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Synergistic Activation of the Germline ε Promoter Mediated by Stat6 and C/EBP β Thomas Mikita, Masae Kurama and Ulrike Schindler This information is current as J Immunol 1998; 161:1822-1828; ; of September 29, 2021. http://www.jimmunol.org/content/161/4/1822 Downloaded from References This article cites 37 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/161/4/1822.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Synergistic Activation of the Germline ⑀ Promoter Mediated by Stat6 and C/EBP␤

Thomas Mikita,1 Masae Kurama, and Ulrike Schindler2

Transcription of the Ig H chain germline transcripts is a prerequisite for class switching. Expression of the ⑀ 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/EBP␤ 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/EBP␤ bind the IL-4 response element simultaneously. The fast dissociation rate apparent when Stat6 binds this DNA element alone is Downloaded from slowed when C/EBP␤ binds at the neighboring site. These data suggest a mechanism whereby C/EBP␤ 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. The Journal of Immunology, 1998, 161: 1822–1828.

ytokine receptor binding initiates a cascade of intracel- cannot be induced in Stat6-deﬁcient mice, and IgE production is

lular signaling events leading to specific changes in gene profoundly impaired (9, 10). http://www.jimmunol.org/ C expression (1). IL-4 is a multifunctional cytokine that Stat6 binding sites have been identified in several of these IL- stimulates changes in many cell types. In the case of T helper cells, 4-responsive genes and are best characterized in the promoters that IL-4 drives the commitment to the Th2 phenotype. Th2 cells pro- govern the expression of the Ig germline ⑀ and ␥ transcripts (11, duce IL-4, IL-5, IL-6, and IL-10 (2, 3), cytokines that predomi- 12). IL-4-induced expression of these genes requires the integrity nantly influence humoral immune responses. IL-4 also plays a crit- of the Stat6 binding site as well as an adjacent site, which is bound ical role in B cell isotype switching and proliferation as well as in by C/EBP (11, 12). In the mouse germline ⑀ promoter, the Stat6 mast cell and eosinophil activity (1, 2). In contrast, IL-12 drives and C/EBP sites are separated by four base pairs; in the human the commitment to the Th1 subset, which increases cytokine se- promoter, the two sites are directly adjacent. Previous studies have by guest on September 29, 2021 cretion predominantly affecting cellular immune responses (2, 3). shown that either one of these composite elements (mouse or hu- Cytokines such as the interleukins and interferons have been man) is able to confer IL-4-induced transcription onto a heterolo- shown to rapidly activate a signaling pathway known as the Janus gous promoter (11, 13). These results indicated that Stat6 and one kinase (Jak)3-STAT pathway. Briefly, STAT proteins are activated or more members of the C/EBP class of proteins may cooperate to through tyrosine phosphorylation by receptor-associated Jak ki- activate transcription from this element by a yet undefined nases following cytokine binding. The activated STAT protein mechanism. dimerizes, translocates to the nucleus, and activates transcription The C/EBP class of proteins consists of several members with via specific DNA response elements (4–6). Currently, there are variable and overlapping cellular expression patterns. Members of seven known members of the STAT family that are characterized and that are activated by different cytokines. In the present study, this family have been shown to play an important role in energy ␣ ␦ ␤ ␥ we focused on Stat6, which is activated upon IL-4 stimulation (7, metabolism (C/EBP ,- ), immune system function (C/EBP ,- , ⑀ ␤ 8). The generation of Stat6-deficient mice has established the re- - ), and development (14–19). C/EBP , like Stat6, has been quirement of this protein in promoting polarization of T helper shown to be expressed in lymphocytic, monocytic, and other cell cells toward the Th2 phenotype. Furthermore, genes that are acti- types known to mediate IL-4 signaling (20, 21). However, unlike ␤ vated in response to IL-4, such as CD23 and MHC class II genes, Stat6, C/EBP 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 Tularik Inc., South San Francisco, CA 94080 this and other C/EBP family members, are brought about by other Received for publication December 15, 1997. Accepted for publication April stimuli (22, 23). 14, 1998. In the current study, we investigate the functional and physical The costs of publication of this article were defrayed in part by the payment of page interaction between Stat6 and several C/EBP proteins. We show charges. This article must therefore be hereby marked advertisement in accordance ␤ with 18 U.S.C. Section 1734 solely to indicate this fact. that Stat6 and C/EBP activate transcription synergistically from the germline ⑀ IL-4-responsive element. The activation domains of 1 Current address: Dr. Tom Mikita, Molecular Oncology, Genentech Inc., Mail Stop 40, One DNA Way, South San Francisco, CA 94080. both proteins are essential for this functional synergy. Moreover, 2 Address correspondence and reprint requests to Dr. Ulrike Schindler, Tularik Inc., Stat6 and C/EBP␤ interact with DNA such that the dissociation Two Corporate Drive, South San Francisco, CA 94080. E-mail address: rate of Stat6 is stabilized when C/EBP␤ is bound at the adjacent [email protected] Ϫ site. These results help to explain the mechanism by which Stat6 3 Abbreviations used in this paper: Jak, Janus kinase; (Ad ), lacking the activation ␤ domain; C/EBP, CAAT/enhancer-binding protein; EMSA, electrophoretic mobility and C/EBP cooperate to synergistically activate transcription shift assay; HEK293, human embryonic kidney 293. from the Ig germline ⑀ promoter.

