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Promoter Mediated by Stat6 and C/EBPß
Tularik Inc., South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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germline
transcript is induced by IL-4 and requires the integrity of a composite
IL-4 response element. The element is bound by the IL-4-inducible
transcription factor Stat6 and one or more members of the
CAAT/enhancer-binding protein (C/EBP) family, a constitutively
expressed class of transcription factors. Here, we show that Stat6 and
C/EBPß cooperate to synergistically activate transcription from the
element. The effect was most pronounced in lymphoid cells, and the
activation domains of both proteins were required to achieve this
synergy. Although other members of the C/EBP family are able to bind
the element, very little cooperativity was seen with C/EBP
and none
with C/EBP
. In fact, C/EBP was able to inhibit IL-4-induced
reporter activity. Stat6 and C/EBPß bind the IL-4 response element
simultaneously. The fast dissociation rate apparent when Stat6 binds
this DNA element alone is slowed when C/EBPß binds at the neighboring
site. These data suggest a mechanism whereby C/EBPß stabilizes Stat6
binding at this element, thereby increasing the likelihood that both of
their activation domains will interact, possibly with other factors, to
activate transcription in an IL-4-dependent
manner. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines such as the interleukins and interferons have been shown to rapidly activate a signaling pathway known as the Janus kinase (Jak)3-STAT pathway. Briefly, STAT proteins are activated through tyrosine phosphorylation by receptor-associated Jak kinases following cytokine binding. The activated STAT protein dimerizes, translocates to the nucleus, and activates transcription via specific DNA response elements (4, 5, 6). Currently, there are seven known members of the STAT family that are characterized and that are activated by different cytokines. In the present study, we focused on Stat6, which is activated upon IL-4 stimulation (7, 8). The generation of Stat6-deficient mice has established the requirement of this protein in promoting polarization of T helper cells toward the Th2 phenotype. Furthermore, genes that are activated in response to IL-4, such as CD23 and MHC class II genes, cannot be induced in Stat6-deficient mice, and IgE production is profoundly impaired (9, 10).
Stat6 binding sites have been identified in several of these
IL-4-responsive genes and are best characterized in the promoters that
govern the expression of the Ig germline and transcripts (11, 12). IL-4-induced expression of these genes requires the integrity of
the Stat6 binding site as well as an adjacent site, which is bound by
C/EBP (11, 12). In the mouse germline promoter, the Stat6 and C/EBP
sites are separated by four base pairs; in the human promoter, the two
sites are directly adjacent. Previous studies have shown that either
one of these composite elements (mouse or human) is able to confer
IL-4-induced transcription onto a heterologous promoter (11, 13). These
results indicated that Stat6 and one or more members of the C/EBP class
of proteins may cooperate to activate transcription from this element
by a yet undefined mechanism.
The C/EBP class of proteins consists of several members with variable
and overlapping cellular expression patterns. Members of this family
have been shown to play an important role in energy metabolism
(C/EBP, -
), immune system function (C/EBPß, -, -), and
development (14, 15, 16, 17, 18, 19). C/EBPß, like Stat6, has been shown to be
expressed in lymphocytic, monocytic, and other cell types known to
mediate IL-4 signaling (20, 21). However, unlike Stat6, C/EBPß is
constitutively present and is not activated upon IL-4 stimulation.
Rather, numerous studies have shown that changes in expression, as well
as posttranslational modification of this and other C/EBP family
members, are brought about by other stimuli (22, 23).
