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* Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, and
Department of Microbiology and Immunology, McGill University, Montreal, Canada; and
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and
NF-AT pathways, resulting in the binding of p50, p65, and c-Rel to the
B1 element and p50, c-Rel, and NF-ATp to the CD28RE element within
the -617 to -209 region of the IRF-4 promoter. Furthermore, mutation
of either the B1 or CD28RE sites blocks Tax-mediated transactivation
of the human IRF-4 promoter in T cells. These experiments constitute
the first detailed analysis of human IRF-4 transcriptional regulation
within the context of HTLV-I infection and transformation of
CD4+ T lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (4), chemokines (RANTES)
(5), cell surface receptors (MHC class I)
(6), and proteins involved in the antiviral defense (PKR,
2'5' oligo A synthetase, IFN-stimulated gene (ISG) (7, 8).
Although the best-characterized members of the IRF family—IRF-1,
IRF-2, and IRF-3—display a fairly ubiquitous pattern of expression in
mammalian cells, expression of IRF-4 is specific to the lymphoid and
myeloid compartments of the immune system (9, 10, 11, 12, 13).
Furthermore, the function of IRF-4 with respect to gene regulation
appears to be as cell type specific as its expression pattern. IRF-4 is
expressed during all stages of B lymphocyte development, where it binds
and transactivates the murine IgL chain enhancer E
2–4
(9) and the IL-1 promoter (14) in
association with the Ets protein PU.1. In B cells, IRF-4 expression is
upregulated by stimulation through CD40 and treatment with IL-4,
resulting in IRF-4-mediated transactivation of IL-4-inducible genes in
conjunction with STAT6 (15). Constitutive IRF-4 expression
in macrophages correlates with repression of the IRF-regulated ISG15
ISRE, in association with IFN consensus sequence binding protein
(ICSBP) or IRF-8 (13). Interestingly, the regulation of
IRF-4 activity in macrophages appears to occur at the level of
cytoplasmic/nuclear localization rather than the level of gene
expression (12).
IRF-4 expression is under stringent control in T lymphocytes; IRF-4 is induced upon T cell activation, an event mimicked by CD3 cross-linking or treatment with PMA/ionomycin and conA (10, 11, 16). Transcriptional regulation of IRF-4 in T cells has been shown to be dysregulated in only one case: IRF-4 is constitutively upregulated at the mRNA and protein levels in T cells infected and transformed with the human T cell leukemia virus type I (HTLV-I) (11, 16). Interestingly, recent studies have identified direct downstream target genes of IRF-4, notably IL-4 (17) and cyclin B1 (18), suggesting the involvement of IRF-4 in these signaling pathways.
At present, between 10 and 20 million people are infected with
HTLV-I, a retroviral agent etiologically associated with ATL, an
aggressive and often fatal malignancy of CD4+ T cells
(19), as well as HTLV-I-associated myelopathy
(HAM)/tropical spastic paraperesis (TSP), a demyelinating neurological
syndrome (20). Infection by HTLV-I transforms T cells in
vitro and in vivo (21, 22), a process that has been
associated with up-regulation of specific cellular genesinvolved in
T cell activation and proliferation during the course of viral
infection (23, 24, 25). Dysregulation of the lymphocyte gene
expression pattern is attributed to the activity of the 40-kDa
HTLV-I-encoded Tax oncoprotein, a key regulator of cellular and viral
gene expression. Tax physically interacts with and modulates the
activity of numerous host signaling components and transcription
factors, resulting in the induction of regulatory genes during the
early stages of HTLV-I infection and transformation
(24, 25, 26). In this study, we provide the first detailed
analysis of the regulation of the human IRF-4 gene within the context
of HTLV-I infection. Our studies establish a link between IRF-4
expression in HTLV-I-infected cells and the presence of the ATL
phenotype. We have characterized several transcriptional regulatory
elements within the human IRF-4 promoter that interact with NF-B,
NF-AT, and stimulating factor 1 (Sp-1) transcription factors to drive
IRF-4 production in HTLV-I transformed T-cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary human T lymphocytes, isolated by Ficoll-density centrifugation from whole blood, were maintained in RPMI 1640 medium (Canadian Life Technologies, Burlington, Ontario, Canada) supplemented with 15% non-heat-inactivated FBS and antibiotics. HTLV-I-infected T cell lines MT2, C8166, and MT4 and T cell lines T4, Jurkat, and CEM were cultured in RPMI 1640 medium (CLT) supplemented with 10% heat-inactivated FBS and antibiotics. PBL extracts from HAM/TSP, ATL patients, and normal donors were obtained from N. Azimi (National Cancer Institute, National Institutes of Health, Bethesda, MD). ATL is characterized by a monoclonal expansion of CD4+ T cells (21). All ATL patients possessed high leukemic cell counts (5- to 6-fold higher PBMC levels compared to normal donors) and at least 50% of peripheral T lymphocytes were leukemic. Leukemic cells were CD4+CD8-CD25+. PMA and ionomycin (Calbiochem, San Diego, CA) were used at a final concentration of 10 ng/ml and 200 nM, respectively, for immunoblot analysis and PMA was used at a final concentration of 50 ng/ml for in vitro kinase assay. FK506 (Sigma-Aldrich, Oakville, Ontario, Canada) was used at a final concentration of 1 µM.
