(Received for publication, December 22, 1994; and in revised form, May 2, 1995)
From the
We have previously reported that the promoter region of the
mouse interleukin-5 (IL-5) gene, extending from a nucleotide position
about -1,200 to +33 relative to the transcription initiation
site, can mediate transcriptional stimulation by phorbol 12-myristate
13-acetate and dibutyryl cAMP (Bt
Interleukin-5 (IL
Mouse Th cells have been classified into two
subsets on the basis of their distinct lymphokine production
patterns(7) . Th1 cells produce IL-2, interferon
We have previously
reported that cAMP has differential effects on the production of Th1-
and Th2-type lymphokines in EL-4 cells, a cell line which produces both
types of lymphokines when stimulated with PMA(16) . Effects of
cAMP on PMA-induced expression of IL-2 and IL-5 genes were opposite; i.e. cAMP activated the IL-5 gene synergistically with PMA,
while it suppressed PMA-induced expression of the IL-2 gene, by
modulating respective promoter activities. Mechanisms for differential
regulation of the IL-2 and IL-5 genes by cAMP in EL-4 cells may be
related to those in Th1/Th2 cells and should provide some insight at
the molecular level.
Transcriptional control of the IL-2 gene has
been extensively studied, and the major regulatory elements have been
shown to reside within a region of approximately 300 bp upstream of the
transcription initiation site(17, 18) . Of several
transcription factors involved in regulation of the IL-2 gene, NF-AT is
essential for transcription of the IL-2 gene upon T cell activation and
plays a major role in various aspects of regulation of the IL-2 gene,
including T cell-specific expression(19) . NF-AT has also been
implicated in the transcriptional regulation of other lymphokine genes,
such as GM-CSF, IL-3, IL-4, and tumor necrosis factor-
On the other hand, details on the
transcriptional control of the IL-5 gene have not been explored at the
molecular level. Within the 5`-flanking region, the IL-5 gene is
associated with the conserved lymphokine elements (CLE)0, CLE1, and
CLE2 which are also found in the promoters of IL-3, GM-CSF, and IL-4
genes, and may have a role in the regulation of coordinated lymphokine
expression (23, 24) (Fig. 1).
Figure 1:
Nucleotide sequence of
the 5`-flanking region encompassing the SphI cleavage site and
the translation initiation site of the IL-5 gene. Numbers are relative
to the transcription initiation site. The nucleotide sequence from
position -1174 to -545 was determined by sequencing both
strands; the sequence further downstream was reported
previously(58, 59) . Locations of sequences that
correspond to CLE0, CLE1, CLE2, NF-1, and AP2 are indicated (bold
character). The transcription initiation site (58) and
TATA-like sequence are indicated. Restriction endonuclease cleavage
sites for NsiI and BclI used for generating mutant
constructs and base substitutions to generate a HindIII site
are also indicated. The IL-5A, IL-5P, IL-5C, and IL-5CLE0 elements are boxed.
Figure 3:
Linker-scanning analysis of the
5`-upstream region of the IL-5 promoter. Serial linker-scanning
mutations were introduced into the 1.2-kb IL-5 promoter, and constructs
obtained were transfected into EL-4 cells and analyzed, as described in Fig. 2. The positions in which substitutions were introduced are
shown schematically on the left panel (SalI and XhoI linkers are indicated by solid boxes). The
1.2-kb promoter region and the positions of the IL-5A, IL-5P, IL-5C,
and IL-5CLE0 elements are indicated. The promoter activity of each
construct in response to PMA (solid bars) or
PMA-Bt
Figure 2:
Deletion analysis of the 5`-upstream
region of the mouse IL-5 promoter. EL-4 cells were transfected with the
serial deletion mutants shown schematically in the left panel.
The cells were stimulated 24 h after transfection with either PMA (solid bars) or PMA and Bt
Next, to identify the region
responsible for inducible expression of the IL-5 promoter in response
to PMA and Bt
Figure 4:
The common sequence between the IL-5P and
NF-AT sites. A, comparison of the mouse and human IL-5
promoter regions. Nucleotide sequence extending from -127 in the
mouse promoter through the ATG is compared with human IL-5 genomic
sequence (60) . The IL-5P, IL-5C, and IL-5CLE0 elements are boxed. Identical nucleotides are indicated by vertical
bars. B, comparison of the nucleotide sequence of IL-5P
with that of the distal NF-AT site of the IL-2 promoter. Gaps are
introduced into the NF-AT site to maximize matches. Differences in the
human sequence are shown below the murine sequence. The purine-rich
sequence and an AP1-like site are indicated by closed circles and shadowed bars,
respectively.
Figure 5:
Binding of nuclear factors to the IL-5P
sequence. A, NFIL-5P is the inducible, sequence-specific
factor which binds to the IL-5P sequence. EMSAs were done with
unstimulated (lane 1) or PMA-Bt
To identify specific bases
critical for NFIL-5P complex formation, segments of IL-5P sequence were
mutated and tested for effects on complex formation (Fig. 6A). The PM1 mutant oligonucleotide with a
mutation in the GGA sequence did not form the NFIL-5P complex, but did
retain the ability to form other minor complexes (Fig. 6B, lane 4). NFIL-5P binding was
gradually restored as mutations were introduced into the segments
closer to the 3` portion of the IL-5P sequence (compare lanes
6, 9, 12, and 15). Competition analyses
gave consistent results (data not shown).