Materials and Methods Results Cell culture and transient transfections Stat6 and C/EBP␤ synergistically activate transcription in an Human embryonic kidney 293 cells and HepG2 cells were grown in IL-4-dependent manner DMEM (Mediatech, Herndon, VA) containing 10% FCS (Mediatech Hern- Previous studies have shown that the minimal IL-4 responsive don, VA). BJAB cells were grown in RPMI medium containing 10% FCS, element in the mouse and human ⑀ promoters is a composite 1mML-glutamine, and 10 ␮M ␤-mercaptoethanol. To measure luciferase activity, HEK293 and HepG2 cells were transfected in six-well plates using element of a Stat6 and a C/EBP binding site (11). This element, calcium phosphate coprecipitation. The amount of DNA used in individual when fused to a truncated promoter, is able to promote tran- transfections is given in the legend of each figure. The medium was scription in an IL-4-dependent manner (11, 13). These obser- changed 15 h posttransfection, and after 42 h the cells were either induced vations indicate that Stat6 and one or more member of the with 10 ng/ml human rIL-4 (R&D Systems, Minneapolis, MN) for6hor left untreated. A control plasmid expressing ␤-galactosidase under the con- C/EBP class of proteins cooperate to activate transcription from trol of the cytomegalovirus promoter was cotransfected to determine the this regulatory element. To study the nature of this cooperativ- transfection efficiency. Luciferase and ␤-galactosidase activity were as- ity, we transiently overexpressed Stat6, C/EBP␣, and C/EBP␤ sayed using the luciferase and ␤-galactosidase assay systems (Promega, in HEK293 cells in the presence of an IL-4-inducible reporter Madison, WI). Stable BJAB cell lines overexpressing human Stat6, C/EBP␣, and C/EBP␤ were generated as described by Tewari and Dixit construct carrying four copies of the composite IL-4 response (24). Resistant clones were selected in 3 mg/ml G418 (Life Technologies, element derived from the human germline ⑀ promoter (13). Pre- Gaithersburg, MD), and positive clones overexpressing the recombinant viously, we showed that HEK293 cells do not express Stat6 proteins were identified by Western analysis using anti-Stat6, anti- (13). However, they do contain all other components of the IL-4 C/EBP␣, or anti-C/EBP␤ Abs. The cells were then transiently transfected signaling pathway, such as the IL-4R and Jak kinases, which are Downloaded from by electroporation using 30 ␮g of the IL-4-inducible reporter plasmid and 10 ␮gofthe␤-galactosidase-expressing control plasmid as described (24). essential for IL-4-induced gene activation. Thus, as Figure 1A After 24 h, cells were stimulated with 10 ng/ml IL-4 (R&D Systems) for shows, no IL-4-dependent activation was observed in the ab- 6 h or left untreated. Luciferase and ␤-galactosidase activity were assayed sence of exogenous Stat6 (Fig. 1A). Overexpression of Stat6 using the corresponding assay systems (Promega). resulted in a 5-fold increase of IL-4-induced luciferase activity Expression constructs (13, Fig. 1, A and B). Overexpression of C/EBP␣ or C/EBP␤