Plasmid construction and mutagenesis
1.2-kbIRF4PRO-PGL3B luc was generated by cloning the human
promoter of IRF-4 (27) into
SacI/BglII digested luciferase reporter plasmid
pGL3 basic (pGL3B) using specific primers (upstream primer
5'-GAGCTCATGAAAATCCCTGGTCCAC-3' and downstream primer
5'-AGATCTTGAGGGCAGCGGTGGGTCCC-3') to amplify the 1189-nt upstream
regulatory element from HEK293 genomic DNA based on the GenBank
sequence U52683. A longer promoter fragment was obtained from Dr. T.
Mak (OCI/Amgen Institute, Toronto, Canada). The following 5' promoter
deletions were generated by restriction enzyme digestion of
1.2-kbIRF4PRO-pGL3B: NotI/SmaI (1 kb);
NotI/HindIII (0.6 kb);
NotI/PstI (0.4 kb);
NotI/StyI (0.2 kb);
NotI/StuI (0.08 kb). Plasmids carrying point
mutations in the B1 and Sp-1 sites were generated by in vitro
mutagenesis (28) using 1.2-kbIRF4PRO-PGL3B as the
template. Subcloning of PCR-amplified fragments into
SacI/BglII digested pGL3B was followed by
NotI/HindI digestion to obtain a 0.6-kb fragment.
The B1 site was mutated to 5'-CTCTGCAAAGCGtcGaaCCCTTCGCACCAG-3'
(nucleotides in lowercase represent point mutations), and the Sp-1 site
was mutated to
5'-CACCAGATTCaaGaTACTACAaGaaCaaCATTTCaaGaaCTGGaaACATCGCTGCAGTTT-3'.
CD28RE was generated by a PstI/StyI
restriction enzyme digest of 0.6-kbIRF-4PRO-pGL3B. pFlag-CMV2
NF-B-inducing kinase (NIK) has been previously described
(29). Expression plasmids for pCMV4-Tax WT, M22, and M47
(30) were provided by Dr. W. C. Greene (Gladstone
Institute, University of California, San Francisco, CA); pCMV4-NF-ATpXS
(31), encoding a truncated, constitutively active form of
NF-ATp, was provided by Dr. S. C. Sun (M. S. Hershey Medical
Center, Pennsylvania State University, Hershey, PA).
Immunoblotting
For preparation of whole-cell lysates, cells were washed twice in PBS and lysed at 4°C in a buffer containing 10 mM Tris-HCl (pH 8), 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40, 0.5 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin (Nonidet P-40 lysis buffer). Equal amounts of whole-cell lysates (25–100 µg) were fractionated by SDS-PAGE on a 10% polyacrylamide gel. After transfer, the Hybond membrane (Amersham, Cleveland, OH) was blocked in 5% milk in PBS for 1 h and probed overnight at 4°C with anti-IRF-4 (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1/500 or for 30 min at room temperature with anti-actin (Chemicon, Temucula, CA) at a dilution of 1/10,000. After four 5-min washes in PBS, membranes were incubated with a peroxidase-conjugated secondary rabbit anti-goat Ab at a dilution of 1/5,000 for IRF-4 and secondary goat anti-mouse at a dilution of 1/15,000 for actin. Reaction was visualized with an ECL detection system as detailed by manufacturer (Amersham).
Transfections and transient coexpression assays
MT2 and Jurkat T cells were transfected with human IRF4-pGL3B-luc reporter plasmids and assayed for luciferase activity. Exponentially growing cells (106) were transfected by Fugene 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) as detailed by the manufacturer using 0.2 µg of luciferase reporter plasmid and 25 ng of pRLTK normalizing plasmid per transfection. Expression plasmids were added in 3-fold molar excess and control pFlag-CMV2 vector was added to each sample to bring the total amount of DNA to 2 µg per sample. Forty-six hours post-transfection, cells were harvested, lysed in 100 µl passive lysis buffer (Promega, San Luis Obispo, CA), and assayed for dual luciferase activity using 5 µL of lysate according to the manufacturer’s instructions (Promega). All firefly luciferase values were normalized to Renilla luciferase to control for transfection efficiency, and results shown represent the average of at least three independent experiments.
Immunoprecipitations and kinase assay
For in vitro kinase assay, whole cell lysates were
immunoprecipitated with IB kinase (IKK) 
-specific, rabbit
polyclonal Ab (Santa Cruz Biotechnology). Immunoprecipitated IKK
complexes were incubated in kinase reaction buffer consisting of 10
µCi [-32P]ATP, 1 mM ATP, 5 mM MgCl2, 1
mM dithiothreitol, 100 mM NaCl, and 50 mM Tris-HCl (pH 8) at 30°C for
30 min with a GST-IBaa 1–55 or GST-2NIBaa
1–55 substrate, which contains two point mutations of serines
32 and 36 to alanine. Following fractionation of samples by SDS-PAGE on
a 10% polyacrylamide gel, the upper half of the gel was transferred to
a nitrocellulose membrane and blotted with IKK-specific mouse
monoclonal antisera (Alexis Biochemicals, San Diego, CA) at a final
concentration of 1 µg/ml; the lower half of the gel was stained with
Coomassie blue for 30 min, destained in 10% methanol/10% acetic acid
for 1 h, dried, and exposed to Biomax XR film (Kodak, Rochester,
NY) for 2 h at room temperature. For IKK- coimmunoprecipitation
assays, whole-cell extracts (1 mg) from Jurkat and MT2 cells were
immunoprecipitated with rabbit polyclonal Tax Ab (a kind gift from Dr.
W. C. Greene) cross-linked to protein A-Sepharose beads (Amersham
Pharmacia Biotech, Uppsala, Sweden) using dimethyl pimelimidate
(Sigma-Aldrich). Extracts were incubated with 30 µl of beads
cross-linked to 1 µg anti-Tax or no Ab for 4 h at 4°C.