In the current study, we investigate the functional and physical
interaction between Stat6 and several C/EBP proteins. We show that
Stat6 and C/EBPß activate transcription synergistically from the
germline IL-4-responsive element. The activation domains of both
proteins are essential for this functional synergy. Moreover, Stat6 and
C/EBPß interact with DNA such that the dissociation rate of Stat6 is
stabilized when C/EBPß is bound at the adjacent site. These results
help to explain the mechanism by which Stat6 and C/EBPß cooperate to
synergistically activate transcription from the Ig germline
promoter.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human embryonic kidney 293 cells and HepG2 cells were grown in
DMEM (Mediatech, Herndon, VA) containing 10% FCS (Mediatech
Herndon, VA). BJAB cells were grown in RPMI medium containing
10% FCS, 1 mM L-glutamine, and 10 µM
ß-mercaptoethanol. To measure luciferase activity, HEK293 and HepG2
cells were transfected in six-well plates using calcium phosphate
coprecipitation. The amount of DNA used in individual transfections is
given in the legend of each figure. The medium was changed 15 h
posttransfection, and after 42 h the cells were either induced
with 10 ng/ml human rIL-4 (R&D Systems, Minneapolis, MN) for
6 h or left untreated. A control plasmid expressing
ß-galactosidase under the control of the cytomegalovirus promoter was
cotransfected to determine the transfection efficiency. Luciferase and
ß-galactosidase activity were assayed using the luciferase and
ß-galactosidase assay systems (Promega, Madison, WI). Stable BJAB
cell lines overexpressing human Stat6, C/EBP, and C/EBPß were
generated as described by Tewari and Dixit (24). Resistant clones were
selected in 3 mg/ml G418 (Life Technologies, Gaithersburg, MD),
and positive clones overexpressing the recombinant proteins were
identified by Western analysis using anti-Stat6, anti-C/EBP,
or anti-C/EBPß Abs. The cells were then transiently transfected
by electroporation using 30 µg of the IL-4-inducible reporter plasmid
and 10 µg of the ß-galactosidase-expressing control plasmid as
described (24). After 24 h, cells were stimulated with 10 ng/ml
IL-4 (R&D Systems) for 6 h or left untreated. Luciferase and
ß-galactosidase activity were assayed using the corresponding assay
systems (Promega).
Expression constructs
Mammalian and baculovirus expression constructs encoding Stat6
and Stat6(Ad-) have been described previously (13, 25). Constructs expressing the C/EBP isoforms were generated as
follows: DNA fragments encoding C/EBP, C/EBPß, and C/EBP were
obtained using the PCR and primers carrying EcoRI sites. The
DNA fragments were subcloned into the EcoRI site of pcDNA3.
The orientation and integrity of the clones were determined by DNA
sequence analysis. The following primer sequences were used to
subclone the C-terminal bzip (basic leucine zipper; DNA-binding domain)
region of human C/EBPß (designated C/EBPß(Ad-) in the
text) into the pcDNA3 expression vector: 5' primer, GCA GAC GAA TTC GCC
ACC ATG GTC AAG AGC AAG GCC AAG AAG; 3' primer, ACG AGC GAA TTC CTA TCA
TCA GCA GTG GCC GGA. The reporter constructs TPU474 (wild type)
and TPU475 (C/EBP site mutated) have been described previously (13).
Purification of C/EBPß, Stat6, and Jak1
C/EBPß was partially purified as follows. The protein was transiently overexpressed in HEK293 cells and nuclear extract was prepared (7, 13). The extract was passed over a heparin sulfate column that had been equilibrated with nuclear extract buffer (7). Bound proteins were eluted with buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 50 mM, 200 mM, 500 mM, and 1000 mM of NaCl. Fractions were assayed for activity using electrophoretic mobility shift assay (EMSA). The 200 mM NaCl fraction contained the highest level of C/EBPß activity and was generally used in subsequent experiments. Recombinant histidine-tagged Stat6 and Jak1 were expressed in High Five cells (Invitrogen, San Diego, CA) using the baculovirus expression system (PharMingen, San Diego, CA). The proteins were purified using nickel chromatography (Qiagen, Valencia, CA) as described previously (25).
In vitro activation of Stat6
Purified Stat6 was activated in vitro using purified Jak1 kinase. Both proteins were expressed in High Five cells. The kinase reaction was conducted as follows: 0.5 µg Jak1 and 1 µg Stat6 were incubated in 10 mM HEPES pH 7.4, 50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 50 µM ATP, and 0.1 mM Na3VO4 for 30 min at room temperature. One microliter of the reaction was used for EMSA.