elevated the basal level of transcription but did not confer IL-4 http://www.jimmunol.org/ Mammalian and baculovirus expression constructs encoding Stat6 and Stat6(AdϪ) have been described previously (13, 25). Constructs expressing responsiveness (Fig. 1A). However, the combination of Stat6 the C/EBP isoforms were generated as follows: DNA fragments encoding and C/EBP␤ resulted in a dramatic increase in IL-4-induced C/EBP␣, C/EBP␤, and C/EBP␥ were obtained using the PCR and primers transcription (11-fold). This increase was significantly greater carrying EcoRI sites. The DNA fragments were subcloned into the EcoRI than the sum of each protein’s individual ability to drive tran- site of pcDNA3. The orientation and integrity of the clones were deter- ␤ mined by DNA sequence analysis. The following primer sequences were scription from this reporter construct. Thus, Stat6 and C/EBP used to subclone the C-terminal bzip (basic leucine zipper; DNA-binding functionally synergize to activate transcription from the human domain) region of human C/EBP␤ (designated C/EBP␤(AdϪ) in the text) ⑀ IL-4 responsive element. Overexpression of Stat6 and into the pcDNA3 expression vector: 5Ј primer, GCA GAC GAA TTC GCC C/EBP␣ also showed an increase in basal and IL-4-induced Ј ACC ATG GTC AAG AGC AAG GCC AAG AAG; 3 primer, ACG AGC transcription (Fig. 1A). However, the fold induction in response by guest on September 29, 2021 GAA TTC CTA TCA TCA GCA GTG GCC GGA. The reporter constructs TPU474 (wild type) and TPU475 (C/EBP site mutated) have been de- to IL-4 was identical to the one observed in the absence of scribed previously (13). C/EBP␣ (Fig. 1B). Hence, the IL-4-dependent synergistic effect seen with C/EBP␤ is far more pronounced than that seen with Purification of C/EBP␤, Stat6, and Jak1 C/EBP␣. C/EBP␤ was partially purified as follows. The protein was transiently over- To further substantiate the functional synergy between Stat6 expressed in HEK293 cells and nuclear extract was prepared (7, 13). The and C/EBP␤, we explored the cooperativity between the two extract was passed over a heparin sulfate column that had been equilibrated with nuclear extract buffer (7). Bound proteins were eluted with buffer (10 proteins in a lymphoid cell line. BJAB cells, a human B cell mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 50 mM, 200 mM, 500 line, have been shown to express Stat6, and IL-4-responsive mM, and 1000 mM of NaCl. Fractions were assayed for activity using genes are properly regulated (unpublished data). Stable cell electrophoretic mobility shift assay (EMSA). The 200 mM NaCl fraction lines that overexpress Stat6, C/EBP␣, or C/EBP␤ were prepared contained the highest level of C/EBP␤ activity and was generally used in subsequent experiments. Recombinant histidine-tagged Stat6 and Jak1 and then transiently transfected with the IL-4-inducible reporter were expressed in High Five cells (Invitrogen, San Diego, CA) using the construct. The parental BJAB cell line showed a 3-fold increase baculovirus expression system (PharMingen, San Diego, CA). The proteins in luciferase activity following IL-4 treatment (Fig. 1B). A 10- were purified using nickel chromatography (Qiagen, Valencia, CA) as de- fold increase was observed in cells that overexpressed Stat6. As scribed previously (25). observed in HEK293 cells, overexpression of C/EBP␣ gave rise In vitro activation of Stat6 to elevated basal as well as IL-4-induced transcription (data not shown), resulting in no increase in IL-4 inducibility when com- Purified Stat6 was activated in vitro using purified Jak1 kinase. Both proteins were expressed in High Five cells. The kinase reaction was conducted pared with the parental cell line (Fig. 1B). In contrast, BJAB as follows: 0.5 ␮g Jak1 and 1 ␮g Stat6 were incubated in 10 mM HEPES cells that overexpressed C/EBP␤ did not show an elevated basal ␮ pH 7.4, 50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2,50 M ATP, and 0.1 level compared with the parental line. However, a 75-fold in- mM Na3VO4 for 30 min at room temperature. One microliter of the reac- crease in luciferase activity was seen in response to IL-4 treat- tion was used for EMSA. ment. This specific increase in reporter activity upon IL-4 treat- DNA binding assays, EMSA, and “trap” experiments ment strongly implies a cooperative interaction between Stat6 ␤ ⑀ Labeling of DNA probes, DNA binding, and EMSAs were performed as and C/EBP to synergistically drive transcription from the previously described (13). Trap experiments were done as follows: Stat6 IL-4 response element. This finding is further supported by us- and C/EBP␤, either alone or in combination, were incubated with 0.1 pmol ing reporter constructs that carried mutations in the C/EBP of labeled probe containing both the Stat6 and C/EBP binding site. The binding site. In HEK293, BJAB (data not shown), or HepG2 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 cells (see below), IL-4-induced promoter activity was com- DNA was added to the reaction mix. Aliquots were taken at various times pletely abolished even in the presence of overexpressed and loaded onto a continuously running native gel. C/EBP␤ when the mutant reporter was used. 1824 IL-4-INDUCED TRANSCRIPTION Downloaded from FIGURE 2. Activation domains of both Stat6 and C/EBP␤ are required for synergistic transcription. HEK293 cells were transfected with the IL- 4-responsive reporter construct (0.75 ␮g) in the presence or absence of the indicated activator constructs (0.75 ␮g each). The full length proteins, Stat6 and C/EBP␤ (␤), and the truncated proteins lacking the activation domain, Stat6(AdϪ) and C/EBP␤(AdϪ), were coexpressed either alone or ␤