Immunocomplexes were washed four times in Nonidet P-40 lysis buffer,
eluted, resolved by SDS-PAGE, and blotted with anti-Tax and
anti-IKK-.
In vivo genomic footprinting
For in vivo methylation by dimethyl sulfate (DMS; Aldrich
Chemical, Milwaukee, WI), exponentially growing MT2 or Jurkat cells
(108) were harvested and resuspended in RPMI 1640/10%
heat-inactivated FBS supplemented with 20 mM HEPES (pH 7.3).
Methylation reaction was performed in the presence of 10 µl/ml
concentrated DMS for 1 min at room temperature. Reaction was stopped by
two washes with ice-cold PBS containing 2% 2-ME. Genomic DNA
extraction was performed as previously described (32). To
obtain naked DNA, cells were first lysed to extract genomic DNA and
then submitted to DMS treatment for 30 s. Genomic DNA resuspended
in 200 µl H2O was treated with 20 µl of piperidine
(Aldrich) for 30 min at 90°C to cleave methylated G (or A) residues.
For each sample, 5 µg of DMS-treated, piperidine-cleaved genomic DNA
was used for ligation-mediated PCR using Vent DNA polymerase (New
Englands Biolabs, Mississauga, Canada), as described elsewhere
(33, 34). To ensure elongation of different fragment
sizes, the PCR amplification step was 2 min for the first cycle and
progressively increased to 10 min in the last cycle, with a total of 18
cycles. A third primer was radiolabeled by end labeling using T4
polynucleotide kinase (Amersham) and [-32P]ATP (ICN
Pharmaceuticals, San Francisco, CA). Two more PCR cycles were performed
to labeled elongated DNA. Each reaction product was phenol-chloroform
extracted and ethanol precipitated prior to electrophoresis on a 7.5%
Explorer sequencing gel (Baker, Phillipsburg, NJ) in 1x TBE at 65 W.
Reactions were visualized by autoradiography using Biomax MR films
(Kodak). For the ligation-mediated PCR, the following sets of primers
were used: for analysis of the noncoding strand of the -600 to -400
region of the IRF-4 promoter, primer 1, 5'-GTCACTTCAATTCACCAGC-3',
Tm 58°C; primer 2, 5'-GCAAAAGGATGTAAGCATGTCAGACACG-3', Tm 63°C;
primer 3, 5'-GTAAGCATGTCAGACACGCAGAGACAGTATTTG-3', Tm 65°C; for
analysis of the noncoding strand of the -460 to -260 region of the
IRF-4 promoter, primer 4, 5'-GTGATGGCCTTGCCGA-3', Tm 60°C; primer
5, 5'-GCAACCTCCACCTCCAGTTCTCTT-3', Tm 63°C; primer 6,
5'-ACCTCCAGTTCTCTTTGGACCATTCCTCC-3', Tm 66°C. Results shown
represent at least three independent experiments.
EMSA
Whole-cell extracts prepared from primary T cells as detailed
above and nuclear extracts prepared from Jurkat and HTLV-I-infected
cells as previously described (35) were subjected to EMSA
using -32P-labeled probes. The binding reaction was
carried out in a 20-µl final volume in binding buffer containing 20
mM HEPES (pH 7.9), 5% glycerol, 0.1 M KCl, 0.2 mM EDTA (pH 8), 0.2 mM
EGTA (pH 8), and 1 µg of poly(dI-dC) using 5–10 µg protein extract
from Jurkat, T4, MT2, or ATL-derived T cells. The following
oligonucleotide probes were used: NF-AT consensus,
5'-CGCCCAAAGAGGAAAATTTGTTTCATA-3'; NF-AT mutant,
5'-CGCCCAAAGCTTAAAATTTGTTTCATA (mutations are indicated
in bold); Sp-1 consensus, 5'-ATTCGATCGGGGCGGGGCGAGC-3'; Sp-1 mutant,
5'-ATTCGATCGGTTCGGGGCGAGC-3'; B1,
5'-CTCTGCAAAGCGAAGTCCCCTTCGCAC-3'; NF-2/Sp-1,
TGGCCAGGGCGGGAAATGGGGGGCGTGTAGCGG-3'; CD28RE,
5'-GCCCTTCGCGGGAAACGGCCCCAGTGACAGTCCCCGAGGCGG-3'. Protein/DNA
complexes were resolved on 5% polyacrylamide-0.5x Tris-borate-EDTA
gels and exposed to Biomax XR film (Kodak) overnight at -80°C.
Supershift analysis was performed for 15 min at room temperature using
1 µg anti-p65, anti-p50, anti-cRel, anti-Sp-1,
anti-NF-ATc3 (Santa Cruz Biotechnology), anti-NF-ATc1 (Affinty
Bioreagents, Golden, CO), and control rabbit or mouse serum or 1 µL
of a 1/10 dilution of anti-NF-ATp (Upstate Biotechnology, Lake
Placid, NY) Ab.
Formaldehyde cross-linking and chromatin immunoprecipitation (chIP) assays
Formaldehyde cross-linking and chIP procedure was modified from
the protocol reported by Boyd et al. (36). Exponentially
growing Jurkat or MT2 cells (2 x 107) were treated
with formaldehyde (Fischer Scientific), which was added directly to the
growth medium at a final concentration of 1% at room temperature with
stirring. The reaction was stopped after 10 min by addition of glycine
at a final concentration of 0.125 M. Suspension cells were harvested,
washed twice in ice-cold PBS, and lysed in SDS lysis buffer (Upstate
Biotechnology) on ice for 10 min. DNA was sonicated to an average
length of 600 bp using 5 sets of 10-s pulses at 30% efficiency,
followed by centrifugation at 13,000 rpm at 4°C. Prior to dilution,
20 µL of lysate was removed for 10% input control. Lysates were
diluted 5-fold in immunoprecipitation buffer (Upstate
Biotechnology) and precleared for 1 h at 4°C with 80 µL of a
50% salmon sperm DNA/protein A agarose slurry (Upstate Biotechnology).