DNA binding assays, EMSA, and "trap" experiments
Labeling of DNA probes, DNA binding, and EMSAs were performed as previously described (13). Trap experiments were done as follows: Stat6 and C/EBPß, either alone or in combination, were incubated with 0.1 pmol of labeled probe containing both the Stat6 and C/EBP binding site. The binding reaction was allowed to reach equilibrium to be reached for 2 min. Then, 20 pmol of unlabled DNA (trap) identical in sequence to the probe DNA was added to the reaction mix. Aliquots were taken at various times and loaded onto a continuously running native gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that the minimal IL-4 responsive
element in the mouse and human promoters is a composite element of
a Stat6 and a C/EBP binding site (11). This element, when fused to a
truncated promoter, is able to promote transcription in an
IL-4-dependent manner (11, 13). These observations indicate that Stat6
and one or more member of the C/EBP class of proteins cooperate to
activate transcription from this regulatory element. To study the
nature of this cooperativity, we transiently overexpressed Stat6,
C/EBP, and C/EBPß in HEK293 cells in the presence of an
IL-4-inducible reporter construct carrying four copies of the composite
IL-4 response element derived from the human germline promoter
(13). Previously, we showed that HEK293 cells do not express Stat6
(13). However, they do contain all other components of the IL-4
signaling pathway, such as the IL-4R and Jak kinases, which are
essential for IL-4-induced gene activation. Thus, as Figure 1
A shows, no IL-4-dependent
activation was observed in the absence of exogenous Stat6 (Fig. 1A). Overexpression of Stat6 resulted in a 5-fold increase
of IL-4-induced luciferase activity (13, Fig. 1, A and
B). Overexpression of C/EBP or C/EBPß elevated the
basal level of transcription but did not confer IL-4 responsiveness
(Fig. 1A). However, the combination of Stat6 and C/EBPß
resulted in a dramatic increase in IL-4-induced transcription
(11-fold). This increase was significantly greater than the sum of each
protein’s individual ability to drive transcription from this reporter
construct. Thus, Stat6 and C/EBPß functionally synergize to activate
transcription from the human IL-4 responsive element.
Overexpression of Stat6 and C/EBP also showed an increase in basal
and IL-4-induced transcription (Fig. 1A). However, the fold
induction in response to IL-4 was identical to the one observed in the
absence of C/EBP (Fig. 1B). Hence, the IL-4-dependent
synergistic effect seen with C/EBPß is far more pronounced than that
seen with C/EBP.
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or C/EBPß were prepared and then transiently transfected with the
IL-4-inducible reporter construct. The parental BJAB cell line showed a
3-fold increase in luciferase activity following IL-4 treatment (Fig. 1
B). A 10-fold increase was observed in cells that
overexpressed Stat6. As observed in HEK293 cells, overexpression of
C/EBP gave rise to elevated basal as well as IL-4-induced
transcription (data not shown), resulting in no increase in IL-4
inducibility when compared with the parental cell line (Fig. 1B). In contrast, BJAB cells that overexpressed C/EBPß did
not show an elevated basal level compared with the parental line.
