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RAPID COMMUNICATION
Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells
By Steve N. Georas, John E. Cumberland, Thomas F. Burke, Rongbing Chen,
Ulrike Schindler, and Vincenzo Casolaro
The differentiation of naive T-helper (Th) cells into cytokinesecreting effector Th cells requires exposure to multiple
signals, including exogenous cytokines. Interleukin-4 (IL-4)
plays a major role in this process by promoting the differentiation of IL-4–secreting Th2 cells. In Th2 cells, IL-4 gene
expression is tightly controlled at the level of transcription
by the coordinated binding of multiple transcription factors
to regulatory elements in the proximal promoter region.
Nuclear factor of activated T cell (NFAT) family members play
a critical role in regulating IL-4 transcription and interact
with up to five sequences (termed P0 through P4) in the IL-4
promoter. The molecular mechanisms by which IL-4 induces
expression of the IL-4 gene are not known, although the
IL-4–activated transcription factor signal transducer and
activator of transcription 6 (Stat6) is required for this effect.
We report here that Stat6 interacts with three binding sites
in the human IL-4 promoter by electrophoretic mobility shift
assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4
transcription, we used Stat6-deficient Jurkat T cells with
different intact IL-4 promoter constructs in cotransfection
assays. We show that, whereas a multimerized response
element from the germline IgE promoter was highly induced
by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4
promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on
promoter context and that this factor promotes IL-4 gene
expression in an indirect manner.
r 1998 by The American Society of Hematology.
I
located in the promoter regions of many IL-4–responsive
genes.21,22 The fundamental role of Stat6 in IL-4–driven responses was demonstrated by the phenotype of Stat6-deficient
mice in which IgE synthesis and Th2 responses were abrogated.23-25 Lederer et al11 discovered a Stat6 binding site in the
mouse IL-4 promoter and found that Stat6 bound this sequence
in EMSA using nuclear extracts from IL-4 induced Th2 cells but
not Th1 cells. We and others identified a corresponding site in
the human IL-4 promoter (2169TTCACAGGAA2160).26,27 Because multimers of these elements were inducible by IL-4 when
linked to heterologous promoters and transfected into Stat6expressing B-cell lines,11,26 it seemed reasonable to conclude
that Stat6 would directly enhance IL-4 transcription in T cells.
However, recent studies have suggested that activation of
IL-4R signaling pathways is not required for IL-4 gene expression in effector T cells. For example, Huang et al28 found that
IL-4 did not enhance transcription driven by a mouse IL-4
promoter construct in anti-CD3–activated Th2 cells, although
the specific role of Stat6 in regulating the intact IL-4 promoter
was not examined in that study. Additionally, Moriggl et al29
reported that a neutralizing anti–IL-4 monoclonal antibody
(MoAb) actually enhanced anti-CD3–induced IL-4 production
NTERLEUKIN-4 (IL-4) is a prototypic immunoregulatory
cytokine.1 By virtue of its ability to induce IgE isotype
switching in B cells, mast cell differentiation, and adhesion
molecule expression, IL-4 plays a central role in many inflammatory responses.2-4 IL-4 is also the primary cytokine promoting the differentiation of naive T cells into cytokine-secreting
T-helper 2 (Th2) cells.5 Cytokine gene expression in Th2 cells is
controlled primarily at the level of gene transcription,6 and
dysregulation of this process is thought to contribute to the
development of allergic diseases.7 Several transcription factors
have recently been implicated in regulating Th2-restricted IL-4
gene expression (Fig 1).8-13 Nuclear factors of activated T cell
(NFAT) are involved in this process by interacting with up to
five sites in the IL-4 promoter (termed P0 through P4).14,15 The
precise role of individual NFAT family members in regulating
IL-4 transcription is currently unknown.16-18
Using T-cell lines derived from NFAT-reporter transgenic
mice, Rincón and Flavell10 found that NFAT transcriptional
activity is preferentially induced in Th2 cells but not in Th1
cells. Growing evidence suggests that Th2-specific NFAT
cofactors may specifically enhance IL-4 transcription in these
cells. For example, Ho et al9 found that the proto-oncogene
c-maf was preferentially expressed in Th2 clones and that c-Maf
acts synergistically with NFATp to induce IL-4 production in
IL-4–negative cells. Additionally, Li-Weber et al12 detected a
multiprotein complex containing C/EBP, NFAT, and AP-1
proteins forming on the P4 element using nuclear extracts from
Th2 but not Th1 cells in electrophoretic mobility shift assays
(EMSA).