in combination, as indicated below the bars. Luciferase and -galactosi- http://www.jimmunol.org/ dase activity was determined 48 h posttransfection in either unstimulated cells or cells that had been stimulated with IL-4 for 6 h.

activation domain of C/EBP ␤ was also an essential domain con- tributing 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 by guest on September 29, 2021 C/EBP␤ lacking the transcription activation domains (Stat6(AdϪ) and C/EBP␤(AdϪ)), 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 Ϫ ␤ Ϫ ␤ (Stat6(Ad ) or C/EBP (Ad )) was overexpressed. Furthermore, FIGURE 1. Stat6 and C/EBP synergistically activate transcription ␤ Ϫ from the IL-4-responsive element of the human germline ⑀ promoter. A, the combination of wild-type Stat6 and C/EBP (Ad )or Ϫ ␤ Transient expression studies using nonlymphoid cells. HEK293 cells were Stat6(Ad ) and wild-type C/EBP resulted in a transcription read- transfected with an IL-4-inducible reporter construct (0.75 ␮g) in the pres- out identical to the single, full length partner alone. The results ence or absence of the indicated activator constructs (0.75 ␮g). Luciferase obtained with both full length proteins are shown for comparison. and ␤-galactosidase activity were determined 48 h posttransfection from These data clearly show that the activation domains of both Stat6 either untreated cells or cells that had been stimulated with IL-4 for 6 h. ␣, and C/EBP␤ are absolutely essential to synergistically activate C/EBP␣; ␤, C/EBP␤. Mean values and SDs of a total of three independent transcription from this IL-4-responsive element. experiments are shown. B, Comparison of IL-4 inducibility in nonlymphoid and lymphoid cells. The ratio of luciferase activity obtained in the Dominant negative effects of Stat6(AdϪ), C/EBP␤(AdϪ), and presence and absence of IL-4 is given and expressed as fold induction. For C/EBP␥ on IL-4-induced transcription HEK293 cells, the values were calculated from the data shown in A. Stably ␣ Previously, we showed that Stat6 lacking the activation domain transfected BJAB cells expressing the indicated activator proteins ( , Ϫ C/EBP␣; ␤, C/EBP␤) were transiently transfected with 30 ␮g of the IL- (Stat6(Ad )) is able to bind DNA and behaves as a potent dom- 4-inducible reporter construct and a control plasmid. Luciferase and ␤-ga- inant negative when transiently expressed in HepG2 cells that con- lactosidase activity was determined from IL-4-treated (6-h stimulation) or tain endogenous Stat6 and C/EBP␤ (13). Based on the observation untreated cells. that C/EBP␤(AdϪ) 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- ␤ Activation domains of both Stat6 and C/EBP are required for 4-induced transcription in HepG2 cells. C/EBP␤(AdϪ) is able to synergistic activation bind DNA as well as the full length protein (data not shown). As Previously we showed that a mutant version of Stat6 lacking the Figure 3 shows, strong activation was seen in response to IL-4 C-terminal 186 amino acids is unable to activate transcription, al- treatment when the reporter construct was transiently transfected though this deletion does not affect IL-4-dependent phosphoryla- into these cells, even in the absence of any added activator. Con- tion or DNA binding afﬁnity. These and other experiments mapped sistent with previous data (13), a reporter carrying a mutation in the transcriptional activation domain to the C-terminal 186 amino the C/EBP binding site could not be activated in response to IL-4 acids of Stat6 (13). Now we wanted to determine whether the stimulation. The Journal of Immunology 1825

