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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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

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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 © 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 inhibition of IL-4 gene transcription by CIITA is due to the competition 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 response 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%

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 electroporated (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 transcription by binding to CBP/p300 (22, 23). Consistent with this, a wild- DNA constructs type E1A, but not a mutant that has deletion of the p300 binding The following DNA constructs were described previously: FLAG-tagged wild-type CIITA (28), antisense CIITA (6), NF-AT1 and NF-AT2 (29), Downloaded from wild-type p300 and p3001–347 (30), CBP (14), E1A wild-type and the mutant lacking the CBP/p300 binding site (31), the luciferase reporter constructs containing the 3-kb or the 157-bp fragment of the IL-4 promoter or the MHC class II E␣ promoter (6), the trimerized NF-AT and the minimal IL-4 promoter (32), and the trimerized AP-1 (33). All CIITA mutants were made using the expression vector pcDNA3 containing a FLAG epitope at the N-terminus. CIITA1–331 containing the acidic and the P/S/T domains was generated using the EcoRI-HindIII fragment of the wild-type gene. http://www.jimmunol.org/ Ј CIITA408–857 was amplified by PCR using primers 5 -TGCTCTAGA CACCGGCGGCCGCGTGAGACACGAGTG-3Ј containing XbaI site at the 5Ј end and 5Ј-TCTTGGTGCTCTGTCATCCCT-3Ј. The PCR product was then digested with XbaI and NcoI and ligated with a fragment encom- passing NcoI to the nucleotide position 2702 of CIITA where the stop codon was introduced. CIITA980–1130 was generated using a fragment from BamHI to the 3Ј end of cDNA for the cloning. Inferior numbers denote amino acids positions. The integrity of the mutants was confirmed by sequencing. by guest on October 2, 2021 EMSA Nuclear extracts from 68-41 T cells were prepared as previously described (34) after5hofstimulation with PMA (25 ng/ml) and ionomycin (1.5 ␮M). Gel-shift reactions were conducted in 20-␮l volumes containing 50 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl (pH 7.5), 5% glycerol, 5–10 ␮g nuclear extract, 1.5 ␮g poly(dI-dC) (Roche, Indianapolis, IN), and 30,000 cpm of 32P end-labeled NF-AT oligonucleotides (5Ј-CGCCCAAAGAG GAAAATTTGTTTCATA-3Ј, Santa Cruz Biotechnology, Santa Cruz, CA). The following protease inhibitors were added to the reactions: 1 mM DTT, 1 mM PMSF, 2 ␮g/ml leupeptin, and 2 ␮g/ml aprotinin. The reactions were incubated on ice for 20 min to allow DNA-protein complexes to form. Reactions were run on a 4.5% nondenaturing polyacrylamide gel at room temperature for3hat150Vin1ϫ TBE. IFN-responsive sequence oligonucleotides (5Ј-TCGAATCTCCACAGTTTCACTTCTGCACCTG 3Ј) were used as a nonspecific competitor.