Immunoprecipitations were carried out by the addition of 4 µg of
mouse monoclonal anti-cRel (Santa Cruz Biotechnology), rabbit
polyclonal anti-p50 (Santa Cruz Biotechnology), rabbit polyclonal
anti-Sp-1 (Santa Cruz Biotechnology), rabbit polyclonal
anti-NF-ATp (Upstate Biotechnology), rabbit polyclonal
anti-IRF-3 (Santa Cruz Biotechnology), or no Ab overnight at 4°C
with rotation. A secondary rabbit anti-mouse Ab was added to the
c-Rel sample 2 h before collection of immune complexes with 60
µL slurry for 1 h at 4°C. Immune complexes were washed for 5
min in low-salt buffer (Upstate Biotechnology), high-salt buffer
(Upstate Biotechnology), LiCl wash buffer (Upstate Biotechnology), and
twice in TE followed by elution of protein/DNA complexes in 1% SDS and
0.1 M NaHCO3. Protein/DNA crosslinks were reversed by
addition of NaCl at a final concentration of 200 mM and incubation at
65°C for 4 h. After proteinase K treatment, samples were
extracted with phenol/chloroform/isoamyl alcohol and precipitated at
-20°C overnight with 95% ethanol/0.5M sodium acetate and 20 µg
glycogen. DNA pellets were resuspended in 50 µL TE and PCR was
performed in the linear range of amplification using primers specific
to the human IRF-4 promoter, corresponding to a 250-bp region
containing the B1, NF-1/Sp-1, and CD28RE sites: primer 1,
5'-GCAACCTCCACCTCCAGTTCTCTTTG-3'; primer 2,
5'-TTCGGGGACTGTCACTGGGGCCGT-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments sought to analyze the pattern of IRF-4
expression in primary T lymphocytes, HTLV-I-infected cell lines, and
PBL isolated from HTLV-I-infected patients. Fig. 1
A demonstrates that IRF-4
protein expression was absent in resting primary T lymphocytes (Fig. 1A, lane 1), but induced after treatment with
PMA/ionomycin (Fig. 1A, lanes 2–6). Induction of
IRF-4 protein in primary T lymphocytes was inhibited by the
immunosuppressive drug FK506 (Fig. 1A, lane 8).
IRF-4 protein expression has been shown to be constitutively produced
in a number of EBV-infected, transformed B cell lines and
HTLV-I-infected, transformed T cell lines (16). Fig. 1B demonstrates IRF-4 overexpression in the
HTLV-I-transformed MT2, C8166, and MT4 cell lines (Fig. 1B,
lanes 1, 2, and 3), whereas IRF-4 is
not detected in noninfected Jurkat, CEM, or T4 cells (Fig. 1B, lanes 4, 5, and 6).
Treatment with anti-CD3/CD28 mAbs, PMA/ionomycin, or Con A did not
further increase IRF-4 levels in HTLV-I-transformed cell lines (data
not shown), indicating that IRF-4 was maximally expressed in these
cells. Analysis of PBL isolated from HTLV-I-infected patients (Fig. 1C) revealed that IRF-4 expression was detected in samples
from patients with the leukemic ATL phenotype (Fig. 1C,
lanes 2–6), whereas IRF-4 was absent from nonleukemic
HAM/TSP patient samples (Fig. 1C, lane 1).
Although a single HAM/TSP sample is shown in Fig. 1, a total of 11
HAM/TSP PBL samples were evaluated and all were negative for IRF-4
expression (data not shown).
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To examine the functional domains of the 1189-bp upstream
nucleotides corresponding to the human IRF-4 promoter (27)
involved in HTLV-I-induced IRF-4 activation, full-length and 5'
promoter deletions were transfected into MT2 cells to analyze relative
promoter activity within the context of HTLV-I-transformed cells (Fig. 2A). Deletion of the -617 to
-367 nucleotides of the IRF-4 promoter resulted in a decrease of
luciferase activity that represented 55% of full-length 1.2-kb
promoter activity in MT2 cells (Fig. 2A, 0.4 kb). Removal of
the adjacent -367 to -209 nucleotides abrogated IRF-4 promoter
luciferase activity in MT2 cells compared to full-length promoter
construct (Fig. 2A, 0.2 and 0.08 kb, respectively); these
results suggest that activation of the IRF-4 gene in HTLV-I-infected
MT2 cells is mediated through the -617 to -209 region of the human
IRF-4 promoter. Sequence evaluation within the -700 to -200 region of
the IRF-4 promoter revealed the presence of multiple consensus binding
sites for NF-B, NF-AT, and several other transcription factors, as
represented schematically in Fig. 2B.