Exogenous IL-4 is a critical stimulus for the effective
differentiation of naive T cells into IL-4–secreting Th2 cells (for
review, see O’Garra5). The mechanism by which IL-4 induces
expression of the IL-4 gene is currently not known. IL-4
interacts with a multichain cell-surface receptor (IL-4R) that is
expressed by several cell types, including B cells, T cells, and
endothelial cells.4,19 IL-4 binding to the IL-4Ra chain induces
different intracellular signals, including Jak-mediated phosphorylation of the transcription factor Stat6.20 Stat6 response
elements share a consensus sequence 58TTCN3/4GAA38 and are
Blood, Vol 92, No 12 (December 15), 1998: pp 4529-4538
From the Divisions of Pulmonary and Critical Care Medicine and
Allergy and Clinical Immunology, The Johns Hopkins University
Asthma and Allergy Center, Baltimore, MD; and Tularik Inc, South San
Francisco, CA.
Submitted July 2, 1998; accepted October 5, 1998.
Supported by Grant No. AI01152 from the National Institutes of
Health and Research Grant No. 056-N from the American Lung
Association.
Address reprint requests to Steve N. Georas, MD, Room 4B.41, The
Johns Hopkins Asthma & Allergy Center, 5501 Hopkins Bayview Circle,
Baltimore, MD 21224; e-mail: [email protected].
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9212-0046$3.00/0
4529
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4530
GEORAS ET AL
Fig 1. The proximal IL-4 promoter (not drawn to scale). NFAT
binding sites (the P elements)
are indicated by open boxes, and
the known Stat6 site is indicated
by the solid box. Transcription
factors implicated in Th2-specific IL-4 gene expression are indicated below their respective
binding sites. A binding site for
GATA-3 has not yet been reported.13 NFAT activity is higher
in effector Th2 cells (‘‘Activated’’
NFAT10) and is shown for simplicity binding only to the P1 NFAT
site.
in committed Th2 cells. Using Stat6-deficient Jurkat T cells in
cotransfection assays, we report here that, although cotransfected Stat6 strongly enhanced transcription driven by a multimerized response element, the human IL-4 promoter was
significantly repressed under similar conditions. The repressive
effects of Stat6 appeared to involve sequence-specific DNA
binding, because a Stat6 DNA binding domain mutant failed to
inhibit the IL-4 promoter. We describe two novel Stat6 binding
sites within the proximal IL-4 promoter and show that Stat6 and
NFAT bind competitively to overlapping nucleotides.
MATERIALS AND METHODS
Plasmid construction. IL-4 promoter constructs were amplified
from human genomic DNA using the polymerase chain reaction (PCR).