Immunoprecipitation and Western blot FIGURE 1. CIITA inhibits p300-mediated activation of the IL-4 promoter. A, Dose-response effect of p300 on the IL-4 promoter. 293T cells ϫ 6 Typically, 1 10 293T cells were transfected for immunoprecipitation were transfected with 1 ␮g of the 748-bp IL-4 promoter-driven luciferase experiments. Cells were harvested 2 days after transfection, washed with and increasing amounts (in micrograms) of the p300 expression vector as 1ϫ PBS, lysed in 200 ␮l of ice-cold lysis buffer (1% Triton X-100, 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 ␮g/ml indicated. B, Adenovirus E1A inhibits p300-mediated activation of the leupeptin, and 1 ␮g/ml aprotinin), and incubated on ice for 1 h with oc- IL-4 promoter. The wild-type or the mutant E1A defective in CBP/p300 casional vortexing. Lysates were clarified through centrifugation at binding was transfected with the IL-4 promoter-driven luciferase and p300 20,800 ϫ g for 30 min at 4°C. The protein concentration was determined expression vector. One microgram of each DNA was used. C, CIITA in- using the Lowry assay (Bio-Rad). p300 coimmunoprecipitation was per- hibits the IL-4 promoter, but activates the MHC class II promoter. The IL-4 formed with the anti-p300 Ab (N-15, Santa Cruz Biotechnology) for 1 h promoter-driven luciferase (left panel) or the E␣ promoter-driven lucif- with occasional rocking. Immune complexes were collected on protein erase (right panel) was transfected with p300 alone or with p300 and A-Sepharose beads, then the beads were washed three times with lysis CIITA together. One microgram of each DNA was used. D, Dose-response buffer, and bound proteins were resolved on SDS-PAGE and Western blot- effect of CIITA. Cells were transfected with 1 ␮g of the IL-4 promoter- ted. NF-AT and CIITA were detected with polyclonal anti-NF-AT Ab (Santa Cruz Biotechnology) and a monoclonal anti-FLAG Ab (M2, anti- driven luciferase reporter and the p300 expression vector. An increasing FLAG, Sigma, St. Louis, MO), respectively. HRP-conjugated Ab (Jackson amount (in micrograms) of DNA encoding CIITA was cotransfected. RLA ImmunoResearch Laboratories, West Grove, PA) was used as a secondary was calculated as described in Materials and Methods. The percent induc- Ab followed by enhanced chemiluminescence assay for visualization tion was calculated using the luciferase activity of cells transfected with (ECL, NEN Life Science Products, Boston, MA). p300 in the absence of CIITA as 100%. The Journal of Immunology 2513 domain, abolished p300-mediated activation of the IL-4 promoter the 157-bp fragment of the IL-4 promoter contains a sufficient (Fig. 1B). cis-acting element(s) for the activation and the repression by p300 We have reported that CIITA inhibits IL-4 gene expression dur- and CIITA, respectively. There are multiple cis-acting elements ing Th1 cell differentiation and in committed Th2 cells (6). Be- within the 157-bp fragment, and studies have demonstrated that cause CIITA interacts with CBP/p300 (23, 24), we examined NF-AT/AP-1 binding sites are the major element required to ac- whether CIITA represses p300-mediated activation of the IL-4 tivate IL-4 gene transcription (32). In addition, NF-AT has been promoter. When the CIITA expression vector was cotransfected shown to interact with CBP/p300 (25, 26). Therefore, we tested with p300, the luciferase activity driven by the IL-4 promoter was whether the NF-AT/AP-1 binding site is responsible for the acti- greatly reduced (Fig. 1C, left panel). This reduction is specific for vation by p300. The luciferase construct driven by the IL-4 min- the IL-4 promoter because the MHC class II promoter was not imal promoter (Ϫ58 to ϩ60) with the trimerized NF-AT/AP-1 inhibited by CIITA (Fig. 1C, right panel). Instead, CIITA together binding site of the IL-4 promoter was transfected with p300. The with p300 activated the MHC class II promoter synergistically, trimerized NF-AT/AP-1 binding site was sufficient to be activated consistent with previous studies (23, 24). The inhibition of the IL-4 by p300 and repressed by CIITA (Fig. 2B, lanes 4–6). Because the promoter by