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B activation in HTLV-I-transformed cells
It has previously been shown that Tax-induced NF-B activation
in HTLV-I-infected T cells occurs at the level of the IKK complex
(37, 38). To compare IKK kinase activity in Jurkat and
HTLV-I-infected T cell lines, in vitro kinase assay was performed using
the N-terminal 55 aa of IB (GST-IB) (Fig. 3A, lanes 1–5) or
S32/36A-substituted IB (GST-2NIB) (Fig. 3A,
lanes 6–10) as substrates for endogenous IKK
immunoprecipitated from Jurkat, PMA-treated Jurkat, and
HTLV-I-transformed T cells. As shown in Fig. 3A, IKK
activity was activated in Jurkat PMA and untreated MT2, C8166, and MT4
cells (Fig. 3A, lanes 2–5) but not in control
Jurkat cells (Fig. 3A, lane 1). Whole-cell
extracts from primary ATL cells were assayed for NF-B activation by
EMSA analysis using an NF-B consensus probe, and the results also
demonstrated constitutive, NF-B-specific DNA binding activity (data
not shown). Coimmunoprecipitation assays using anti-Tax Ab were
performed in control Jurkat and HTLV-I-transformed MT2 cells (Fig. 3B). As previously demonstrated (38), Tax
immunoprecipitation coprecipitated IKK-/NEMO in MT2 cells (Fig. 3B, lane 4) but not in Jurkat cells (Fig. 3B, lane 2).
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NF-AT transcription factors play a pivotal role in inducible gene
expression during the immune response. At least five structurally
related subsets of NF-AT proteins differentially expressed in various
classes of immune and nonimmune cells (39) have been
implicated in the direct regulation of cytokines, cell surface
receptors, and transcription factors (39, 40, 41).
HTLV-I-transformed and Tax-expressing T cell lines are characterized by
constitutively dephosphorylated, active NF-AT proteins in the nucleus
(42); a role for NF-AT proteins in mediating inducible
gene expression during the course of HTLV-I infection in T lymphocytes
has been established through analysis of the IL-2 CD28RE enhancer
element, which identified constitutive protein-DNA complexes containing
NF-AT proteins (43). To assess NF-AT DNA binding activity
in HTLV-I-infected MT2 and ATL cells (ATL2, Fig. 1
C,
lane 3), EMSA was performed using a consensus NF-AT
oligonucleotide probe. An inducible protein-DNA complex was detected in
MT2 and ATL2 cells (Fig. 4, A
and B, lane 2) but not in control Jurkat cells
(Fig. 4, A and B, lane 1). Supershift
analysis using NF-AT-specific Abs revealed that inducible complexes
were composed of NF-ATp (NF-AT1/NF-AT-c2) in MT2 extracts (Fig. 4A, lane 4) and NF-ATp/NF-ATc (NF-AT2/NF-ATc1) in
ATL2 extracts (Fig. 4B, lanes 3 and
4). Binding specificity of these complexes was confirmed by
competition using 100-fold molar excess of NF-AT consensus
oligonucleotide (Fig. 4, A and B, lane
11) and the corresponding mutant NF-AT probe (Fig. 4, A
and B, lane 12). Thus, in HTLV-I-transformed MT2
and ATL2 cells, NF-AT DNA binding activity is constitutive.
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B1 and B2 sites in
Jurkat and MT2 cells
Activation of IRF-4 expression by NF-B was studied by in vivo
genomic footprinting in HTLV-I-transformed cells. Primers were designed
to analyze in vivo protein-DNA interactions in the -600 to -400
region of the IRF-4 promoter containing the B1 and B2 binding
sites (primers 1, 2, and 3, Fig. 2B). Analysis was performed
on the noncoding strand because the B1 and B2 sites are G rich on
this strand. Protection of the B1 site was observed in MT2 cells but
not in control Jurkat cells (Fig. 5A, compare lanes 1
and 3) and comparison of the in vivo and naked DNA pattern
obtained from MT2 cells revealed decreased methylation of -429G,
-430G, and -431 G residues (Fig. 5A, lanes 2
and 3). Modifications with respect to the methylation
pattern within the B1 site of the IRF-4 promoter are represented by
densitometric scanning in Fig. 5A, upper right
panel, with arrows indicating the position of the three G residues
showing decreased methylation in MT2 cells. In contrast, no occupancy
of the B2 site was observed by in vivo genomic footprinting or EMSA
(data not shown) in Jurkat or MT2 cells. Interestingly, analysis of
this region also showed significant modification at the Ets-1 binding
site (-483 to -492). Significant hypermethylation of -487G and
-493G residues were observed specifically in MT2 cells, suggesting
protein-DNA binding at this site in MT2 cells (Fig. 5A,
compare lanes 2 and 3). Densitometry scanning of
in vivo footprinting within the Ets-1 site of the IRF-4 promoter is
represented in Fig. 5A, lower right panel, where
the arrows represent G residues showing increased methylation in vivo
in MT2 cells.
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B1 site in HTLV-I-infected
cells, EMSA experiments were performed using a probe corresponding to
the -447 to -421 region of the human IRF-4 promoter (Fig. 2B). Analysis of DNA binding activity in nuclear extracts
from MT2 cells and whole-cell extracts from ATL2 cells revealed subtle
differences in complex composition. Anti-c-Rel Abs supershifted the
majority of the upper complex detected in HTLV-I-infected MT2 cells
(Fig. 5B, upper panel, lane 3).
Supershift using anti-p50 revealed the upper complex to be a
p50/c-Rel heterodimer (Fig. 5B, upper panel,
lane 5). In ATL2, the upper complex did not react with
anti-c-Rel Abs (Fig. 5B, lower panel,
lane 3) but shifted with anti-p65 and p50 Abs (Fig. 5B, lower panel, lanes 5 and
6), indicating that the upper complex was a heterodimer of
p65/p50 and the middle complex was a p50 homodimer. Complex NS was
designated nonspecific, based on the failure of 100-fold molar excess
of cold B1 probe or antisera and consensus probes to NF-B, NFAT,
Sp-1, Ap-1, or Ets proteins to specifically compete for formation of
this complex (data not shown). B1 EMSA was also performed using
whole-cell extracts derived from primary human T lymphocytes, which
demonstrated inducible, specific binding to the B1 site upon T cell
stimulation with PMA/ionomycin (data not shown).