A 25-bp 58 primer annealing 265 bp upstream from the transcription
start site (tss) according to Otsuka et al30 was used with a 25-bp 38
primer ending at position 165. PCR products were sequenced using the
dideoxy method and then ligated into the HindIII and Xba I sites of
pCAT Basic (Promega, Madison, WI) to yield pCAT 265. The reporter
C/EBP-N4 luc contains 4 copies of the composite C/EBP/Stat6
response element from the germline e promoter fused to a thymidine
kinase (TK) minimal promoter driving the firefly luciferase gene.21 The
wild-type Stat6 expression vector (TPU 388) and the DNA-binding
domain mutant vector (TPU522, in which the 3 amino acids VVI at
positions 411 to 413 were replaced by EAA), both driven by the
cytomegalovirus (CMV) promoter, have been described.21
Cell lines and transfections. Jurkat T cells (a kind gift of Dr Jack
Strominger, Harvard University, Cambridge, MA) were maintained in
complete medium (RPMI 1640 supplemented with 10% heatinactivated fetal calf serum [FCS; Life Technologies, Gaithersburg,
MD] and 50 µg/mL gentamycin [Life Technologies]). As previously
reported, these cells constitutively express IL-4 mRNA.31 HepG2 cells
were obtained from the ATCC (Rockville, MD) and maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% FCS and 50 µg/mL gentamycin. In cotransfection experiments, 3
3 106 cells were transfected with 1 µg reporter, 2 µg expression or
empty vector as a control, and 7 µL Superfect (Qiagen, Valencia, CA) in
5 mL complete medium and allowed to recover for 24 hours. Cells were
then stimulated with combinations of the following agonists as indicated in the text for the last 18 hours: 1 µmol/L calcium ionophore
(A23187; Calbiochem, San Diego, CA), 25 ng/mL phorbol-12-myristate13-acetate (PMA; Calbiochem), and IL-4 (10 or 50 ng/mL; Peprotech,
Rocky Hill, NJ). Cells were lysed by three freeze-thaw cycles and
reporter gene expression was determined either by measuring CAT
enzyme levels using a sensitive enzyme-linked immunosorbent assay
(ELISA; Boehringer Mannheim, Indianapolis, IN) or by assaying for
luciferase activity using standard techniques (Analytic Luminescence
Laboratories, Sparks, MD). Cell extracts were normalized for protein
content using the Bradford technique (Bio-Rad, Hercules, CA) before
assays for reporter gene expression.
EMSA. The following 30-bp oligonucleotides and their complements
were synthesized (mutations in the P2 oligonucleotide are indicated as
lowercase letters, and the Stat6 consensus sequence is underlined): 58ATTGCTGAAACCGAGGGAAAATGAGTTTACAT- TG-38 (P0 269 to
236); 58-TGAGTTTACATTGGAAATTTTCGTTACACCAGATTG-38 (P1
292 to 260); 58-TCTGATTTCACAGGAACATTTTACCTGTTT-38 (P2
wt 2175 to 2146); 58-gagac-TTTCACAGGAACATTTTACCTGTTT-38
(P2 m1); 58-TCTGAgggacCAGGAACATTTTACCTGTTT-38 (P2 m2);
58-TCTGATTTCAgctcgACATTTTACCTGTTT-38 (P2 m3); 58-TCTGATTTCACAGGActcggTTACCTGTTT-38 (P2 m4); 58-TCTGATTTCACAGGAACATTTgcgtt-TGTTT-38 (P2 m5); 58-TCTGATTTCACAGGAACATTTTTACCcaccg-38 (P2 m6); 58-AATCAGACCAATAGGAAAATGAAACCTTTTTAA-38 (P3 2201 to 2169); and 58-AGTTTCAGCATAGGAAATTACACCATAATTTGC-38 (P4 2248 to 2216).
The Bcl-6 oligonucleotide (B6BS: 58-GAAAATTCCTAGAAAGCATA-38; donated by Dr Riccardo Dalla-Favera, Columbia University,
New York, NY) has been described.32 Nuclear extracts were obtained
from 5 3 106 Jurkat cells treated without or with IL-4 (20 ng/mL for 20
minutes; Peprotech) using the method of Schrieber et al.33 EMSAs were
performed using 5 µg nuclear protein, 0.8 µg poly (dG-dC) (Amersham
Pharmacia Biotech, Piscataway, NJ), and [g32P] end-labeled probe in a
final volume of 10 µL per reaction. Free probes and protein-DNA
complexes were resolved by 5% polyacrylamide gel electrophoresis
(PAGE) with 0.53 TBE. In antibody experiments, extracts were
incubated at room temperature with 1 µL of the following antisera for 30
minutes after the addition of labeled probe: anti-Stat6 (Santa Cruz
Biotech, Santa Cruz, CA), N70-6 (anti–Bcl-632; donated by Dr Riccardo
Dalla-Favera), or isotype-matched control antisera.