lutely dependent on the integrity of the C/EBP binding site adjacent to the Stat6 site of this IL-4-responsive element. Overexpression of either Stat6(AdϪ) or C/EBP␤(AdϪ) com- pletely suppressed IL-4-induced activation of the wild-type reporter construct. These experiments clearly support the observations that both proteins, Stat6 and C/EBP␤, 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/EBP␤ (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(AdϪ)or C/EBP␤(AdϪ). To further investigate the inhibitory effects of Downloaded from C/EBP␥ on IL-4-induced transcription from this reporter element, we overexpressed increasing amounts of C/EBP␤ 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/EBP␤. This observation raises the possibility that the balance between http://www.jimmunol.org/ C/EBP␤ and C/EBP␥ may determine the extent of germline ⑀ transcription in B cells following IL-4 treatment.

Both Stat6 and C/EBP␤ 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 by guest on September 29, 2021 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/EBP␤, 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 FIGURE 3. A, Dominant negative effect of Stat6 derivatives and C/EBP cells (data not shown). Therefore, the protein was purified to ho- isoforms. HepG2 cells expressing endogenous Stat6 and C/EBP were tran- mogeneity and activated in vitro using recombinant Jak1, which ␮ siently transfected with the IL-4-inducible reporter construct (0.75 g) in also had been expressed and purified from insect cells. Human the absence or presence of the indicated activator constructs (0.75 ␮g): ␤, Ϫ C/EBP␤ was overexpressed in HEK293 cells and partially puri- C/EBP␤; ␥, C/EBP␥; (Ad ), proteins lacking the activation domain. wt, wild-type reporter; mutant, reporter construct carrying mutations in the fied. The proteins were then incubated with radiolabeled DNA C/EBP binding site (see Fig. 4). B, C/EBP␥ represses both endogenous and probes carrying either both protein binding sites or mutations exogenous C/EBP␤ activity. HepG2 cells were transfected with the IL-4- within either one of the two sites (Fig. 4). Both proteins, Stat6 and inducible reporter construct in the absence (Ϫ) or presence (ϩ) of over- C/EBP␤, bound the wild-type sequence independently, resulting in expressed C/EBP␥ (0.75 ␮g each). Increasing amounts (0.025, 0.08, and the formation of complex A (mediated by C/EBP␤) and complex 0.25 ␮g) of C/EBP␤ were cotransfected as indicated. For both panels, B (mediated by Stat6). When the two proteins were mixed in the luciferase and ␤-galactosidase activity were determined 48 h posttransfec- presence of the wild-type probe, an additional, more slowly mi- tion in either unstimulated cells or IL-4-treated cells (6 h of IL-4 treat- grating complex C was observed. Using Abs directed against either ment). Mean values and SDs of a total of three independent experiments Stat6 or C/EBP␤ we could show that both proteins are present in are shown. complex C. These experiments were done in the presence of excess probe. Hence, the relative amount of material present in complex Overexpression of C/EBP␤ in the presence of the wild-type re- C suggests that some degree of DNA binding cooperativity exists porter resulted in a significant increase of both basal and IL-4- for these two proteins. induced promoter activity. These results are consistent with the The lanes (DNA Probe II) in Figure 4 show that a mutation in observations made in HEK 293 cells. In contrast, no IL-4-specific the C/EBP binding site eliminates C/EBP␤ binding without affect- increase was seen with the mutant reporter in the presence of ex- ing Stat6 binding. Similarly, Stat6 binding is abolished if the probe ogenous C/EBP␤, although a slight elevation in basal expression carries a mutation in the Stat6 binding site, but C/EBP␤ binding is was observed. These data clearly show that the increase in IL-4- unaffected (DNA Probe III). Formation of complex C is not seen induced activation mediated by overexpressed C/EBP␤ is abso- with either one of the mutant probes. 1826 IL-4-INDUCED TRANSCRIPTION

plex 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/EBP␤, 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/EBP␤ has a much slower dissociation rate. Even after 12 min, some C/EBP␤ 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/EBP␤ or a change in the conformation of the DNA that stabilizes the binding of Stat6 when C/EBP␤ 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; Downloaded from e.g., truncated proteins (Stat6 or C/EBP␤) lacking the activation domain also resulted in more stable protein/DNA complexes (data not shown).