The requirement for NF-B in Tax-mediated transactivation of the
IRF-4 promoter was further investigated through luciferase analysis
performed in Jurkat T cells (Fig. 5C). Overexpression of
NF-B-inducing kinase resulted in a 3-fold induction of the 0.6-kb
IRF-4 promoter construct (Fig. 5C). The mutant Tax M47,
deficient in the activation of the CREB/ATF pathway, retained the
ability to transactivate 0.6-kbIRF4PRO-LUC similar to wild-type levels
(Fig. 5C), whereas Tax M22, which is defective in
NF-B activation, failed to stimulate the 0.6-kb IRF-4
promoter. Together, these results implicate NF-B signaling as a
necessary component of Tax-mediated activation of the IRF-4 promoter,
as demonstrated through constitutive in vivo occupancy of the B1
site in HTLV-I-infected cells.
Analysis of the Sp-1/NF-2 site in MT2 cells
A second set of primers for genomic footprinting was
designed to analyse the noncoding strand of the -260 to -460 region
of the IRF-4 promoter containing the CD28RE/NF-1, Sp-1/NF-2, and Ap-1
consensus binding sites (primers 4, 5, and 6, Fig. 2B).
Using these primers, protection of several G residues within and
adjacent to the Sp-1/NF-2 site (-385G to -413G) was observed in MT2
cells compared to the DNA control (Fig. 6A, lanes 2 and
3). Protection of the -388, -396 to -398, -402, -410,
-412, and -413 G residues within and around the Sp-1/NF-2 site was
specific to HTLV-I-transformed cells and was not detected in control
Jurkat cells (Fig. 6A, compare lanes 1 and
3). Changes in methylation within this site of the IRF-4
promoter in MT2 cells was further analyzed by densitometric scanning
(Fig. 6A, right panel), with arrows indicating
the eight G residues within and adjacent to NF-2/Sp-1 that exhibit a
decrease in methylation in MT2 cells.
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B). EMSA analysis revealed two inducible complexes binding
to the Sp-1/NF-2 probe in MT2 cells compared to control T4 cells (Fig. 6B, left panel, lanes 1 and
2). Competition analysis demonstrated that inducible
complexes were diminished by a consensus Sp-1 oligonucleotide (Fig. 6B, left panel, lane 6) but not with
the corresponding mutant oligonucleotide (Fig. 6B,
left panel, lane 7); furthermore, supershift with
Sp-1 Ab demonstrated that the upper complex was specifically displaced
(Fig. 6B, left panel, lanes 3 and
4). MT2-specific complexes were not removed by competition
with heterologous NF-AT or Ets oligonucleotides, nor were they
supershifted with NF-AT or Ets specific Abs (data not shown). EMSA with
Sp-1/NF-2 was also performed with primary ATL2 cell extracts (Fig. 6B, right panel) and primary T lymphocytes
treated with PMA/ionomycin (data not shown) with the same results.
These data indicate that occupancy of the Sp-1/NF2 region of the IRF-4
promoter in HTLV-I cells is mediated through Sp-1 binding to at least
two adjacent sites. Analysis of in vivo and in vitro occupancy of the CD28RE site in MT2 cells
The CD28-response enhancer element (CD28RE) plays an important
role in the regulation of gene expression during the T cell activation
response. CD28RE mediates activation of several lymphoid-specific genes
such as IL-2, GM-CSF, and CD40 ligand (44, 45, 46), through
binding of numerous transcriptional activators, including Fos/Jun
(AP-1), NF-B, and NF-AT proteins. Furthermore, the IL-2 CD28RE is a
direct target for HTLV-I Tax-mediated trans-activation
(39, 43, 47). IRF-4 promoter deletion analysis in MT2
cells indicated that the CD28RE element may function in activation of
IRF-4 expression in HTLV-I-infected cells (Fig. 2A).
Consistent hypermethylation of -270G residue adjacent to the CD28RE
site was observed in MT2 cells but not in Jurkat cells, suggesting
binding of one or several transcription factors involved in IRF-4
regulation near this site (Fig. 7A, lane 3).
Hypermethylation of the IRF-4 promoter adjacent to the CD28RE site was
quantified by densitometric scanning (Fig. 7A, right
panel); the arrow represents the -270 G residue and demonstrates
a reproducible increase in methylation that is specific to MT2 cells.
Further analysis of the coding strand would have been necessary to
provide a detailed characterization of promoter occupancy in the CD28RE
region (including the NF-1 site) of the IRF-4 promoter by in vivo
genomic footprinting. However, PCR amplification was not successful
using antisense primers because of the fact that the highly GC-rich
region impaired PCR-mediated elongation. Because of the low abundance
of G residues in the noncoding strand of the CD28RE site, no
modification of the methylation pattern was detected in either Jurkat
cells or in MT2 cells (Fig. 7A, lanes 1 and
3) compared to naked DNA.