CIITA is dose dependent (Figs. 1D and 4), and the NF-AT/AP-1 site is a composite site for both NF-AT and AP-1 luciferase activity was not affected when the antisense CIITA was binding, it is necessary to distinguish whether the activation is cotransfected (Fig. 4, lane 7). These data indicate that p300 acti- mediated by NF-AT or AP-1. p300 did not activate the promoter vates the IL-4 promoter in nonlymphoid cells, and CIITA inhibits driven by the trimerized AP-1 binding sites or the minimal IL-4 p300-mediated activation of the IL-4 promoter. promoter without the NF-AT binding site (Fig. 2B, lanes 7–12). Together, these data demonstrate that the NF-AT binding site is the The NF-AT binding site of the IL-4 promoter is sufficient to primary target of p300 and CIITA. Downloaded from mediate the activation by p300 CIITA inhibits NF-AT-mediated activation of the IL-4 promoter CBP/p300 activates transcription by interacting with other transcription factors; thus, we wanted to examine which proteins are It has been shown that NF-AT and CBP/p300 activate the IL-2 recognized by CBP/p300 on the IL-4 promoter. To do this, we promoter or a synthetic promoter containing the NF-AT binding delineated a cis-acting element(s) of the IL-4 promoter that medi- site (25, 26). Moreover, our results, shown in Fig. 2, indicate that ates the activation by p300. Luciferase reporters driven by three the NF-AT binding site of the IL-4 promoter is sufficient to be http://www.jimmunol.org/ different lengths of the IL-4 promoter, 3 kb, 748 bp, and 157 bp, activated by p300. Therefore, we examined whether protein-pro- were tested. As shown in Fig. 2A, all three promoters were activated tein interaction between NF-AT and p300 mediates activation of at comparable levels when p300 was cotransfected (lanes 2, 5, and 8). the IL-4 promoter. When 293T cells were transfected with the This activation was repressed by CIITA (lanes 3, 6, and 9). Therefore, NF-AT1 or the NF-AT2 expression vector, the IL-4 promoter was activated significantly (Fig. 3A, lanes 2 and 4), and this activation was inhibited by CIITA (Fig. 3A, lanes 3 and 5). Cotransfection of NF-AT1 or NF-AT2 with p300 further enhanced the luciferase activities, demonstrating the synergistic effect of NF-AT and p300 by guest on October 2, 2021 interaction (Fig. 3A, lanes 8 and 10). Again, CIITA inhibited the activation by NF-AT and p300 (Fig. 3A, lanes 9 and 11). Next, we investigated whether CIITA can inhibit NF-AT-mediated activation of the IL-4 promoter in T cells. We used the 68-41 Th1 cell hybridoma for the transfections. 68-41 cells were transfected with DNA, followed by stimulation with PMA and ionomycin. Consistent with data from 293T cells, the IL-4 promoter was activated by NF-AT1 or NF-AT2, and this activation was inhibited by CIITA (Fig. 3B, left panel). The addition of CBP and NF-AT augmented the luciferase activity, and it was repressed by CIITA (Fig. 3B, right panel). Therefore, CIITA inhibits IL-4 gene transcription activated by the interaction of NF-AT and CBP/p300 in both nonlymphoid and T cells. CIITA and NF-AT compete for p300 binding The binding of NF-AT to its DNA motif is required to activate the IL-4 promoter. Therefore, we asked whether CIITA inhibits NF-AT binding to the DNA template, causing repression of the IL-4 promoter. To test this, EMSA was performed using nuclear extracts prepared from 68-41 cells stably transfected with the CI- ITA expression vector or the control DNA. The DNA binding activity of NF-AT was comparable regardless of CIITA expression, suggesting that CIITA does not modulate the DNA binding ability of NF-AT (Fig. 4A). Studies have shown that NF-AT and CIITA bind to the same regions of CBP/p300 (23–26). These observations raise the possi- FIGURE 2. The NF-AT binding site is sufficient to mediate activation bility that the inhibition of IL-4 gene transcription by CIITA is and repression. Luciferase reporters driven by different lengths of the IL-4 caused by competition between CIITA and NF-AT for CBP/p300 promoter (A) or other promoters (B) were cotransfected with p300 or with binding. To test this hypothesis, we used two experimental ap- p300 and CIITA. One microgram of each DNA was used. proaches. First, IL-4 promoter activity was measured to determine 2514 INHIBITION OF IL-4 GENE TRANSCRIPTION BY CIITA Downloaded from FIGURE 3. CIITA inhibits NF-AT-mediated activation of the IL-4 promoter in 293T (A) and 68-41 (B) T cell hybridomas. The 748-bp IL-4 promoter-driven luciferase was transfected with different combinations of DNA encoding NF-AT1, NF-AT2, CIITA, p300, or CBP as indicated. For 293T cells 1 ␮g of each DNA was used for transfection. 