However, a 75-fold increase in luciferase activity was seen in response
to IL-4 treatment. This specific increase in reporter activity upon
IL-4 treatment strongly implies a cooperative interaction between Stat6
and C/EBPß to synergistically drive transcription from the IL-4
response element. This finding is further supported by using reporter
constructs that carried mutations in the C/EBP binding site. In HEK293,
BJAB (data not shown), or HepG2 cells (see below), IL-4-induced
promoter activity was completely abolished even in the presence of
overexpressed C/EBPß when the mutant reporter was used. Activation domains of both Stat6 and C/EBP ß are required for synergistic activation
Previously we showed that a mutant version of Stat6 lacking the
C-terminal 186 amino acids is unable to activate transcription,
although this deletion does not affect IL-4-dependent phosphorylation
or DNA binding affinity. These and other experiments mapped the
transcriptional activation domain to the C-terminal 186 amino acids of
Stat6 (13). Now we wanted to determine whether the activation domain of
C/EBP ß was also an essential domain contributing to the ability of
these two proteins to drive transcription from the IL-4-responsive
element. Figure 2 shows the results
obtained with transiently transfected HEK293 cells. In these
experiments, we expressed the truncated versions of Stat6 and C/EBPß
lacking the transcription activation domains (Stat6(Ad-)
and C/EBPß(Ad-)), either alone or in combination with
the full length form of the other protein. No IL-4-induced
transcription was observed when either one of the two truncated
proteins (Stat6(Ad-) or C/EBPß(Ad-)) was
overexpressed. Furthermore, the combination of wild-type Stat6 and
C/EBPß(Ad-) or Stat6(Ad-) and wild-type
C/EBPß resulted in a transcription readout identical to the single,
full length partner alone. The results obtained with both full length
proteins are shown for comparison. These data clearly show that the
activation domains of both Stat6 and C/EBPß are absolutely essential
to synergistically activate transcription from this IL-4-responsive
element.
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on IL-4-induced transcription
Previously, we showed that Stat6 lacking the activation domain
(Stat6(Ad-)) is able to bind DNA and behaves as a potent
dominant negative when transiently expressed in HepG2 cells that
contain endogenous Stat6 and C/EBPß (13). Based on the observation
that C/EBPß(Ad-) is unable to cooperate with Stat6 to
activate transcription (Fig. 2), we were interested to see whether
expression of this protein would also have a dominant negative effect
on IL-4-induced transcription in HepG2 cells.
C/EBPß(Ad-) is able to bind DNA as well as the full
length protein (data not shown). As Figure 3 shows, strong activation was seen in
response to IL-4 treatment when the reporter construct was transiently
transfected into these cells, even in the absence of any added
activator. Consistent with previous data (13), a reporter carrying a
mutation in the C/EBP binding site could not be activated in response
to IL-4 stimulation.
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Overexpression of either Stat6(Ad-) or C/EBPß(Ad-) completely suppressed IL-4-induced activation of the wild-type reporter construct. These experiments clearly support the observations that both proteins, Stat6 and C/EBPß, have to be present to drive IL-4-induced transcription and that the integrity of both activation domains is required for this function.
C/EBP is a ubiquitously expressed member of the C/EBP family of
proteins (26). It lacks an activation domain, and it has been
implicated in suppression of transcription at genes known to be
activated by other members of the C/EBP class of proteins, including
C/EBPß (18). C/EBP is able to bind to the site of the IL-4
response element of the promoter (data not shown). Hence, we were
interested to see what effect C/EBP may have on IL-4-induced
promoter activity. Figure 3A shows that overexpression of
C/EBP also suppressed IL-4-induced transcription, identical to the
dominant negative effects seen with Stat6(Ad-) or
C/EBPß(Ad-). To further investigate the inhibitory
effects of C/EBP on IL-4-induced transcription from this reporter
element, we overexpressed increasing amounts of C/EBPß in the
presence or absence of C/EBP in HepG2 cells. Figure 3B
shows that C/EBP suppresses both the endogenous IL-4 response as
well as the elevated IL-4 response mediated by overexpressed C/EBPß.
This observation raises the possibility that the balance between
C/EBPß and C/EBP may determine the extent of germline
transcription in B cells following IL-4 treatment.