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B, lane 2). Supershift analysis with
NF-B Abs identified c-Rel and p50 (Fig. 7B, lanes
3 and 4) but not p65 (Fig. 7B, lane
5) binding in MT2 cells. Because the CD28RE binds several
transcription factors, other competition analyses were performed,
demonstrating that the NF-AT-specific oligonucleotide (Fig. 7B, lane 6) but not the mutant oligonucleotide
removed a specific complex on the CD28RE element in HTLV-I-infected MT2
nuclear extracts (Fig. 7B, lane 7). Supershift
with NF-AT-specific Abs identified NF-ATp bound to the CD28RE in MT2
cells (Fig. 7B, lane 8). However, complexes bound
to the IRF-4 CD28RE did not react with Abs to AP-1, Sp-1, or Ets (data
not shown), indicating that the IRF-4 CD28RE binds both NF-B (c-Rel
and p50) and NF-AT (NF-ATp) in HTLV-I-infected cells. Using whole cell
extracts from primary human T lymphocytes, CD28RE-specific EMSA
demonstrated inducible and specific binding in whole-cell extracts
stimulated with PMA/ionomycin, suggesting that the IRF-4 CD28RE is
occupied in primary, activated T lymphocytes (data not shown). To
complement footprinting studies, EMSA experiments were performed using
DNA probes corresponding to each potential transcription factor
consensus sequence identified within the -367 to -209 region of the
IRF-4 promoter. In accordance with in vivo footprinting data, novel
protein-DNA complexes were not detected using DNA probes within this
region when comparing resting cells to HTLV-I-infected cells.
To further examine the potential synergy between NF-B and NF-AT in
IRF-4 promoter regulation, the 0.4-kb IRF-4 promoter construct lacking
the B1 and Sp-1 elements while retaining the CD28RE site was
analyzed in coexpression studies; CD28RE was induced 4-fold following
NIK expression (Fig. 7C) while expression of NF-ATpXS—a
constitutively active form of NF-ATp—induced the 0.4-kb promoter
3-fold. Coexpression of both NIK and NF-ATpXS together induced
7-fold induction of the 0.4-kb IRF-4 promoter fragment (Fig. 7C), confirming that NF-B and NF-AT synergize to
stimulate the CD28RE element. Wild-type Tax and Tax M47 transactivated
the 0.4-kb IRF-4 promoter 14- and 8-fold, respectively (Fig. 7C), while Tax M22 induced the 0.4-kb IRF-4 promoter
3-fold.
Mutation of B1 or CD28RE abrogates Tax-induced IRF-4 promoter
activity
To demonstrate the importance of B1, Sp-1, and CD28RE in the
regulation of IRF-4 activity, the 0.6-kb IRF-4 promoter was mutated or
deleted at these specific sites. Mutation of B1 blocked IRF-4
promoter activity in response to Tax expression (Fig. 8A), confirming the
requirement for this site for Tax-mediated IRF-4 activation. Although
mutation of the Sp-1 element reduced Tax-mediated IRF-4 fold activation
25% compared to the wild-type promoter, deletion of the CD28RE
resulted in a complete loss of IRF-4 promoter activity in response to
Tax (Fig. 8A). These data indicate that de novo IRF-4
induction in response to Tax expression requires both the B1 and
CD28RE sites of the human IRF-4 promoter.
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B1, Sp-1/NF-2, and
CD28RE elements of the IRF-4 promoter in HTLV-I-infected MT2 cells,
formaldehyde cross-linking and chIP assays (33) were
performed in IRF-4-expressing, HTLV-I-infected MT2 (Fig. 8B,
upper panel) and control Jurkat (Fig. 8B,
lower panel) cells. A 250-bp sequence, corresponding to the
B1 through CD28RE sites of the IRF-4 promoter, was PCR amplified
from DNA immunoprecipitated with Abs to NF-ATp, c-Rel, p-50, and Sp-1
(Fig. 8B, lanes 4–7), to a degree significantly
above control IRF-3 or no Ab samples. (Fig. 8B, lanes
2 and 4). Pulldown of IRF-4-specific regulatory
sequences was limited to MT2 cells and was absent from control Jurkat
cells, which demonstrated constitutive binding of specific
transcription factors to the B1, Sp-1/NF-2, and CD28RE sites in
HTLV-I-infected cells. These results are summarized in a schematic
representation of human IRF-4 promoter occupancy in HTLV-I-infected
cells (Fig. 8C). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B1, Sp-1, and CD28RE enhancer elements. Transient coexpression
assays indicate that two of the sites, B1 and CD28RE, are absolutely
essential for Tax-mediated trans-activation of the human
IRF-4 promoter T cells.
Gene knockout analysis has demonstrated that c-Rel is essential for
IRF-4 production in primary murine B and T lymphocytes
(48). IRF-4 expression was completely blocked in response
to Ag-mimetic CD3/CD28 stimulation in c-Rel-deficient lymphocytes, and
c-Rel was shown to bind B enhancers within the murine IRF-4 promoter
(48). Our data extend these studies and establish an
important role for NF-B in HTLV-I-induced activation of the human
IRF-4 promoter in ATL cells. Tax-mediated activation of the IKK complex
in HTLV-I-transformed cells correlates with constitutive in vivo
occupancy at the B1 site of the IRF-4 promoter that is not occupied
in control Jurkat cells. B1 is a classical NF-B sequence that
binds p65/p50 or c-Rel/p50 complexes in primary ATL and MT2 extracts,
respectively. Furthermore, abrogation of IRF-4 promoter activity in
response to Tax M22 expression indicates that NF-B activity is
absolutely required for Tax-mediated IRF-4 gene induction in
HTLV-I-infected T lymphocytes.
