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 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 e Promoter Mediated by Stat6 and C/EBPb 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 e 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/EBPb cooperate to synergistically activate transcription from the e 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/EBPa and none with C/EBPg. In fact, C/EBPg was able to inhibit IL-4-induced reporter activity. Stat6 and C/EBPb 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/EBPb binds at the neighboring site. These data suggest a mechanism whereby C/EBPb 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 e and g 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 e 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 a d b g we focused on Stat6, which is activated upon IL-4 stimulation (7, metabolism (C/EBP ,- ), immune system function (C/EBP ,- , e b 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 b 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 b with 18 U.S.C. Section 1734 solely to indicate this fact. that Stat6 and C/EBP activate transcription synergistically from the germline e 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/EBPb 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/EBPb is bound at the adjacent [email protected] 2 site. These results help to explain the mechanism by which Stat6 3 Abbreviations used in this paper: Jak, Janus kinase; (Ad ), lacking the activation b 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 e promoter. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 1823 Materials and Methods Results Cell culture and transient transfections Stat6 and C/EBPb 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 e promoters is a composite 1mML-glutamine, and 10 mM b-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 b-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 b-galactosidase activity were as- ity, we transiently overexpressed Stat6, C/EBPa, and C/EBPb sayed using the luciferase and b-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/EBPa, and C/EBPb 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 e 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).