Both Stat6 and C/EBPß bind the human promoter
IL-4-responsive element
It has often been shown that adjacently bound transcription
factors that functionally cooperate to activate transcription also
physically associate to some extent, either in the presence or absence
of their cognate DNA elements (27, 28). In an attempt to determine the
mechanism underlying the functional synergy observed between Stat6 and
C/EBPß, we explored whether the two proteins facilitate each other in
their interaction with the IL-4-responsive element. Human Stat6
overexpressed in insect cells does not bind DNA, indicating that it
does not become tyrosine phosphorylated in these cells (data not
shown). Therefore, the protein was purified to homogeneity and
activated in vitro using recombinant Jak1, which also had been
expressed and purified from insect cells. Human C/EBPß was
overexpressed in HEK293 cells and partially purified. The proteins were
then incubated with radiolabeled DNA probes carrying either both
protein binding sites or mutations within either one of the two sites
(Fig. 4). Both proteins, Stat6 and
C/EBPß, bound the wild-type sequence independently, resulting in the
formation of complex A (mediated by C/EBPß) and complex B (mediated
by Stat6). When the two proteins were mixed in the presence of the
wild-type probe, an additional, more slowly migrating complex C was
observed. Using Abs directed against either Stat6 or C/EBPß we could
show that both proteins are present in complex C. These experiments
were done in the presence of excess probe. Hence, the relative amount
of material present in complex C suggests that some degree of DNA
binding cooperativity exists for these two proteins.
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show that a mutation in the
C/EBP binding site eliminates C/EBPß binding without affecting Stat6
binding. Similarly, Stat6 binding is abolished if the probe carries a
mutation in the Stat6 binding site, but C/EBPß binding is unaffected
(DNA Probe III). Formation of complex C is not seen with either one of
the mutant probes. In an attempt to show a physical association between these two proteins in the absence of DNA, we performed coimmunoprecipitation experiments with cellular extracts that contained Stat6 and C/EBPß. These experiments did not show a physical association between the two proteins in either the presence or absence of IL-4 treatment (data not shown).
The presence of C/EBPß reduces the fast dissociation rate of
Stat6 bound to the IL-4-responsive element
We next investigated whether the dissociation rate between Stat6
and the IL-4-responsive element would be effected when C/EBPß is
bound to the adjacent site. A slower dissociation rate in the double
occupancy situation would be indicative of a cooperative interaction
between these two proteins while bound to DNA and might contribute to
the functional synergy seen in our reporter assays. Band shift
experiments, like those shown in Figure 4, are equilibrium measurements
of the proteins affinity for its DNA binding site. To determine the
dissociation rate of Stat6 and C/EBPß when bound to DNA, we
investigated the stability of the protein/DNA complexes, as shown in
Figure 5. Stat6 and C/EBPß were
incubated either separately or together with the labeled probe.
After a brief incubation period to allow the protein-DNA binding
equilibrium to be reached with the labeled probe, an excess of
unlabeled oligonucleotides (trap) carrying the same DNA sequence as the
probe was added to the reaction mix. The time-dependent disappearance
of the radiolabeled protein-DNA complex reflects the dissociation rate
of the individual protein and the bound DNA probe (29). This can be
visualized because subsequent rebinding events are far more likely to
occur to the unlabeled DNA fragments, which are in vast excess over the
labeled probe. Several repeats of this experiment showed that in the
absence of C/EBPß, Stat6 rapidly dissociates from its cognate DNA
sequence; after 0.5 min, the majority of Stat6 was released from the
radiolabeled oligonucleotide (Fig. 5, left panel). In
contrast, C/EBPß has a much slower dissociation rate. Even after 12
min, some C/EBPß still remained bound to the radiolabeled
oligonucleotide (right panel). When both proteins bind the
same DNA probe (state of double occupancy), the dissociation rate of
Stat6 from complex C is reduced. Complex C is still visible after 2 min
(middle panel). The increased stability of Stat6 in the
double occupancy complex suggests some physical contact between Stat6
and C/EBPß or a change in the conformation of the DNA that stabilizes
the binding of Stat6 when C/EBPß is bound at the adjacent site.