The CD28RE site of the IRF-4 promoter functions as a more complex regulatory element, and our results are consistent with previous studies examining the CD28RE within the context of the IL-2 promoter. The CD28RE is essential for IL-2 production by CD3/CD28 stimulation (43, 49) and functions as a Tax-responsive element in HTLV-I-induced IL-2 production (46, 47). CD28RE binds several different transcriptional regulatory proteins (46, 50, 51, 52). In the present study, the IRF-4 CD28RE interacted with c-Rel and p50, as demonstrated through EMSA analysis and chIP in HTLV-I-infected cells. Furthermore, we have correlated constitutive NF-AT DNA binding activity in the MT2 cell line and primary leukemic ATL cells with occupancy of the IRF-4 CD28RE by NF-ATp in HTLV-I-infected cells. Overexpression of NF-ATp in Jurkat T cells synergizes with NIK to activate the CD28RE of the human IRF-4 promoter in the reporter gene assay, indicating that these two factors are involved in IRF-4 CD28RE trans-activation.
HTLV-I Tax-mediated induction of cellular gene expression is required
during the early stages of viral pathogenesis for the establishment of
infection. Hence, HTLV-I infection of a mature CD4+ T
lymphocyte is directly associated with up-regulation of numerous
cellular genes involved in T cell activation and proliferation, such as
IL-2, IL-2R, IL-15, c-fos, and Fas ligand (23, 53, 54, 55).
The classical example of this phenomenon is HTLV-I-associated induction
of the IL-2 gene, a hallmark of the early phase of HTLV-I infection
(22, 56, 57). IL-2, as well as IL-15, induction by the Tax
oncoprotein is believed to initiate an autocrine/paracrine loop that
drives the polyclonal proliferation of HTLV-I-infected cells (26, 58, 59). IL-dependent proliferation during the early stages of
infection may facilitate the accumulation of multiple genetic mutations
that contribute to the transition from immortalization to
transformation (26, 58, 59).
Regulation of the IRF-4 gene in T lymphocytes displays striking
similarities to that of the IL-2 promoter. IRF-4 expression is
inducible through stimuli that mimic TCR engagement by Ag, such as
PMA/ionomycin, con A, or anti-CD3/CD28 treatment (10, 11, 18). Induction of IRF-4 expression in primary T cells is also
blocked by treatment with the immunosuppressive drug FK506, which
suggests that changes in intracellular calcium concentration—an early
feature of T cell activation—is required for IRF-4 induction. The
parallels between IL-2 and IRF-4 expression imply that their continuous
production within the context of HTLV-I infection may involve common
mechanisms. Like IL-2, IRF-4 expression in HTLV-I-transformed cells is
Tax dependent, and transient transfection of the tax gene
induces low levels of IRF-4 expression in Jurkat T cells
(11). In this study we have demonstrated that
overexpression of Tax in Jurkat T cells stimulates transcriptional
activity within the -617 to -209 region of the human IRF-4 promoter
through the B1, Sp-1, and CD28RE sites.
Continuous production of IRF-4 in HTLV-I-transformed cells suggests that IRF-4 may contribute to virus-induced leukemogenesis. In fact, studies examining the physiological role of IRF-4 in T cells are consistent with such an interpretation. Knockout analysis revealed that the IRF-4 gene is essential for the function and homeostasis of mature B and T lymphocytes. Although mice lacking IRF-4 displayed a normal distribution of mature B and T cells in the periphery at 4–5 wk of age, the lymphocyte population was severely impaired at the level of late-stage activation events. IRF-4-/- T lymphocytes maintained normal early activation responses such as calcium influx and expression of CD25 and CD69, but exhibited a dramatic reduction with respect to proliferation and cytokine production in response to anti-CD3 stimulation, con A, or bacterial superantigen staphylococcal enterotoxin A treatment (60). This phenotype could not be rescued by exogenous IL-2 treatment, which suggests that IRF-4 may be involved during the later stages of the T cell activation response. A role for IRF-4 expression in the development of certain lymphomas was further emphasized through a link to multiple myeloma, a hyperproliferative disorder of terminally differentiated B cells. Multiple myeloma has been associated with a t(6;14)(p25;q32) translocation that juxtaposes the IgH chain locus regulatory region to the IRF-4 coding sequence (61). As a result, MUM/IRF-4 is overexpressed, an event that has been implicated in leukemogenesis because IRF-4 overexpression is capable of transforming Rat-1 fibroblasts in vitro (62). Tax-driven IRF-4 expression in HTLV-I-infected cells suggests a role for IRF-4 in reprogramming T cell gene expression. Constitutive IRF-4 expression in HTLV-I-infected cells may reprogram T cell-specific gene expression; further studies will identify and characterize IRF-4 target genes in an effort to further characterize the role of the IRF-4 transcription factor in HTLV-I pathogenesis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John Hiscott, Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T 1E2. E-mail address: john.hiscott{at}mcgill.ca
3 Abbreviations used in this paper: IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; ATL, adult T cell leukemia; DBD, DNA binding domain; HTLV-1, human T cell leukemia virus type I; HAM, HTLV-I-associated myelopathy; TSP, tropical spastic paraperesis; DMS, dimethyl sulfate; chIP, chromatin immunoprecipitation; Sp-1, stimulating factor 1; PKR, dsRNA activated protein kinase; ISG, IFN-stimulated gene; NIK, NF-B-inducing kinase; ICSBP, IFN consensus binding protein; IKK, IB kinase.
Received for publication April 11, 2002. Accepted for publication July 17, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene regulatory elements. Cell 54:903.[Medline]
-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes. Proc. Natl. Acad. Sci. USA 87:3743.B by the tax transforming protein of HTLV-1: hijacking cellular IB kinases. Oncogene 18:6948.[Medline]