Discussion http://www.jimmunol.org/ ␤ FIGURE 4. Both Stat6 and C/EBP bind the IL-4-responsive element ⑀ of the human ⑀ promoter. EMSA: the DNA sequence of the probes used in Activation of the promoter in B lymphocytes triggers the recom- each lane are shown. Probe I represents the wild-type sequence; probes II bination event leading to class switching and expression of the IgE and III carry mutations in the C/EBP and Stat6 binding sites, respectively. isotype. The ⑀ promoter is activated in response to multiple sig- Mutations are given in lower case, unchanged nucleotides are represented nals, the most crucial being IL-4 (11). The IL-4 response element by hyphens. Purified Stat6 was activated in vitro with Jak1; C/EBP␤ (␤) has previously been characterized and shown to contain binding was partially purified from transiently transfected HEK293 cells. The two sites for both Stat6 and C/EBP (11, 13). In this study, we explored proteins were incubated with the DNA either together or separately as possible mechanisms underlying the functional synergy seen be- ␤ ␤ indicated above each lane. Abs directed against C/EBP (anti ) or Stat6 tween Stat6 and C/EBP in driving IL-4-induced expression of the by guest on September 29, 2021 (anti Stat6) were included in the binding reactions to probe complex C for germline ⑀ sterile transcript. We investigated whether Stat6 would the presence of Stat6 and C/EBP␤. Complex A is mediated by C/EBP␤, complex B by Stat6. 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 In an attempt to show a physical association between these two Western blots or gel shift experiments (data not shown). These proteins in the absence of DNA, we performed coimmunoprecipi- data are substantiated by the observation that very low luciferase tation experiments with cellular extracts that contained Stat6 and activity was obtained with a C/EBP-dependent reporter construct C/EBP␤. These experiments did not show a physical association in the absence of C/EBP overexpression. Hence, transfection stud- between the two proteins in either the presence or absence of IL-4 ies in HEK293 cells allowed us to determine the relative contri- treatment (data not shown). bution of each protein (Stat6 or C/EBP) to activate transcription from the IL-4-dependent reporter. These experiments clearly show ␤ The presence of C/EBP reduces the fast dissociation rate of that overexpression of Stat6 alone is sufficient to drive a low level ⑀ Stat6 bound to the IL-4-responsive element of IL-4-induced expression. In contrast, overexpression of We next investigated whether the dissociation rate between Stat6 C/EBP␣ or C/EBP␤ elevates basal expression, but does not trigger and the ⑀ IL-4-responsive element would be effected when IL-4-dependent transcription. However, both C/EBP isoforms, C/EBP␤ is bound to the adjacent site. A slower dissociation rate in when expressed in the presence of Stat6, greatly increase the over- the double occupancy situation would be indicative of a coopera- all luciferase activity in response to IL-4 treatment, but C/EBP␤ is tive interaction between these two proteins while bound to DNA significantly more effective than C/EBP␣. The increase in IL-4- and might contribute to the functional synergy seen in our reporter induced transcription seen when both Stat6 and C/EBP␤ were assays. Band shift experiments, like those shown in Figure 4, are overexpressed is far greater than the sum of each protein’s indi- equilibrium measurements of the proteins affinity for its DNA vidual ability to drive transcription from this reporter. This su- binding site. To determine the dissociation rate of Stat6 and peradditive effect is a defining feature of transcriptional synergy C/EBP␤ when bound to DNA, we investigated the stability of the (27, 28). These results were most dramatic with C/EBP␤ in the protein/DNA complexes, as shown in Figure 5. Stat6 and C/EBP␤ human B cell line BJAB, suggesting that C/EBP␤ is limiting in were incubated either separately or together with the labeled ⑀ these cells and that C/EBP␤ rather than C/EBP␣ cooperates with probe. After a brief incubation period to allow the protein-DNA Stat6 to drive IL-4-induced germline ⑀ expression in B cells. binding equilibrium to be reached with the labeled probe, an ex- The functional synergy between Stat6 and C/EBP␤ requires the cess of unlabeled oligonucleotides (trap) carrying the same DNA activation domains of both proteins. Deletion of either the Stat6 or sequence as the probe was added to the reaction mix. The time- C/EBP␤ activation domain results in a protein that can still bind to dependent disappearance of the radiolabeled protein-DNA com- the element, but can no longer partner with the other protein to The Journal of Immunology 1827