68-41 T cells were electroporated with 10 ␮g of the reporter and 20 ␮g of each expression vector, followed by stimulation with PMA and ionomycin. http://www.jimmunol.org/ the effect of CIITA dose. Second, using the same transfected cells protein-protein interactions among NF-AT, CIITA, and p300 were assessed. 293T cells were cotransfected with a constant amount of vector expressing p300 and NF-AT1, and a luciferase reporter driven by the IL-4 promoter. As indicated in Fig. 4B, an increasing amount of the CIITA expression vector was cotransfected. After by guest on October 2, 2021 cells were harvested, we measured the luciferase activity and performed immunoprecipitation assays. The luciferase activity was greatly enhanced when p300 and NF-AT1 were cotransfected, confirming the synergistic interaction FIGURE 4. CIITA competes with NF-AT to bind p300. A, NF-AT between p300 and NF-AT1 (Fig. 4B, lanes 1 and 2). This activa- DNA binding activity is not altered by CIITA expression. 68-41 T cells tion of the IL-4 promoter by p300 and NF-AT1 was repressed by were stably transfected with the control DNA or the CIITA expression CIITA in a dose-dependent manner (Fig. 4B, lanes 3–6). The in- vector. Cells were then treated with PMA and ionomycin for 5 h, and hibition is specific, because antisense CIITA at the highest dose nuclear extracts were prepared. DNA binding reactions were conducted did not inhibit the IL-4 promoter (Fig. 4B, lane 7). using different amounts of extracts as indicated. The specific and nonspe- If the inhibitory effect is due to the competition of CIITA with cific competitors are 100-fold molar excesses of unlabeled NF-AT and NF-AT to bind p300, we should be able to detect more CIITA but IFN-responsive sequence oligonucleotides, respectively. B, The IL-4 promoter activity was inhibited by CIITA in a dose-dependent manner. 293T less NF-AT protein bound by p300 as the CIITA level increases. cells were transfected with 1 ␮g of the 748-bp IL-4 promoter-driven lu- To test this, coimmunoprecipitation experiments were conducted ciferase reporter construct, and 3 ␮g each of p300 and NF-AT expression using an anti-p300 Ab followed by Western blotting using an anti- vectors. An increasing amount (in micrograms) of CIITA expression vector NF-AT Ab. The level of NF-AT protein coimmunoprecipitated by was cotransfected as indicated. The empty expression vector was used as p300 was diminished as the amount of the CIITA expression vec- a filler to maintain a constant amount of total plasmid DNA in all trans- tor increased for transfection (Fig. 4C, upper panel). The protein level fections. The RLA shown in this figure was normalized using the luciferase of transfected NF-AT remained constant (Fig. 4C, lower panel). value transfected with p300 as 1. C, In vivo competition between CIITA The membrane was then stripped and reprobed with an anti-FLAG and NF-AT for p300 binding. Whole-cell extracts were prepared from the Ab to detect CIITA. As expected, the level of CIITA protein bound same transfected cells as those in B. The immunoprecipitation was per- to p300 was increased as more CIITA protein was expressed. formed using an anti-p300 Ab followed by Western blotting using an anti- NF-AT and an anti-FLAG Ab to detect NF-AT and CIITA, respectively Taken together, our data demonstrate that the inhibition of IL-4 (upper panel). As a control, total lysates were blotted with the same Abs to gene transcription by CIITA is at least partly due to the competi- monitor the expression level of NF-AT and CIITA protein (lower panel). tion between NF-AT and CIITA for CBP/p300 binding.