Recombinant proteins. A recombinant fragment of murine NFATp
(including 298 amino acids of the DNA binding domain [DBD] that is
highly conserved among different NFAT family members34) was
expressed as a hexahistidine-tagged protein and extracted as described.35 The NFATp expression vector was kindly donated by Dr
Anjana Rao (Harvard University). Recombinant full-length, in vitro
phosphorylated Stat6 has been described.21
Statistical analysis. All transfections were performed in duplicate
using cells of similar passage number. Average results of the indicated
numbers of independent experiments were analyzed using the paired
Student’s t-test (Statview II Software; SAS Institute, San Francisco,
CA), and a P value less than .05 was considered to be statistically
significant.
RESULTS
The IL-4R signaling pathway is intact in Jurkat T cells. To
investigate the role of Stat6 in regulating transcription of the
intact IL-4 promoter, we studied Jurkat T cells transiently
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Stat6 INHIBITS THE INTERLEUKIN-4 PROMOTER
transfected with different IL-4 promoter reporter constructs. In
preliminary experiments, we did not detect immunoreactive
Stat6 in nuclear extracts from IL-4–activated Jurkat cells in
EMSA, suggesting that the cells used in these experiments
express negligible levels of this factor (not shown). Consistent
with this result, IL-4 stimulation alone did not induce a
full-length IL-4 promoter construct (not shown) or the reporter
construct C/EBP-N4 luc (which contains 4 copies of the IgE
C/EBP/Stat6 element driving the firefly luciferase gene; Fig
2A). However, as previously reported,21 C/EBP-N4 luc was
strongly induced by IL-4 in Stat6-expressing HepG2 cells
(Fig 2B).
To verify that the IL-4R signaling pathway was otherwise
functional in Jurkat cells, we first analyzed the inducibility of
C/EBP-N4 luc in cells cotransfected with a full-length Stat6
expression vector. Activity of C/EBP-N4 luc is strongly dependent on the coordinated binding of C/EBP and Stat6 to adjacent
sites.21 Figure 2A shows that C/EBP-N4 luc was highly induced
by IL-4 in Stat6-expressing Jurkat cells, but not in control cells
cotransfected with the corresponding empty vector. This result
confirms the known expression of C/EBP proteins by Jurkat
cells.36 Consistent with previous findings,21 C/EBP-N4 luc was
induced by IL-4 in HepG2 cells in the absence of exogenous
Stat6 (Fig 2B). Thus, although Jurkat cells do not constitutively
express functional Stat6 protein, cotransfected Stat6 is highly
induced by IL-4 in these cells.
4531
Stat6 represses transcription driven by the intact IL-4
promoter. We next analyzed the ability of Stat6 to transactivate the intact IL-4 promoter. Figure 3 shows that a full-length
human IL-4 promoter construct (pCAT 265) was consistently
inhibited by IL-4 in Stat6-cotransfected Jurkat cells. To determine whether unstimulated Jurkat cells lacked an activationinduced Stat6 cofactor or whether Stat6 needed a further
activation signal, which is necessary for IL-4 transcription, we
next analyzed the effects of IL-4 in activated cells cotransfected
with Stat6 and pCAT 265. As previously reported,37,38 a
calcium-dependent signal alone was sufficient to maximally
induce the IL-4 promoter (Fig 3). PMA downregulated IL-4
promoter activity, which we previously found was due to the
displacement of NFATp from the human P1 sequence by
induced nuclear NF-kB heterodimers.37 Interestingly, IL-4
consistently inhibited calcium-induced promoter activity in
Stat6-cotransfected cells and almost completely repressed the
promoter in combination with PMA (Fig 4). Thus, even in
conjunction with activation of intracellular calcium and PKCsignaling pathways, Stat6 inhibited transcription driven by the
full-length IL-4 promoter.