Figure 6:
Effects of mutations in the IL-5P sequence
on the NFIL-5P binding and IL-5 promoter activity. A, location
of the nucleotides substituted in PM1 to PM5. The sequences are aligned
with the wild type IL-5P sequence, and identical bases are denoted by dashes. B, EMSAs using a panel of mutant IL-5P
probes. Identical amounts of each probe depicted in panel A were incubated with nuclear extracts from EL-4 cells stimulated
with PMA and Bt
The IL-5 gene is expressed mainly in activated T
cells. To determine the cell-type specificity of the NFIL-5P complex,
EMSAs were done using nuclear extracts from a variety of cell types
either unstimulated or stimulated with PMA, A23187, and
Bt
Figure 7:
Cell type distribution of the NFIL-5P
complex. Nuclear extracts were prepared from EL-4 (lanes 1 and 2), Jurkat (lanes 3 and 4), Ba/F3 (lanes
5 and 6), NIH3T3 (lanes 7 and 8), and
COS-7 cells (lanes 9 and 10) untreated (lanes
1, 3, 5, 7, and 9) or treated
for 3 h either with PMA and Bt
In EMSAs using
nuclear extracts from EL-4 cells stimulated with PMA and
Bt
Figure 8:
Relationship between NFIL-5P and NF-AT. A, nuclear extracts from EL-4 cells stimulated with PMA and
Bt
To determine whether an
NF-AT component is involved in the NFIL-5P complex, supershift EMSAs
were done (Fig. 8C). Both NFIL-5P and NF-AT complexes (lanes 1-3 and 5-7, respectively, arrow) from nuclear extracts stimulated with PMA and
Bt
We then examined
whether recombinant NF-AT would bind to the IL-5P site. It has been
shown that the preexisting cytoplasmic component of NF-AT is
heterogenous (33) and in fact is composed of a family of
homologous proteins(34, 35) . We recently isolated
cDNA clones encoding two of these, namely NF-ATc and
NF-ATx(26) . EMSAs were done using extracts from recombinant
NF-ATx- or NF-ATc-transfected COS-7 cells. As shown in Fig. 8C, both NF-ATx and NF-ATc bound to the IL-5P, in
combination with AP1 purified from Jurkat cells (left panel, lanes 4 and 6, upper and lower
bands, respectively). The mock transfected COS-7 cells showed no
specific band (left panel, lanes 1 and 2).
These results indicate that IL-5P can be recognized by different forms
of the cytoplasmic component of NF-AT. The mobility of the complex
formed with NF-ATx was slightly slower than that formed with NF-ATc (lanes 7 and 8), and the binding specificity of
NF-ATx and NF-ATc to the IL-5P and NF-AT sites was somewhat different, i.e. NF-ATc but not NF-ATx can bind to the NF-AT site without
AP1, while neither bound to the IL-5P without AP1 (compare left and right panels, lane 5). Taken together, these
results strongly suggest that NFIL-5P is related to, if not identical
to, NF-AT.
We have previously reported that both PMA and cAMP are
required for the activation of the IL-5 promoter(16) . In the
present work, we analyzed regulatory elements in the promoter which
were necessary to respond to these stimuli. By deletion and mutation
analyses, we identified four cis-regulatory elements,
designated IL-5A, IL-5P, IL-5C, and IL-5CLE0, which are important for
the full activity of the IL-5 promoter in response to PMA and cAMP. We
showed that the IL-5P, IL-5C, and IL-5CLE0 elements but not the IL-5A
element were necessary to respond weakly to PMA alone (Fig. 3).
The sequences within the former three elements are highly conserved
between human and mouse IL-5 genes (Fig. 4A), and these
elements are also crucial for the function of the human IL-5 promoter
(data not shown). Therefore, the regulatory mechanism of the IL-5 gene
is conserved between the two species.
Although IL-5A elicits
activation of the IL-5 promoter in response to PMA and cAMP, it shares
no homology with the cAMP response element (CRE). EMSA with an
oligonucleotide probe containing the IL-5A element showed several
sequence-specific complexes distinct from cAMP response element-binding
protein (CREB).
The antisense
strand sequence of the center of the IL-5C element, 5`-AGATAG-3`
(-71
The IL-5CLE0 element includes the CLE0 motif, which also
occurs in the promoter proximal regions of the GM-CSF and IL-4 genes.
The CLE0 motifs of these genes are also crucial for their promoter
activities(24, 43, 44) . The sequence of
these motifs is identical between humans and mice, although it is
somewhat different among lymphokines(24) . Thus, the CLE0 motif
may be involved in the coordinated and/or specific expression of these
genes and related studies are ongoing. Interestingly, a recent report
indicated that a similar region of the mouse IL-5 promoter is
functionally important in D10.G4.1 (Th2 cell clone) and an inducible
nuclear factor bound to this region is detected only in D10.G4.1 but
not in HDK1 (Th1 cell clone)(45) .