Interestingly, the increased residence time of Stat6 in the double
occupancy complex was independent of the integrity of the activation
domain; e.g., truncated proteins (Stat6 or C/EBPß) lacking the
activation domain also resulted in more stable protein/DNA complexes
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter in B lymphocytes triggers the
recombination event leading to class switching and expression of the
IgE isotype. The promoter is activated in response to multiple
signals, the most crucial being IL-4 (11). The IL-4 response element
has previously been characterized and shown to contain binding sites
for both Stat6 and C/EBP (11, 13). In this study, we explored possible
mechanisms underlying the functional synergy seen between Stat6 and
C/EBP in driving IL-4-induced expression of the germline sterile
transcript. We investigated whether Stat6 would cooperate with
different members of the C/EBP family. Previously, we showed that
HEK293 cells lack Stat6 (13). Furthermore, only low amounts of C/EBP
proteins can be detected in these cells using Western blots or gel
shift experiments (data not shown). These data are substantiated by the
observation that very low luciferase activity was obtained with a
C/EBP-dependent reporter construct in the absence of C/EBP
overexpression. Hence, transfection studies in HEK293 cells allowed us
to determine the relative contribution of each protein (Stat6 or C/EBP)
to activate transcription from the IL-4-dependent reporter. These
experiments clearly show that overexpression of Stat6 alone is
sufficient to drive a low level of IL-4-induced expression. In
contrast, overexpression of C/EBP or C/EBPß elevates basal
expression, but does not trigger IL-4-dependent transcription. However,
both C/EBP isoforms, when expressed in the presence of Stat6, greatly
increase the overall luciferase activity in response to IL-4 treatment,
but C/EBPß is significantly more effective than C/EBP. The
increase in IL-4-induced transcription seen when both Stat6 and
C/EBPß were overexpressed is far greater than the sum of each
protein’s individual ability to drive transcription from this
reporter. This superadditive effect is a defining feature of
transcriptional synergy (27, 28). These results were most dramatic with
C/EBPß in the human B cell line BJAB, suggesting that C/EBPß is
limiting in these cells and that C/EBPß rather than C/EBP
cooperates with Stat6 to drive IL-4-induced germline expression in
B cells.
The functional synergy between Stat6 and C/EBPß requires the
activation domains of both proteins. Deletion of either the Stat6 or
C/EBPß activation domain results in a protein that can still bind to
the element, but can no longer partner with the other protein to
synergistically activate transcription. These data were further
substantiated by overexpression studies in HepG2 cells. HepG2 cells
express both Stat6 and C/EBP and are able to activate our
IL-4-inducible reporter (13). Overexpression of either protein lacking
an activation domain suppressed promoter activity, arguing for an
almost equivalent role of C/EBPß and Stat6 in driving transcription
from the IL-4-responsive element. A dominant negative effect was
also observed with C/EBP, a naturally occurring C/EBP isoform that
lacks an activation domain. These results are particularly interesting
as this C/EBP family member has been implicated as playing a negative
regulatory role in antagonizing the function of other C/EBP proteins
(18, 20). Hence, the balance between C/EBPß and C/EBP may
determine the amount of sterile transcript produced in response to IL-4
stimulation.
While no naturally occurring splice variant of Stat6 lacking the
C-terminal activation domain has been yet characterized, such naturally
occurring splice variants have been observed for Stats 1, 3, 4, and 5
(30, 31, 32). Studies with these STAT proteins and additional cooperating
factors highlight similarities and differences when compared with our
observations with Stat6 and C/EBPß. The naturally occurring splice
variant of Stat3, designated Stat3ß, is missing most of the
C-terminal activation domain. Yet, both the full length form of Stat3
(Stat3) and Stat3ß can cooperate with c-Jun to activate
transcription from an IL-6-responsive element taken from the rat
2-macroglobulin gene (30). Similarly, both the full
length form of Stat4 (Stat4) and a splice variant lacking the
C-terminal activation domain (Stat4ß) can cooperate with c-Jun to
activate transcription from an IL-12-responsive element taken from the
IRF1 promoter (X. Xu and T. Hoey, unpublished observations). In
both these examples, activation function appears to be provided by
c-Jun, which binds at an adjacent site in each element (29, 30).
Yet at other STAT-activated promoter elements, the activation domains
of these proteins are clearly required (31, 32). In the case of
IFN--induced gene expression, activation is mediated by the
multiprotein complex IFN-stimulated gene factor-3, which is
composed of Stat1, Stat2, and the nuclear protein p48. Here, both the
full length Stat1 protein (Stat1) or the variant (Stat1ß) lacking
the activation domain can participate in the complex, because all
transactivation depends on Stat2 (33), whereas in the case of
IFN--induced gene expression, only the full length form of Stat1 is
capable of activating transcription (31). Hence, there appear to be
multiple mechanisms that allow STAT proteins to cooperate with other
transcription factors. Specific interactions are probably dictated by
the individual promoter context (31).