To map the IL-4 promoter element(s) necessary for Stat6mediated transcriptional repression, we used a smaller promoter
construct in additional cotransfection experiments. This construct (pCAT 145) contains 145 bp of the human promoter,
including the P1 and P0 NFAT elements, but lacks the previ-
Fig 2. The IL-4 receptor signaling pathway is intact in Jurkat T cells. (A) The luciferase reporter construct C/EBP-N4 luc (see text) was
transiently transfected into Jurkat cells together with a control (Empty) or Stat6 expression vector. Cells were then stimulated without (h) or
with (j) IL-4 (50 ng/mL) for 18 hours before assays for reporter gene expression. In the absence of either cotransfected Stat6 or IL-4 stimulation,
C/EBP-N4 luc is not active in Jurkat cells, but it is highly inducible by IL-4 in cells expressing Stat6. (B) Consistent with the known expression of
Stat6 by Hep G2 cells,21 C/EBP-N4 luc was induced by IL-4 in these cells, but its activity was further increased by overexpressing Stat6 (note the
different scales). Results are the mean 6 SEM of two (B) or three (A) independent experiments.
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4532
GEORAS ET AL
Fig 3. Stat6 inhibits transcription driven by the
intact IL-4 promoter. pCAT 265, which contains all of
the known NFAT P elements including the P2 Stat6
site (solid box), was transfected into unstimulated
Jurkat cells together with a control (Empty) or a
Stat6 expression vector, and the cells were incubated without (h) or with (j) IL-4 (50 ng/mL) for 18
hours before assays for reporter gene expression by
ELISA. Results are expressed relative to the constitutive activity of pCAT 265 without IL-4 and are the
mean 6 SEM of four independent experiments. IL-4
significantly downregulated promoter activity only
in Stat6-cotransfected cells.
ously described Stat6 binding site (2169TTCACAGGAA2160).
Interestingly, pCAT145 activity was significantly inhibited by
IL-4 in both resting and stimulated Stat6 cotransfected cells
(Fig 5). Importantly, an expression vector encoding a Stat6
DNA binding domain mutant (TPU522, see Materials and
Methods) did not inhibit pCAT145 activity in either resting or
activated cells (Fig 5, right side). These results suggested that
Stat6-induced repression involved binding sites located downstream of the known Stat6 sequence and that the DNA binding
ability of Stat6 was required for this effect.
The human IL-4 promoter contains multiple overlapping
Stat6 and NFAT binding sites. The previously described Stat6
binding site is contained within the P2 NFAT element (Fig 6).
We and others have shown that this element can support the
cooperative binding of NFAT and AP-1 proteins.39,40 As shown
in Fig 6, the 38-half of the Stat6 site (58GAA38) overlaps the
58-end of the NFAT site (58GGAA38). In view of our functional
data showing repression of a construct lacking the P2 element
(Fig 5), we speculated that Stat6 could interact with additional P
elements from the IL-4 promoter. To test this hypothesis, we
analyzed the ability of recombinant Stat6 to bind similar length
oligonucleotides including the five known IL-4 P elements by
EMSA. Figure 7 shows that recombinant Stat6 bound 30-bp
oligonucleotides containing the P1, P2, and P4 NFAT sites, but
not the P0 or P3 sites.
Based on sequence homology and mutational analysis (Fig
6), we predicted that Stat6 and NFAT would bind competitively
to overlapping sites in the IL-4 promoter. To test this hypothesis
and to exclude any combinatorial interactions of these factors
on the IL-4 promoter, we analyzed the effect of Stat6 on the
ability of the NFATp DBD (see Materials and Methods) to
interact with oligonucleotides containing the P1 and P2 elements in EMSA. As expected, NFATp readily bound both
probes (Fig 8). Interestingly, increasing amounts of Stat6
displaced NFATp from its cognate sites on both oligonucleotides. Displacement of NFAT from the P1 element by Stat6 may
provide an explanation for the repressive effects of Stat6 on
pCAT 145.