The fourth cis-regulatory element, IL-5P, was investigated in detail
because of its similarity to the NF-AT-binding site. We first
identified an inducible nuclear factor (NFIL-5P) bound to the IL-5P
element. Next, we demonstrated that NFIL-5P binding is required for in vivo functioning of this element by showing that mutations
in IL-5P which prevent this interaction also inhibit promoter activity (Fig. 6). Finally, we showed that NFIL-5P is similar to the
transcription factor NF-AT in several respects: the sequence
specificity of NFIL-5P binding is closely correlated with that of the
NF-AT (Fig. 6B),(46, 47) ; an excess
NF-AT oligonucleotide inhibits NFIL-5P binding to IL-5P (Fig. 8A); the formation of both complexes is inhibited
by CsA and CHX (Fig. 8B); the presence or absence of
NFIL-5P in the several cell lines tested is correlated with that of
NF-AT (Fig. 7); an antiserum raised against NF-ATp supershifts
both complexes (Fig. 8C); and finally, recombinant
NF-ATx and NF-ATc expressed in COS-7 cells in combination with AP1 can
bind to the IL-5P site, as well as to the NF-AT site, with mobilities
similar to those seen with stimulated EL-4 and Jurkat cell nuclear
extracts (Fig. 8D).
Though NF-AT binding is induced
by PMA alone in EL-4 cells, it is inhibited by CsA which is known to
block Ca
Recent cloning of the cytoplasmic
components of NF-AT has revealed that they are encoded by at least
three members of related clones containing the Rel-homology
domain(26, 34, 35) . Moreover, mRNA of each
NF-AT clone has distinct tissue distribution and induction
characteristics. Considering the diversity of the AP1 family, it is
tempting to speculate that distinct NF-AT complexes composed of
different combinations of members of the cytoplasmic component of NF-AT
and AP1 families are formed depending on signals and cell types.
Indeed, the NFIL-5P complexes with different mobilities were induced
depending on the stimulation given (Fig. 5B). We
demonstrated that IL-5P can be bound by multiple NF-AT complexes and
the mobilities of complexes containing NF-ATx and NF-ATc were slightly
different (Fig. 8D). Whether NF-ATp, NF-ATx, NF-ATc,
and other members of the NF-AT family are induced differently by PMA
and cAMP remains to be clarified.
We found that the NF-AT site is
the target of the inhibitory action of cAMP on the IL-2 promoter in
EL-4 cells(22) . However, NF-AT binding to this site increased
with the addition of cAMP. Thus, it appears that IL-5P and the IL-2
promoter NF-AT site, though they are closely related, are regulated
oppositely by cAMP. PMA and cAMP may induce distinct NF-AT-related
complexes which have different transcriptional activation functions on
the IL-5P and NF-AT sites. Furthermore, PMA and cAMP may differentially
affect the components of the NF-AT-related complexes by protein
modifications to either promote or to inhibit their transcriptional
activation functions. For instance, certain members of the AP1 family
have been shown to be regulated not only at transcriptional but at
post-translational levels in response to PMA and cAMP(53) . At
present, we cannot distinguish between these possibilities.
Nevertheless, the identification of IL-5P and NFIL-5P provides a system
for exploring the mechanism for differential regulation through
NF-AT-related sites.
It is important to note that the cAMP effect on
the IL-5 promoter was not limited to the IL-5P site. The regulation of
many genes by cAMP is mediated by either the AP2 or CREB/activating
transcription factor family of nuclear
proteins(54, 55, 56) . However, we did not
find CRE or the AP2 binding sequence within the elements which we
identified in this study. Since all four regulatory elements were
required for maximal activity of the IL-5 promoter responding to PMA
and cAMP, they may function cooperatively, as was suggested for another
system(57) . Thus, the effect of cAMP may be exerted at the
level of assembly of a functional transcription machinery including
factors bound to these elements.
In summary, we have delineated the cis-regulatory elements essential for expression of the IL-5
gene and obtained the first evidence for an NF-AT related factor
regulating expression of the gene. The results presented here narrow
down the elements involved in the mechanisms underlying the
differential effect by cAMP on expression of the IL-5 and IL-2 genes in
EL-4 cells. It will be interesting to check whether the same elements
are involved in the differential lymphokine expression by Th1 and Th2
cells.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
D14461[GenBank® Link].
We thank N. Koyano-Nakagawa, H. Yssel, D. Wylie, and
M. Ohara for critical reading of the manuscript and for helpful
discussions, L. Tsuruta and J. Nishida for helpful discussions, and D.