This is the first example of Stat6 functionally synergizing with
another transcription factor to activate transcription from an
IL-4-responsive element. In contrast, C/EBPß has been shown to
functionally synergize with a large number of transcription factors,
including those playing important roles in immune cell function, such
as NF-
B, AML1, Myb, and PU.1 (34, 35, 36, 37). In several of these examples,
C/EBPß has been shown to associate physically in some way with the
other cooperating factor. These and other examples of transcription
factors physically associating to mediate transcriptional synergy, as
well as the proximity between the Stat6 and C/EBP binding sites that is
conserved in both the mouse and human promoter, prompted us to
search for a physical association between C/EBPß and Stat6.
Our DNA binding studies showed that both proteins are able to
bind the promoter element independently. However, based on mobility
shift experiments conducted in the presence of excess DNA, simultaneous
binding of both proteins appeared to be favored over single occupancy,
suggesting some degree of facilitated DNA binding. To further
investigate the possibility for a direct interaction between these two
proteins, we measured the dissociation rates of Stat6 and C/EBPß when
bound to this DNA element. These experiments showed that in the double
occupancy state, where both Stat6 and C/EBP are bound to the probe, the
dissociation rate of Stat6 is slower than when it binds the probe
alone. This increased stability of Stat6 binding under double occupancy
conditions must be the result of some physical contact with the
adjacently bound C/EBPß, or possibly it is the result of a change in
the conformation of the DNA that stabilizes the binding of Stat6 when
C/EBPß binds the adjacent site. Although we and others (11) have
clearly shown that most isoforms of C/EBP are able to interact with the
IL-4 response element of the promoter, it remains to be determined
whether C/EBPß binds to this site in vivo.
The requirement for the activation domains of both Stat6 and C/EBPß
secures the IL-4 responsiveness of the germline promoter. Yet, it
is unclear how these activation domains interact to synergistically
activate transcription. Stat6(Ad-) also exhibited a
reduced dissociation rate from this element when C/EBPß was bound
(data not shown). Similarly, C/EBPß(Ad-) also stabilized
Stat6 binding, indicating that this effect is not due to the activation
domains. Thus, while the change in dissociation rate may contribute to
the synergistic effect seen in transcriptional activation, it is
clearly not the main component. A more likely mechanism could be that
both activation domains interact to adopt a unique conformation that
allows them to cooperate with the basal transcription machinery more
effectively than they can separately. Another possibility is that the
two activation domains independently interact with other proteins in
the basal machinery to achieve the same result. Alternatively, both of
these mechanisms could be operative simultaneously. Either way, the
decrease in dissociation rate of Stat6 in the presence of C/EBPß is
likely to increase the chances that the two activation domains will
interact (either with themselves, or with additional factors) to
activate transcription. Furthermore, the superadditive effect on
transcription activation ensures the strong induction of gene
expression at this immunologically important locus.
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Acknowledgments |
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clone and Dr.
Steven McKnight for the human C/EBP and C/EBPß clones and Abs
directed against different C/EBP isoforms. We also thank Keith
Williamson for DNA sequencing, Carla Daniel for providing purified
Stat6 and Jak1 proteins and for helpful discussions, and Drs. Cao
Zhaodan, Greg Peterson, and Tim Hoey for critical comments on the
manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Ulrike Schindler, Tularik Inc., Two Corporate Drive, South San Francisco, CA 94080. E-mail address:
3 Abbreviations used in this paper: Jak, Janus kinase; (Ad-), lacking the activation domain; C/EBP, CAAT/enhancer-binding protein; EMSA, electrophoretic mobility shift assay; HEK293, human embryonic kidney 293.
Received for publication December 15, 1997. Accepted for publication April 14, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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