DISCUSSION
The molecular basis by which IL-4 induces the production of
Th2 cytokines is currently not known. Experiments with
Stat6-deficient mice have conclusively demonstrated a requirement for this IL-4–inducible transcription factor during the
differentiation of naive T cells into Th2 cells.23-25 However,
recent studies have found that activation of the IL-4R-signaling
pathway is not required for the induction of IL-4 gene expression in committed Th2 cells.28,29,41 Additionally, Stat6 can
repress IL-4 gene expression in Th1 cells by binding a cell-type
specific silencer in the 38 untranslated region (UTR).42 Thus, the
precise role of Stat6 in regulating IL-4 expression is currently
not clear.
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Stat6 INHIBITS THE INTERLEUKIN-4 PROMOTER
4533
Fig 4. Stat6 does not synergize with other signals
to activate the IL-4 promoter. Methods were similar
to those described in Fig 3, except that cells were
also stimulated with calcium ionophore (L), PMA
(j), both (O), or no agonists (h) with or without IL-4
as indicated. In the presence of IL-4, reporter activity
was consistently decreased in Stat6-cotransfected
cells for each condition examined. Note that the
combination of PMA and IL-4 almost completely
repressed the promoter. Results are from one experiment performed in duplicate and are representative
of three.
Th2-restricted IL-4 gene expression is thought to be controlled at the level of transcription by the coordinated interactions of transcription factors binding to a proximal promoter
region. Regulatory elements within the promoter have been
shown not only to bind nuclear factors unique to Th2 cells, but
also to be preferentially induced in Th2 cells.8-13 The observation that Stat6 can interact with a consensus sequence from both
the mouse11 and human26 IL-4 promoters suggested that this
Fig 5. Stat6 inhibits a minimal IL-4 promoter
construct. Jurkat cells were cotransfected with a
minimal promoter construct lacking the known P2
Stat6 element (pCAT 145) with or without a wildtype (wt) or DNA-binding domain mutant (DBD mut)
Stat6 expression vector as indicated. Cells were
stimulated for 18 hours with calcium ionophore (L)
with or without IL-4 as indicated, followed by cell
lysis and analysis of reporter gene expression by
ELISA. Results are expressed relative to CAT production in unstimulated cells and are the mean 6 SEM of
four (DBD mut) or seven (wt) independent experiments. *P F .05.
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4534
GEORAS ET AL
B
Fig 6. The P2 element contains overlapping binding sites for Stat6 and NFATp. (A) Alignment of the human IL-4 P2 NFAT element with
canonical binding sites for NFAT (overline) and Stat6 (underline). Note that the 38-end of the Stat6 sequence overlaps the 58-end of the NFAT site.
(B) The ability of recombinant Stat6 to interact with wild-type (wt) and mutated probes (see Materials and Methods) was determined by EMSA.
Stat6 no longer bound the m2-m4 oligonucleotides, confirming the overlapping nature of the Stat6 and NFAT binding sites.
factor might provide a direct link between IL-4R activation and
IL-4 gene expression. However, a conclusion of our studies is
that Stat6 might only facilitate IL-4 gene expression in T cells in
an indirect fashion.
This report, showing for the first time the specific effect of
Stat6 on the intact IL-4 promoter, contains several novel
observations. First, we found that, although IL-4 receptormediated signals are faithfully transduced in Jurkat T cells (Fig
2), Stat6 was unable to transactivate the proximal human IL-4
promoter in these cells (Figs 3 through 5). Together with
previous studies showing that IL-4 can induce multimers of the
P2 Stat6 site linked to heterologous promoters,11,26,28 our
findings suggest that the transactivation potential of Stat6 is
dependent on promoter context. This conclusion is in keeping
with recent analyses of Stat6 and the germline IgE43 and
b-casein genes44 and with the study of Huang et al,28 who found
that Stat6 differentially regulated multimers of the P2 Stat6
element in M12 B cells depending on the minimal promoter
construct used.