Ligget at DNAX Research Institute and the staff at the Laboratory of
Molecular Genetics, The Institute of Medical Science, The University of
Tokyo (IMSUT) for synthesizing oligonucleotides. Computational time was
provided by the Human Genome Center, IMSUT.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cAMP) in mouse thymoma
EL-4 cells. Here, we describe identification of four cis-regulatory elements necessary for full activity of the
IL-5 promoter, using deletion and mutation analyses. We designated
these elements as IL-5A (-948
-933), IL-5P
(-117
-92), IL-5C (-74
-56), and
IL-5CLE0 (-55
-38). We found that IL-5P bears
homology to the binding site for the nuclear factor of activated T
cells (NF-AT) and interacted with protein factors in nuclear extracts
prepared from EL-4 cells stimulated with phorbol 12-myristate
13-acetate and Bt
cAMP (designated NFIL-5P). NFIL-5P complex
was inhibited in the presence of an excess NF-AT and AP1
oligonucleotides and supershifted by antisera raised against NF-ATp,
c-Fos, and c-Jun. It thus seems likely that an NF-AT-related factor is
involved in the regulation of IL-5 gene transcription.
(
)-5) is primarily a T
cell-derived lymphokine with multiple regulatory functions, including
stimulation of growth and differentiation, on eosinophils (1, 2) and, in the mouse, on B cells(3) . The
IL-5 gene is located on mouse chromosome 11 and human chromosome 5,
within a cluster of the IL-3, granulocyte-macrophage colony stimulating
factor (GM-CSF), IL-4, and IL-13 genes (4, 5, 6) which are commonly expressed in T
helper 2 (Th2) cells.
, and
lymphotoxin and promote cell-mediated immunity, whereas Th2 cells
produce IL-4, IL-5, IL-6, and IL-10 and promote humoral
immunity(8) . Polarized Th1 and Th2 responses have been shown
to be a feature of a number of disease states in both mice and humans,
and a proper balance between the two subsets is crucial for the
effective responses(9, 10, 11) . Despite
their physiological significance, molecular mechanisms which account
for the difference in lymphokine gene expression in Th1 and Th2 cells
are not well understood. One mechanism may entail differences in
transcription factors interacting with cis-acting elements in
lymphokine genes. Another mechanism may involve differences in signal
transduction pathways between Th1 and Th2 cells(12) . There are
reports suggesting that Th1 and Th2 cells use different signal
transduction pathways; several groups have demonstrated that compounds
which elevate intracellular cAMP levels have differential effects on
lymphokine production in Th1 and Th2
cells(13, 14, 15) .
, hence, a
role in the coordinated expression of these genes was
suggested(20, 21) . We also have data that NF-AT is a
major target of the inhibitory action of cAMP on the IL-2
promoter(22) .
In this study,
we identified four cis-regulatory elements which are necessary
for full activity of the IL-5 promoter in response to PMA and
BtcAMP stimulation in the mouse thymoma line EL-4. We
showed that one element, the IL-5P element, shares homology with the
binding site of NF-AT of the IL-2 gene and interacts with the inducible
nuclear factor NFIL-5P which is closely related to the transcription
factor NF-AT.
Cells
The mouse thymoma cell line EL-4 TB6 was
maintained in RPMI 1640 medium supplemented with 2 mM
glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, 50
µM 2-mercaptoethanol, and 5% fetal bovine serum under 5%
CO. Jurkat, Ba/F3, and COS-7 cells were cultured in the
same medium containing 10% fetal bovine serum. Recombinant mIL-3 (1
ng/ml) was additionally supplemented for maintenance of Ba/F3 cells.
NIH3T3 cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum and the antibiotics
described above.
Antibodies
Anti-mouse NF-ATp (NF-ATp), a
rabbit polyclonal antiserum raised against recombinant NF-ATp, was
purchased from Upstate Biotechnology Incorporated. The Fos antibody
(
Fos), a rabbit polyclonal IgG reactive with c-Fos, Fos B, Fra-1,
and Fra-2 and Jun antibody (
Jun), a rabbit polyclonal IgG reactive
with c-Jun, Jun B, and Jun D and their cognate peptides purchased from
Santa Cruz Biotechnology.
DNA Sequencing
The DNA sequence of both strands of
the region extending from position -1174 to -545 was
determined using an Applied Biosystems 373A DNA sequencer.
Plasmid Constructs
Plasmids pUC00Luc and
pmIL5Luc(1.2) were described previously(16) . pmIL5Luc(1.2) was
linearized by SalI digestion and a series of nested 5`
deletions was generated by BAL-31 exonuclease treatment. Then, 7-bp SalI linkers were attached and the SalI-HindIII fragments were inserted into pUC00Luc.
The 5` border of each deletion mutant was determined by DNA sequencing
and was named according to its 5`-end point. To generate pmIL5Luc(1.8),
the 5`-EcoRI site of the pmIL5EHE (16) was replaced by
the SalI site and the 1.8-kb SalI-HindIII
fragment was inserted into pUC00Luc. The linker-scanning mutants were
generated by replacing nucleotide sequences in various positions in the
1.2-kb IL-5 promoter with a 7-bp SalI or 6-bp XhoI
linker sequences, using polymerase chain reaction (PCR). The resulting
linker-scanning mutants contained 3-7-bp substitutions out of 6
or 7 bp at various positions in the context of the 1.2-kb IL-5 promoter (Fig. 3). These mutants were named according to the positions at
which substitution linkers were introduced. To make the linker-scanning
mutants LS971/965, PCR was performed with pmIL5Luc(1.2) as the
template, M13 universal primer ((M13(-47)), and 1SA, an antisense
strand oligonucleotide primer containing a SalI recognition
site at nucleotide position -972 (Table 1). The resulting
PCR product was digested with SacI (at the polylinker region)
and SalI and was inserted into the SacI-SalI
site of p964, a deletion mutant with a 5`-end point of -964 (Fig. 2). LS946/940, LS939/933, LS110/104, LS103/97, LS91/85,
LS69/63, and LS57/51 were generated by essentially the same procedure.
