binding conditions and nuclear extract preparations described in this study.
In vivo footprinting of the IL-2 promoter indicates the presence of a footprint over the ARRE-2 in resting T cells (17). This
footprint undergoes activation-specific changes likely corresponding to
replacement of constitutively bound factors such as ILF by the NFAT
multi-protein complex.
ILF mainly binds to the IL-2 promoter in resting T cells during a period of transcriptional silence. The temporal binding pattern of ILF would be consistent with a role on recruiting cellular factors to the IL-2 promoter that are required for rapid transcriptional activation by other factors upon T cell activation. For example, ILF binding in resting T cells may help to recruit basal transcription factors to the IL-2 promoter enhancing the eventual formation of the transcriptional initiation complex. ILF binding activity can also be detected in nuclear extract prepared from activated Jurkat extract. In order for IL-2 transcriptional activation to occur, the NFAT complex must bind to the IL-2 promoter thereby replacing ILF. ILF may have no functional role in activated T cells when NFAT catalyzes IL-2 transcriptional activation or the interchangeable binding of ILF and the NFAT complex may occur in activated T cells to regulate IL-2 transcriptional levels.
The mechanism by which ILF functions to modulate IL-2 gene expression
remains unclear. A number of DNA binding proteins including members of
the winged-helix family of transcription factors have been shown to
induce topological changes in their target sequences including DNA
bending (30). Bending of DNA can in theory influence transcription by
altering DNA binding of factors or by inducing interaction between
DNA-bound proteins. However, we were unable to detect ILF binding of
IL-2 ARRE-2 DNA (data not shown). Winged-helix factors have also been
demonstrated to regulate chromatin remodeling. One study reveals that
mutation of the winged-helix protein HNF-3 site in the
hepatocyte-specific serum albumin enhancer results in alterations in
chromatin structure as well as inhibiting serum albumin expression
(29). In addition, structural studies of the winged-helix domain of the
transcription factor HNF-3 demonstrates similarity with histone-like
domains (27). Thus, it is reasonable to hypothesize that ILF may alter
the chromatin structure of the IL-2 gene in T cells to facilitate
transcriptional activation by the NFAT proteins. Since examination of
chromatin structure of the IL-2 promoter strongly suggests the presence
of a DNase I-hypersensitive site immediately upstream of the IL-2
ARRE-2 region (15), it is possible that the binding of factors such as
ILF may be responsible. Further studies will be required to determine
the mechanism by which ILF regulates IL-2 gene expression.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U58196[GenBank], U58197[GenBank], and U58198[GenBank].
We thank Sharon Johnson for the preparation of this manuscript and Anthony Cancel for preparation of the figures.