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Binding Protein (CBP)/P300 NF-AT to Bind the Coactivator CREB Gene Transcription by Competing with MHC Class II Transactivator I

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Binding Protein (CBP)/P300 NF-AT to Bind the Coactivator CREB Gene Transcription by Competing with MHC Class II Transactivator I MHC Class II Transactivator Inhibits IL-4 Gene Transcription by Competing with NF-AT to Bind the Coactivator CREB Binding Protein (CBP)/p300 This information is current as of October 2, 2021. Tyler J. Sisk, Tania Gourley, Stacey Roys and Cheong-Hee Chang J Immunol 2000; 165:2511-2517; ; doi: 10.4049/jimmunol.165.5.2511 http://www.jimmunol.org/content/165/5/2511 Downloaded from References This article cites 43 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/165/5/2511.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on October 2, 2021 *average 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. MHC Class II Transactivator Inhibits IL-4 Gene Transcription by Competing with NF-AT to Bind the Coactivator CREB Binding Protein (CBP)/p3001 Tyler J. Sisk, Tania Gourley, Stacey Roys, and Cheong-Hee Chang2 The MHC class II transactivator (CIITA) activates the expression of multiple genes involved in Ag presentation, but inhibits Th2-type cytokine production, including IL-4, during Th1 cell differentiation. Th1 cells derived from CIITA-deficient mice pro- duce both Th1- and Th2-type cytokines, and the introduction of CIITA to Th2 cells down-regulates Th2-type cytokine gene transcription. Here we show that the IL-4 promoter is regulated by multiple protein-protein interactions among CIITA, NF-AT, and coactivator CBP/p300. The introduction of CBP/p300 and NF-AT enhances the IL-4 promoter activity, and this activation was repressed by CIITA. Furthermore, our data show that CIITA competes with NF-AT to bind CBP/p300 and that this competition dramatically influences transcriptional activation of the IL-4 promoter. We identified two domains of CIITA that interact with two Downloaded from distinct domains of CBP/p300 that are also recognized by NF-AT. CIITA mutants that retain the ability to interact with CBP/p300 are sufficient to inhibit NF-AT-mediated IL-4 gene expression. The Journal of Immunology, 2000, 165: 2511–2517. lass II transactivator (CIITA)3 is a critical transcription fac- tional level (13–19). Depending on the context, specific transcrip- tor activating genes involved in Ag presentation, such as tion factors can either cooperate or interfere with each other. An- C MHC class II, invariant chain, and H-2M genes (1–4). The other function of CBP/p300 appears to be the modification of http://www.jimmunol.org/ deficiency in CIITA results in immunodeficiency in both human and chromatin structure. Intrinsic histone acetyltransferase activity has mouse (4, 5). Recently, we have demonstrated that CIITA inhibits the been found in CBP/p300, which is considered to play a crucial role expression of the IL-4 gene during CD4 T cell differentiation, and in transcription from tightly packed chromatin (20). CBP/p300 introduction of CIITA to Th2 cells is sufficient to down-regulate IL-4 also acetylates transcription factors (21, 22). GATA-1 and p53 are production (6). CIITA is not a DNA binding protein. The exact mech- acetylated by CBP/p300, and the DNA binding ability and trans- anism of CIITA action is not known, but the interaction of CIITA activation potential of these transcription factors are greatly en- with sequence-specific DNA binding proteins as well as the basal hanced upon acetylation. transcriptional machinery may be required for its function (7–10). In MHC class II-specific transcription, CBP/p300 functions as a Therefore, it is not surprising to observe the different outcome of coactivator. CBP/p300 binds to CIITA and stimulates MHC class by guest on October 2, 2021 CIITA function with a different set of regulatory factors depending on II promoter activity (23, 24). Adenovirus E1A inhibits MHC class the target promoter. II transcription by interacting directly with CBP/p300 (24). E1A Cyclic AMP response element binding protein (CREB) binding interacts with the same CBP/p300 region that binds CIITA, sug- protein (CBP) and p300 are highly homologous nuclear proteins gesting that E1A interferes with MHC class II gene expression by originally identified for their ability to interact with the transcrip- targeting interactions between CIITA and coactivators (24). CBP/ tion factor CREB and with adenovirus E1A proteins, respectively p300 is also a coactivator for NF-AT-mediated transcription of the (reviewed in Ref. 11). CBP/p300 also plays a critical role during IL-2 promoter and the synthetic promoter