For the latter five mutants, p161, a deletion mutant whose 5`-end point
is -161, was used as the template to minimize PCR-derived
sequence and a sequence further upstream was attached using the SacI-NsiI (at nucleotide position -141)
fragment of pmIL5Luc(1.2). To obtain LS954/949 and LS932/927, a pair of
PCR amplifications was performed for each construct, as
described(25) . Fragments upstream of the introduced mutations
were generated using pmIL5Luc(1.2) as the template and M13(-47),
and primers containing the XhoI recognition sites at the
5`-ends of the antisense strand oligonucleotides (Table 1). To
generate downstream fragments, we used sense-strand oligonucleotide
primers containing the XhoI linker sequence at the 5`-ends to
overlap with the XhoI site of the upstream fragment, and
antisense-strand oligonucleotide primer C1A (Table 1). The
resulting upstream and downstream fragments were digested with SacI and XhoI, XhoI and BclI (at
nucleotide position -800), respectively, and ligated between the SacI and BclI sites of pmIL5Luc(1.2). LS123/118,
LS117/112, LS80/74, LS62/57, LS37/32 were generated essentially by the
same procedure used for LS955/949 and LS933/927. For these
constructions, we used p161 as the template and GLprimer2, a primer
corresponding to the antisense strand of the luciferase structural gene
(Promega Corp., Madison, WI) instead of C1A to generate downstream
fragments. The resulting PCR fragments were cloned into the NsiI and HindIII (at nucleotide position +33)
sites of pmIL5Luc(1.2). To generate pM1 to pM4 containing mutations in
the IL-5P site, two steps of PCR were performed as
described(25) . First, fragments upstream and downstream of
point mutations were generated, using the same procedure as for
LS124/118, except for use of sense and antisense oligonucleotides (Table 2, PM1 to PM4) used in the electrophoretic mobility shift
assays (EMSAs) instead of primers containing the XhoI linker
sequence. Second step PCR amplifications were done using one-tenth of
the fragments generated by first step PCR and M13(-47) and
GLprimer2. The resulting fragments were inserted into the NsiI
and HindIII sites of pmIL5Luc(1.2). To increase fidelity, PCR
amplifications were performed for 15 cycles (four cycles with annealing
at 45 °C and 11 cycles at 65 °C) with a large amount (100 ng)
of template. The PCR-derived sequences of each construct were confirmed
by DNA sequencing. The expression plasmids of NF-ATc and NF-ATx were
constructed by inserting corresponding cDNAs downstream of the SR
promoter in pME18S(26) .
cAMP stimulation (cross-hatched bars) was
measured. Results are expressed as the percentage of activity of the
wild type promoter in response to PMA-Bt
cAMP stimulation
and represent the means ± S.D. of at least three independent
experiments with at least two different DNA
preparations.
cAMP (cross-hatched
bars), cell lysates were prepared 16 h later, and tested for
luciferase activity, as described under ``Materials and
Methods.'' Results represent the mean value of duplicate
transfections. Two other independent experiments gave similar results.
The regions responding to PMA and Bt
cAMP are indicated by shadowed boxes.
Transfections
Transient transfections were done
using a DEAE-dextran procedure, as described previously(16) .
At 24 h after transfection, cells were stimulated with 1 mM
BtcAMP (Sigma) and 10 ng/ml PMA (Calbiochem). After another
16 h, the cells were harvested for luciferase assays. Luciferase
activity was determined using the luciferase assay substrate (Promega)
with a luminometer (Berthold, Postfach, Germany). Protein concentration
in an aliquot of each sample was measured using BCA reagents (Pierce),
and the results were calculated as (relative light
unit-background)/µg protein.
Preparation of Extracts and EMSAs
Cells were
stimulated for 3 h with various drugs, as described in figure legends,
and nuclear extracts were prepared either by the method of Dignam et al.(27) or as described(28) . EMSAs were
done using the double-stranded oligonucleotides given in Table 2.
Each single-stranded oligonucleotide was purified on denaturing
polyacrylamide gels before annealing. The annealed oligonucleotides
were P-labeled with Klenow fragment and purified on a 12%
polyacrylamide gels. The DNA-binding reactions were performed at room
temperature for 30 min with 3-5 µg of nuclear extracts, 0.5
µg of poly(dI-dC), 10 mM HEPES, pH 7.9, 10% glycerol, 1
mM EDTA, 1 mM dithiothreitol, 100 mM KCl,
and 50,000 counts/min of probe (
0.5 ng) in a total volume of 10
µl. The samples were resolved on a 4% nondenaturing polyacrylamide
gel at 120 V in 1
Tris-glycine-EDTA buffer, and the results
were visualized by autoradiography. Extracts from transfected COS-7
cells were prepared as described(26) .