Second, we found that transcription driven by two deletion
constructs of the human IL-4 promoter was consistently inhibited by IL-4 in Stat6-expressing Jurkat T cells. Interestingly,
recent studies suggest that IL-4 can inhibit IL-4 gene expression
in a negative feedback fashion. For example, Moriggl et al29
found that committed Th2 cells secreted more IL-4 when
restimulated with anti-CD3 in the presence of a neutralizing
anti–IL-4 MoAb. Additionally, IL-4 seemed to inhibit anti-CD3–
induced transcription driven by the full-length mouse IL-4
A
Fig 7. The IL-4 promoter contains multiple Stat6
binding sites. (A) The ability of recombinant Stat6 to
bind oligonucleotides containing the human P elements was determined by EMSA. Stat6 bound the
P1, P2, and P4 probes (solid arrow). An additional
slowly migrating complex (open arrow) formed on
the P1 oligonucleotide (and occasionally on the P2
probe; see Fig 8). The relative migration of the free
probes, which were of similar length (see Materials
and Methods) and radiolabeled with similar specific
activity, is not shown in this figure. (B) Alignment of
the P elements that supported Stat6 binding with
the composite C/EBP-Stat6 site from the germline
IgE promoter. The P2 oligonucleotide is shown in
opposite orientation than in Fig 2. Binding sites for
NFAT, Stat6, and C/EBP are indicated by boxes.
C/EBP proteins may interact with the P0, P1, and P4
NFAT elements, although the precise nucleotide binding sites have been reported only for the P4 sequence.36
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Stat6 INHIBITS THE INTERLEUKIN-4 PROMOTER
4535
Fig 8. Stat6 binds competitively with NFAT to the
P1 and P2 elements. (A) The ability of a recombinant
fragment of NFATp (see Materials and Methods) to
bind oligonucleotide probes containing the P1 and
P2 elements in the presence of increasing concentrations of recombinant Stat6 was determined by EMSA.
The relative mobilities of each factor are indicated.
Serial twofold dilutions of Stat6 were examined
against a constant amount of NFATp. n.s., nonspecific. (B) The relative intensities of observed bands
were analyzed by densitometry and expressed relative to intensity of the NFAT complex for each
oligonucleotide in the absence of Stat6 (which was
defined as 1).
promoter in a Th2 clone.28 Taken together, these results suggest
that activated Stat6 might downregulate IL-4 expression in
effector Th cells and emphasize the need to distinguish between
Th2 differentiation and IL-4 gene expression (see below).
Third, we have identified two novel Stat6 binding sites
(within the P1 and P4 elements) in the IL-4 promoter. Although
these sites do not contain the consensus Stat6 binding site
(58-TTCN4GAA-38) defined by binding site selection assays, a
significant fraction (10/42) of sequences selected by Stat6 in
these assays contained single nucleotide substitutions within the
dyad half-sites.45 Thus the ability of Stat6 to bind the P1
oligonucleotide (58-TTCN4GTA-38) is not surprising. The reason why Stat6 bound the P4 element but not the P0 element is
less apparent, because they contain similar noncanonical dyad
half sites, although they do differ in the spacer region.
Fourth, the demonstration that Stat6 and NFAT can bind
competitively to the IL-4 promoter provides evidence of a
previously unreported interaction between these two factors.
Given the fundamental role of NFAT family members in
regulating IL-4 gene expression, this observation provides a
plausible explanation for the observed inhibitory effects of Stat6
on transcription driven by the IL-4 promoter. This would be
similar to the recently described antagonism of NF-kB by Stat6
in the E-selectin promoter.46 Because the P1 element in
particular plays a major role in activating IL-4 transcription,8,47
competition by Stat6 for NFAT binding to this element might
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4536
have particularly repressive effects. Alternatively, we cannot
exclude the possibility that Stat6 might actively repress the
basal transcription complex or that it might also bind to and
titrate away from the promoter a factor necessary for maximal
IL-4 transcription.