FIGURE 5. The dissociation rate of Stat6 bound to the ⑀ IL-4-responsive element is reduced when C/EBP␤ binds to the adjacent site. Stat6 and C/EBP␤, either alone or in combination, are incubated with 0.1 pmol of the probe carrying binding sites for both Stat6 and C/EBP (probe I, Fig. 4). The reaction was allowed to reach equilibrium be- fore 20 pmol of unlabled DNA (trap) carrying the same sequence as the probe was added to the reaction mix. Aliquots were taken at the times indicated above each lane and loaded onto a continuously running native gel. A, B, and C mark the positions of the individual protein/DNA complexes: A, C/EBP␤ complex; B, Stat6 complex; C, both proteins. Downloaded from http://www.jimmunol.org/ synergistically activate transcription. These data were further sub- of IFN-␥-induced gene expression, only the full length form of stantiated by overexpression studies in HepG2 cells. HepG2 cells Stat1 is capable of activating transcription (31). Hence, there ap- express both Stat6 and C/EBP and are able to activate our IL-4- pear to be multiple mechanisms that allow STAT proteins to co- inducible reporter (13). Overexpression of either protein lacking an operate with other transcription factors. Speciﬁc interactions are activation domain suppressed promoter activity, arguing for an al- probably dictated by the individual promoter context (31). most equivalent role of C/EBP␤ and Stat6 in driving transcription This is the ﬁrst example of Stat6 functionally synergizing with from the ⑀ IL-4-responsive element. A dominant negative effect another transcription factor to activate transcription from an IL-4- was also observed with C/EBP␥, a naturally occurring C/EBP iso- responsive element. In contrast, C/EBP␤ has been shown to func- by guest on September 29, 2021 form that lacks an activation domain. These results are particularly tionally synergize with a large number of transcription factors, interesting as this C/EBP family member has been implicated as including those playing important roles in immune cell function, playing a negative regulatory role in antagonizing the function of such as NF-␬B, AML1, Myb, and PU.1 (34–37). In several of other C/EBP proteins (18, 20). Hence, the balance between these examples, C/EBP␤ has been shown to associate physically in ␤ ␥ C/EBP and C/EBP may determine the amount of sterile tran- some way with the other cooperating factor. These and other ex- script produced in response to IL-4 stimulation. amples of transcription factors physically associating to mediate While no naturally occurring splice variant of Stat6 lacking the transcriptional synergy, as well as the proximity between the Stat6 C-terminal activation domain has been yet characterized, such nat- and C/EBP binding sites that is conserved in both the mouse and urally occurring splice variants have been observed for Stats 1, 3, human ⑀ promoter, prompted us to search for a physical associa- 4, and 5 (30–32). Studies with these STAT proteins and additional tion between C/EBP␤ and Stat6. cooperating factors highlight similarities and differences when Our DNA binding studies showed that both proteins are able compared with our observations with Stat6 and C/EBP␤. The nat- to bind the ⑀ promoter element independently. However, based urally occurring splice variant of Stat3, designated Stat3␤, is miss- on mobility shift experiments conducted in the presence of ex- ing most of the C-terminal activation domain. Yet, both the full cess DNA, simultaneous binding of both proteins appeared to length form of Stat3 (Stat3␣) and Stat3␤ can cooperate with c-Jun to activate transcription from an IL-6-responsive element taken be favored over single occupancy, suggesting some degree of ␣ facilitated DNA binding. To further investigate the possibility from the rat 2-macroglobulin gene (30). Similarly, both the full length form of Stat4 (Stat4␣) and a splice variant lacking the C- for a direct interaction between these two proteins, we mea- ␤ terminal activation domain (Stat4␤) can cooperate with c-Jun to sured the dissociation rates of Stat6 and C/EBP when bound to activate transcription from an IL-12-responsive element taken this DNA element. These experiments showed that in the dou- from the IRF1 promoter (X. Xu and T. Hoey, unpublished obser- ble occupancy state, where both Stat6 and C/EBP are bound to vations). In both these examples, activation function appears to be the probe, the dissociation rate of Stat6 is slower than when it provided by c-Jun, which binds at an adjacent site in each element binds the probe alone. This increased stability of Stat6 binding (29, 30). Yet at other STAT-activated promoter elements, the ac- under double occupancy conditions must be the result of some tivation domains of these proteins are clearly required (31, 32). In physical contact with the adjacently bound C/EBP␤, or possibly the case of IFN-␣-induced gene expression, activation is mediated it is the result of a change in the conformation of the DNA that by the multiprotein complex IFN-stimulated gene factor-3, which stabilizes the binding of Stat6 when C/EBP␤ binds the adjacent is composed of Stat1, Stat2, and the nuclear protein p48. Here, site. Although we and others (11) have clearly shown that most both the full length Stat1 protein (Stat1␣) or the variant (Stat1␤) isoforms of C/EBP are able to interact with the IL-4 response lacking the activation domain can participate in the complex, be- element of the ⑀ promoter, it remains to be determined whether cause all transactivation depends on Stat2 (33), whereas in the case C/EBP␤ binds to this site in vivo. 1828 IL-4-INDUCED TRANSCRIPTION