Functional Analysis of the 5`-Flanking Region of the
IL-5 Gene
We have previously reported that the 5`-flanking
region of the IL-5 promoter extending from the SphI site at a
nucleotide position about -1,200 to +33 relative to the
transcription initiation site mediated induction by PMA and
BtcAMP in EL-4 cells(16) . Since only a partial
nucleotide sequence has been reported, we first determined the complete
nucleotide sequence of this region and found a number of potential
regulatory elements (Fig. 1).
cAMP, the luciferase reporter plasmids
carrying sequential 5`-deletions of the mouse IL-5 promoter were
transiently transfected into EL-4 cells. The plasmid pUC00Luc, which
has no upstream sequence derived from the IL-5 gene(16) ,
showed an almost negligible response to PMA or a combination of PMA and
Bt
cAMP (Fig. 2). Removal of the 5` sequence up to
position 964 did not affect the promoter activity responding to PMA and
Bt
cAMP, but deletions beyond this position through
-932 did reduce the response by 50%. Further deletions up to
position -140 led to no further reduction. Remarkably, the
subsequent removal of nucleotides -140 through -80
diminished the promoter activity by 90%. Thus, the response to a
combination of PMA and Bt
cAMP appears to decline in two
stages, at the -964 to -933 (region I) and at the
-140 to -80 (region II) (Fig. 2). On the other hand,
the IL-5 promoter responded weakly to PMA alone, and removal of the 5`
sequences up to position -140 did not affect promoter activity
responding to PMA, while deletions beyond this position almost
completely abolished the response. Thus, the sequences between
-140 to -80 (region II) of the IL-5 promoter are
required for a response, albeit a weak one, to the PMA signal.
Four Cis-regulatory Elements Are Required for Full
Activity of the IL-5 Promoter
To further delineate regulatory
elements in the IL-5 promoter, we dissected regions shown to be
important for promoter activity. Using the linker-scanning method, we
replaced the sequence in various positions between -972 and
-932, and between -124 and -31 in the 1.2-kb IL-5
promoter with a 7-bp SalI or 6-bp XhoI linker
sequence (see ``Materials and Methods''). We did not
introduce mutations around -140, because we found that in further
deletion analysis, the proximal regulatory region is located between
-127 and -103 (data not shown). Consistent with results of
the 5`-deletion analysis, mutations introduced between -948 and
-933 (designated IL-5A) reduced promoter activity by 60% in
response to PMA and BtcAMP but not to PMA alone (Fig. 3). Mutations introduced in the region extending from
-117 to -92 (designated as IL-5P) reduced promoter activity
by about 80%, in response to a combination of PMA and
Bt
cAMP as well as to PMA alone. Mutation analyses allowed
for definition of third and fourth elements, extending from -74
to -56 (designated IL-5C) and from -55 to -38
(designated IL-5CLE0), respectively. The IL-5C and IL-5CLE0 elements
are located in close proximity, and mutation analysis has heretofore
shown no clear boundary between them. However, we defined IL-5CLE0 in
analogy with CLE0 of the GM-CSF gene, and the remaining sequence as
IL-5C. These two elements are the most crucial for promoter activity,
because mutations in these regions almost completely abolished promoter
activity in response to PMA as well as to PMA and Bt
cAMP.
Characterization of IL-5P
Deletion and mutation
analyses revealed that downstream sequences of -124 are critical
for IL-5 promoter activity. Comparison of the mouse and human IL-5
promoters showed a high degree of homology within this region (Fig. 4A). Interestingly, closer inspection revealed
that IL-5P is homologous to the binding site for NF-AT (Fig. 4B). NF-AT has also been implicated in
transcriptional regulation of other lymphokine
genes(20, 21) . We recently noted that the NF-AT site
is a major target of the inhibitory action of cAMP on the IL-2
promoter(22) . Thus, we speculate that IL-5P may be involved in
coordinated and differential regulation of the IL-5 gene and focused on
further characterization of IL-5P.
To identify nuclear factors that
can mediate inducibility through IL-5P, EMSAs were done using a probe
corresponding to the region extending from -119 to ;89 of the
IL-5 promoter (Table 2). Nuclear extracts prepared from EL-4
cells stimulated with a combination of PMA and BtcAMP
formed several complexes with the IL-5P probe (Fig. 5A). The major complex (designated NFIL-5P)
appeared only when we used extracts prepared from stimulated cells
(compare lanes 1 and 2), and this complex formation
was specifically inhibited in a dose-dependent manner by excess
unlabeled IL-5P oligonucleotide (lanes 2-5) but not by
the Sp1 oligonucleotide (lanes 6-8). Other minor
complexes with different mobilities could be detected, but appearance
was variable, depending on different nuclear extract preparations, and
they did not seem to be specific for IL-5P because binding was also
inhibited by the Sp1 oligonucleotide (lanes 6-8).
cAMP stimulated (lanes 2-8) EL-4 nuclear extracts in the presence of
increasing amounts of unlabeled IL-5P (lanes 3-5) or Sp1 (lanes 6-8) oligonucleotides, as indicated. B,
regulation of NFIL-5P binding activity. Nuclear extracts from EL-4
cells untreated (lanes 1 and 5) or stimulated with
either PMA (lanes 2 and 6), Bt
cAMP (lanes 3 and 7), or the combination of both (lanes 4 and 8) were incubated with the IL-5P (lanes 1-4) or Sp1 (lanes 5-8) probes.