Stat6 might require a coactivator not expressed by Jurkat
cells to maximally transactivate the IL-4 promoter. For example, Stat6 cooperates with both C/EBP21 and NF-kB/Rel
proteins48 to promote germline IgE transcription. The fact that
C/EBP-N4 luc is highly induced (Fig 2) argues that the inability
of Stat6 to activate IL-4 transcription in our experiments is not
due to the lack of C/EBP proteins and confirms the known
expression of C/EBP in Jurkat cells.36 Additionally, we have
shown that PMA-activated Jurkat cells contain abundant nuclear
NF-kB.37 We cannot formally exclude the requirement for as yet
unidentified Stat6 coactivators necessary for IL-4 transcription,
although the studies by Huang et al28 argue against the existence
of such factors in a differentiated Th2 clone. Another possibility
is that Stat6 might require additional posttranslational modification to achieve full transcriptional competency in Jurkat cells.
However, the observations that (1) PMA enhanced the repressive effects of Stat6 (Fig 4) and (2) C/EBP-N4 luc was inducible
by IL-4 in Stat6-cotransfected Jurkat cells (Fig 2) argue against
this explanation.
Finally, it is possible that other factors inhibit the transactivation potential of Stat6 on the IL-4 promoter. For example, it has
recently been suggested that Bcl-6, a transcriptional repressor
deregulated in many lymphomas,32,49 can specifically inhibit the
ability of Stat6 to transactivate gene expression. In fact,
Bcl-6–deficient mice were found to have enhanced IL-4 production and Th2 responses.50 To exclude the possibility that Bcl-6
was inhibiting transactivation of the IL-4 promoter by Stat6 in
Jurkat cells, we assayed for Bcl-6 binding to both the P2
element and a consensus Bcl-6 site using Jurkat nuclear extracts
in EMSA. Using conditions known to support Bcl-6 binding,32
we did not detect Bcl-6 using two specific antisera (not shown).
Additionally, IL-4 gene expression was not inhibited in Jurkat
cells expressing inducible Bcl-6 protein (Dr Riccardo DallaFavera, personal communication, Spring 1998). Thus, we
conclude that the inability of Stat6 to activate IL-4 transcription
in Jurkat T cells is not due to the presence of the specific Stat6
antagonist Bcl-6.
The IL-4R transduces other signals in addition to the
Jak-mediated tyrosine phosphorylation of Stat6. For example,
the I4R motif of the IL-4Ra subunit leads to the IRS-1–
dependent phosphorylation of the nonhistone chromosomal
protein HMGI/Y.51 Phosphorylation of HMGI/Y in B cells
inhibits its ability to bind DNA and results in the derepression of
germline IgE transcription.43,52 HMGI/Y has recently been
shown to downregulate the IL-4 promoter by competing with
NFAT for binding to the P1 element.53 Thus, activation of this
IL-4R signaling pathway would be expected to de-repress the
IL-4 promoter. However, our observation that Stat6 also displaces NFAT from the P1 element (Fig 8) might explain why
this did not occur in our experiments.
It is worth emphasizing that IL-4–dependent differentiation
of naive Th cells into effector Th2 cells involves prolonged
GEORAS ET AL
exposure to multiple concomitant signals emanating from the
T-cell receptor, costimulatory molecules, and possibly other
APC-derived cytokines.5,54-56 The precise role of Stat6 in this
process requires further study. We have shown that Stat6 does
not directly transactivate the IL-4 promoter, which it actually
represses in cells transcribing the IL-4 gene. Together with
recent analyses of committed Th cells,28,29 our results suggest
that Stat6 may rather facilitate the acquisition of an IL-4–
producing phenotype in differentiating Th cells in an indirect
fashion. In this regard, investigating the regulation of other
lineage-specific Th2 transcription factors by Stat6 may be
helpful.
ACKNOWLEDGMENT
The authors thank Dr Riccardo Dalla-Favera for assistance with the
Bcl-6 experiments, Dr Anjana Rao for the NFATp expression vector,
and Dr Marcia Wills-Karp (Johns Hopkins University, Baltimore, MD)
for helpful suggestions.
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1998 92: 4529-4538
Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells
Steve N. Georas, John E. Cumberland, Thomas F. Burke, Rongbing Chen, Ulrike Schindler and Vincenzo
Casolaro
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