containing the NF-AT development, and the deficiency of CBP/p300 is fatal (12). The binding sites (25, 26). However, a role for CBP/p300 in IL-4 gene ability of CBP/p300 to interact with multiple, signal-dependent transcription has not been demonstrated. transcription factors, CREB, STATs, MyoD, nuclear hormone re- The observation that CBP/p300 interacts with both CIITA and ceptors, and the basal transcriptional machinery, has led to the NF-AT raises the possibility of competitive interactions of two proposal that these coactivators function as signal integrators by transcription factors for CBP/p300 binding to regulate gene ex- coordinating complex signal transduction events at the transcrip- pression. We tested this hypothesis to determine whether the in- hibition of IL-4 gene transcription by CIITA is due to the compe- tition between NF-AT and CIITA for CBP/p300 binding. Here, we Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109 demonstrate that an interaction of CBP/p300 with NF-AT can lead Received for publication April 4, 2000. Accepted for publication June 13, 2000. to dramatic activation of the IL-4 promoter. Furthermore, our data The costs of publication of this article were defrayed in part by the payment of page indicate that CIITA interferes with NF-AT binding to CBP/p300, charges. This article must therefore be hereby marked advertisement in accordance resulting in the down-regulation of IL-4 gene transcription. This with 18 U.S.C. Section 1734 solely to indicate this fact. study provides further insight into the mechanisms by which IL-4 1 This work was supported in part by National Institutes of Health Grant AI41510 gene transcription is controlled by multiple protein-protein interactions. (to C.-H.C.). 2 Address correspondence and reprint requests to Dr. Cheong-Hee Chang, Department of Microbiology and Immunology, 6606 Medical Science Building II, University of Michigan Medical School, Ann Arbor, MI 48109. E-mail address: heechang@ Materials and Methods umich.edu Cell culture and transfections 3 Abbreviations used in this paper: CIITA, class II transactivator; CREB, cAMP re- sponse element binding protein; CBP, CREB binding protein; RLA, relative lucif- Both 293T human embryonic kidney epithelial cells and the 68-41 T cell erase activity. hybridoma were maintained in Clicks medium supplemented with 10% Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 2512 INHIBITION OF IL-4 GENE TRANSCRIPTION BY CIITA FBS, 2 mM glutamine, 100 ␮g/ml of penicillin, and 100 ␮g/ml of strep- Results tomycin. To generate stable transfectants of 68-41 T cells (27), cells were CIITA inhibits CBP/p300-mediated activation of the IL-4 electroporated with DNA encoding CIITA or neomycin gene and selected with 1 mg/ml of G418 (Life Technologies, Gaithersburg, MD). For the promoter 7 transient transfection of 68-41 cells, 1 ϫ 10 cells were mixed with 10–30 The significance of CBP/p300 has been demonstrated for the reg- ␮g of DNA as described in the figure legends. Cells were then electropo- rated (0.25 kV and 960 ␮F) using Gene Pulser (Bio-Rad, Hercules, CA) ulation of transcription, including genes encoding molecules that followed by stimulation with PMA (25 ng/ml) and ionomycin (1.5 ␮M) are essential for the proper immune response such as MHC class II overnight. Cells were harvested and analyzed for luciferase and ␤-galac- and IL-4 (23–26). Therefore, we first tested whether p300 can tosidase activity as previously described (6). activate the IL-4 promoter. 293T human embryonic kidney epi- 293T cells were transfected using a standard calcium phosphate method thelial cells were transfected with the 3.0-kb IL-4 promoter-driven with 2.5 ϫ 105 cells and 1 ␮g of DNA unless indicated otherwise. Cells were analyzed for luciferase activity 2 days after transfection. The CMV luciferase and the p300 expression vector or the empty control promoter-driven ␤-galactosidase expression vector was cotransfected in all vector. As shown in Fig. 1A, the luciferase activity was enhanced transfections, and luciferase values were normalized to ␤-galactosidase ac- by cotransfecting p300 in a dose-dependent manner, indicating that tivity as described previously (6). Relative luciferase activity (RLA) was p300 is sufficient to activate the IL-4 promoter in the absence of T calculated using the luciferase activity of cells transfected with the reporter DNA alone as 1 unless noted. Values in all transfections represent the cell-specific transcription factors. Studies have demonstrated that average of at least three independent experiments. the adenovirus E1A protein inhibits CBP/p300-mediated transcrip- tion by binding to CBP/p300 (22, 23).
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