The positions of the NFIL-5P complexes induced by PMA (II),
Bt
cAMP (I), and combination of both (III)
and the Sp1 complex (arrowhead) are
indicated.
When EMSAs were done with nuclear extracts from EL-4 cells
stimulated with different combinations of PMA and BtcAMP,
the NFIL-5P complex was induced by either PMA or Bt
cAMP.
The combination of both agents intensified the band (Fig. 5B, lanes 2-4). Interestingly, the
NFIL-5P complex migrated with a different mobility, depending on the
stimulation given. The control Sp1 binding was not altered by any of
these stimuli (lanes 5-8).
cAMP. For each binding reaction, the
competition was performed with 20 ng of unlabeled self or wild type
oligonucleotides. The NFIL-5P complex is indicated by the arrow. C, functional analysis of site-specific
mutations in the IL-5P site. Mutations corresponding to those analyzed
by EMSAs in panel B were introduced into the 1.2-kb IL-5
promoter. The wild type pmIL5Luc(1.2) and mutant constructs (pM1 through pM4 and LS103/97) were transiently
transfected into EL-4 cells, and luciferase activity was assayed at 16
h after stimulation with PMA and Bt
cAMP, as described under
``Materials and Methods.'' Results are expressed as a
percentage of the activity of the wild type (shadowed bars),
and represent the means ± S.D. of at least three independent
experiments with at least two different DNA preparations. Relative
intensity of NFIL-5P band elicited by each probe (B) was
quantitated by PhosphorImage analyzer and plotted on the same graph (cross-hatched bars).
To search for a possible
correlation between the in vitro formation of the NFIL-5P
complex and in vivo activity of the IL-5 promoter, we
introduced site-specific mutations corresponding to mutations
introduced into the IL-5P probe for the EMSAs described above, in the
context of the 1.2-kb IL-5 promoter. Constructs were transiently
transfected into EL-4 cells and analyzed for luciferase activity (Fig. 6C). The results are shown along with the
intensity of the NFIL-5P band elicited by each probe (Fig. 6B), as determined by PhosphorImage analyzer
(Molecular Dynamics). Introduction of PM1, PM2, PM3, and PM5 mutations
decreased IL-5 promoter activity by about 80%, in response to PMA and
BtcAMP, whereas the PM4 mutation caused only a 50% decrease
in promoter activity. Therefore, the in vivo activity of the
IL-5 promoter correlated with the binding activity of NFIL-5P to the
IL-5P site, i.e. the bases which were required for formation
of the NFIL-5P complex in vitro were the same as those
required for promoter activity. These results strongly suggest that
NFIL-5P is the factor which regulates the IL-5 promoter through the
IL-5P sequence.
cAMP (Fig. 7). The NFIL-5P complex appeared only
in the stimulated EL-4, Jurkat T cell line, and pro-B cell line Ba/F3
cells (left panel, lanes 2, 4, and 6, respectively), while the AP1 complex was detected in all
cell types tested (right panel).
cAMP (lanes 2, 8, and 10) or with PMA, A23187, and
Bt
cAMP (lanes 4 and 6). EMSAs were done
using IL-5P probe, as described under ``Materials and
Methods.''
NFIL-5P Is Related to NF-AT
We next asked whether
NFIL-5P and NF-AT were related. NF-AT is composed of a pre-existing
cytoplasmic component, referred to as NF-ATp (29) or
NF-ATc(30) , which is translocated into the nucleus in response
to a calcium signal, and a newly synthesized nuclear component, which
belongs to the AP1 family (30, 31) .
cAMP, NFIL-5P and NF-AT complexes migrated with a similar
mobility (Fig. 8A, lanes 1 and 7).
NFIL-5P complex formation was completely inhibited by excess unlabeled
NF-AT as well as by IL-5P oligonucleotides (lanes 2 and 5) but not by those mutated in GGA sequences (lanes 3 and 6). As expected, AP1 oligonucleotide also inhibited
NFIL-5P complex formation (lane 4). Conversely, NF-AT binding
was inhibited specifically by both excess unlabeled NF-AT and IL-5P
oligonucleotides, indicating that NFIL-5P is an NF-AT-related complex (lanes 7-11).