The requirement for the activation domains of both Stat6 and 13. Mikita, T., D. Campbell, P. Wu, K. Williamson, and U. Schindler. 1996. Re- C/EBP␤ secures the IL-4 responsiveness of the germline ⑀ pro- quirements for interleukin-4-induced gene expression and functional characterization of Stat6. Mol. Cell. Biol. 16:5811. moter. Yet, it is unclear how these activation domains interact to 14. Wang, N.-D., M. J. Finegold, A. Bradley, C. N. Ou, S. V. Abdelsayed, synergistically activate transcription. Stat6(AdϪ) also exhibited a M. D. Wilde, L. R. Taylor, D. R. Wilson, and G. J. Darlington. 1995. Impaired reduced dissociation rate from this element when C/EBP␤ was energy homeostasis in C/EBP a knockout mice. Science 269:1108. Ϫ 15. Cao, Z., R. M. Umek, and S. L. McKnight. 1991. Regulated expression of three bound (data not shown). Similarly, C/EBP␤(Ad ) also stabilized C/EBP isoforms during adipose conversion of 3T3–L1 cells. Genes Dev. 5:1538. Stat6 binding, indicating that this effect is not due to the activation 16. Tanaka, T., S. Akira, K. Yoshida, M. Umemoto, Y. Yoneda, N. Shirafuji, H. Fujiwara, S. Suematsu, N. Yoshida, and T. Kishimoto. 1995. Targeted dis- domains. Thus, while the change in dissociation rate may contrib- ruption of the NF-IL6 gene discloses its essential role in bacteria killing and ute to the synergistic effect seen in transcriptional activation, it is tumor cytotoxicity by macrophages. Cell 80:353. clearly not the main component. A more likely mechanism could 17. Antonson, P., B. Stellan, R. Yamanaka, and K. G. Xanthopoulos. 1996. A novel human CCAAT/enhancer binding protein gene, C/EBP⑀, is expressed in cells of be that both activation domains interact to adopt a unique confor- lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to mation that allows them to cooperate with the basal transcription the T-cell receptor ␣/␦ locus. Genomics 35:30. machinery more effectively than they can separately. Another pos- 18. Cooper, C., A. Henderson, S. Artandi, N. Avitahl, and K. Calame. 1995. Ig/EBP (C/EBP gamma) is a transdominant negative inhibitor of C/EBP family transcrip- sibility is that the two activation domains independently interact tional activators. Nucleic Acids Res. 23:4371. with other proteins in the basal machinery to achieve the same 19. Screpanti, I., L. Romani, P. Musiani, A. Modesti, E. Fattori, D. Lazzaro, C. Sellitto, S. Scarpa, D. Bellavia, G. Lattanzio, F. Bistoni, L. Frati, R. Cortese, result. Alternatively, both of these mechanisms could be operative A. Gulino, G. Ciliberto, F. Constanti, and V. Poli. 1995. Lymphoproliferative simultaneously. Either way, the decrease in dissociation rate of disorder and imbalanced T-helper response in C/EBP ␤ deﬁcient mice. EMBO J. Stat6 in the presence of C/EBP␤ is likely to increase the chances 14:1932.

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