cAMP were incubated with the IL-5P (lanes
1-6) or the NF-AT (lanes 7-11) probes in the
presence of 20 ng of the unlabeled competitor oligonucleotides. The left and right arrowheads indicate positions of
NFIL-5P and NF-AT complexes, respectively. B, nuclear extracts
were prepared from EL-4 cells stimulated with PMA and
Bt
cAMP in the presence of different concentrations of CsA
or CHX, as indicated. EMSAs were performed with IL-5P, NF-AT, and Sp1
probes. The positions of NFIL-5P, NF-AT, and Sp1 complexes are
indicated by arrowheads. C, the NFIL-5P complex
contains NF-ATp and AP1. Nuclear extracts from EL-4 cells stimulated
with PMA and Bt
cAMP were incubated without antiserum (lanes 1, 5, and 9), with 0.2 µl of an
antiserum to the NF-ATp (lanes 3, 7, and 11), or with non-immune serum (lanes 2, 6,
and 10), followed by binding assays with IL-5P, NF-AT, and Sp1
probes. Nuclear extracts were also incubated without (lanes 13 and 18) or with 1 µl of antibodies raised against
c-Fos (lanes 14 and 15) and c-Jun (lanes 16 and 17) in the presence (lanes 15 and 17) or absence (lanes 14 and 16) of their
cognate peptides, followed by binding assays with IL-5P probe. D, nuclear extracts were prepared from Jurkat cells stimulated
with PMA and A23187 (lane 7) and EL-4 cells stimulated with
PMA and Bt
cAMP (lane 8). Cytosolic extracts were
prepared from COS-7 cells transfected with vector alone (lanes 1 and 2) or expression vector containing cDNA encoding
NF-ATx (lanes 3 and 4) or NF-ATc (lanes 5 and 6). EMSAs were done with IL-5P and NF-AT probes (left and right panels, respectively) using above
extracts in the absence(-) or presence (+) of purified
AP1.
NF-AT binding is known to be inhibited
by either cyclosporin A (CsA) (30) or the protein synthesis
inhibitor cycloheximide (CHX)(19, 32) . We then tested
sensitivity of the NFIL-5P complex to CsA and CHX; treatment of CsA
inhibited the induction of NFIL-5P complex in cells stimulated with PMA
and BtcAMP, in a dose-dependent manner similar to that seen
for NF-AT binding (Fig. 8B, top and central panels, lanes 2 and 3). Induction of
both the NFIL-5P and NF-AT complexes was almost completely inhibited by
treatment with 10 µg of CHX (lanes 4 and 5). This
inhibition was not caused by nonspecific effects of these reagents on
nuclear proteins, since there was no change in Sp1 binding, under the
same conditions (bottom panel).
cAMP were supershifted by an antiserum raised against
NF-ATp (lanes 3 and 7, respectively, arrowhead). This antiserum did not supershift the Sp1 complex (lanes 9-11), indicating these supershifts are specific.
Antibodies raised against c-Fos and c-Jun which can recognize several
members of the AP1 family also supershifted the NFIL-5P complex (lanes 14 and 16, respectively).
(
)The 3` portion of the IL-5A
element, 5`-TGGGTCAAGGCCA-3`(-940
-928), has a close
sequence similarity to the NF-1 consensus binding sequence,
5`-TGGNNNNNNGCCA-3` (36, 37) (Fig. 1). However,
it is unlikely that NF-1 is involved in regulation through the IL-5A
element, since LS932/927, in which the NF-1 site is mutated, showed
activity similar to that of the wild type promoter.
-66), matches the GATA consensus sequence,
5`-WGATAR-3`. Indeed, the mutant LS69/63 in which the GATA-binding site
was destroyed lost promoter activity almost completely (Fig. 3).
So far, four members of GATA family transcription factors, GATA-1
through GATA-4, have been reported, each GATA factor has a somewhat
distinctive pattern of expression and is involved in cell-specific
expression of certain genes(38, 39) . Among them,
GATA-3 is the predominant GATA protein present in T cells (40, 41) and is involved in the regulation of a number
of T cell-specific genes, including T cell receptor genes (42) . We are currently testing the role of the GATA family
transcription factors in regulation of the IL-5 gene through this
element.
-dependent pathway. Two possible explanations
for this discrepancy were raised and but are yet to be proven: either
calcineurin is activated by other mechanisms in EL-4 cells or the
resting Ca
concentration is sufficient to activate
calcineurin in EL-4 cells(48) . Our in vitro result
indicating that NFIL-5P is inhibited by CsA appears to disagree with a
report showing that the level of IL-5 mRNA induced by PMA was not
inhibited CsA(49) . However, in the subline of EL-4 cells of
our use only low level of IL-5 mRNA is induced with PMA alone, and the
combination of PMA and cAMP synergistically augments the level of
expression(16) . This maximal level expression of IL-5 mRNA was
inhibited by addition of CsA.
In addition, other reports (50, 51, 52) and our unpublished
results
(
)also indicate that CsA inhibits the
production of IL-5 from several T cell lines both in human and mouse
systems. Thus, the disagreement may be caused from the difference of
cell line and stimuli adopted.
cAMP, dibutyryl cAMP; NF-AT,
nuclear factor of activated T cells; EMSA, electrophoretic mobility
shift assay; CsA, cyclosporin A; CHX, cycloheximide; Th, helper T; bp,
base pair(s); kb, kilobase; CLE, conserved lymphokine element; PCR,
polymerase chain reaction; CRE, cAMP response element.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.