CIITA mutants capable of interacting with CBP/p300 inhibit because it cannot compete with NF-AT to bind CBP/p300. Unexpect- IL-4 gene expression edly, a CIITA mutant lacking the acidic domain inhibited the IL-4 The acidic domain of CIITA is shown to interact with the CH3 promoter at a comparable level as the wild type (data not shown). This domain of CBP/p300 (23, 24). This predicts that a CIITA mutant result indicates that the mutant may be able to interact with CBP/p300 lacking the acidic domain should not repress the IL-4 promoter, through another domain. To identify the domain, we divided CIITA The Journal of Immunology 2515

into three regions (Fig. 5A); CIITA1–331 containing the acidic and the scription factors, including NF-AT (11, 25, 26). 293T cells were proline-serine-threonine rich domain, CIITA408–857 with the GTP transfected with CIITA mutants with either the full-length p3001–2414 binding domain, and CIITA980–1130 that has leucine-rich repeats. or p3001–347 followed by immunoblot. CIITA408–857 was able to Each mutant was then tested for the repression function on the IL-4 interact with both the wild-type and the amino-terminal regions of promoter that was activated by NF-AT2 and CBP/p300. The three p300 (Fig. 5C, lanes 1 and 2). CIITA980–1130 containing leucine- mutants showed different degrees of inhibition, although none of them rich repeats did not interact with p300 (Fig. 5C, lane 3). The wild- was able to activate the MHC class II promoter (Fig. 5B). CIITA1–331 type CIITA also interacted with N-terminus as well as the full- mutant repressed better than the wild type, while CIITA980–1130 or the length p300 (Fig. 5D). These results together with the previous antisense CIITA could not repress the IL-4 promoter (Fig. 5B, middle studies imply bivalent interaction between CIITA and CBP/p300: panel, lanes 9, 11, and 12). Interestingly, CIITA408–857 also behaved the acidic domain of CIITA with the CH3 region of CBP/p300, and as a repressor (Fig. 5B, lane 10). A similar pattern of repression was the GTP binding domain of CIITA with N-terminus CBP/p300. observed in T cells as well (Fig. 5B, right panel). These data dem- Not surprisingly, both regions of CBP/p300 interacting with CI- onstrate that either the acidic or the GTP binding domain of CIITA is ITA overlap with NF-AT recognition sites. sufﬁcient for the repression of IL-4 gene expression. We next determined which region of p300 is recognized by CIITA . The CH3 domain is already identiﬁed as an inter- 408–857 Discussion action site for the acidic domain of CIITA. Therefore, we thought The proper regulation of IL-4 gene expression during Th2 cell that p300 containing N-terminal 347 aa would be a reasonable 1–347 differentiation is critical to a host to regulate the immune response candidate, because N-terminus p300 is recognized by other tran-

upon infection. We have shown previously that CIITA plays a role Downloaded from in the regulation of Th cell differentiation (6). The CIITA gene is expressed in developing Th1 cells, but not in Th2 cells. The de- ﬁciency of CIITA during Th1 cell differentiation allows activation of the IL-4 gene, and the introduction of CIITA to Th2 cells down- regulates IL-4 gene transcription. Here, our data showed that the

inhibition of IL-4 by CIITA is at least in part due to the compe- http://www.jimmunol.org/ tition between CIITA and NF-AT to bind a limited amount of the coactivators CBP/p300. NF-AT is an essential transcription factor regulating IL-4 gene transcription. There are four NF-AT genes encoding the cytoplasmic subunit, NF-AT1, NF-AT2, NF-AT3, and NF-AT4, although at the protein level NF-AT1 and NF-AT2 are expressed in peripheral T cells and T cell lines (35). In vitro, all can bind to and activate multiple cytokine genes, but the studies in vivo suggest functional differences among NF-AT family by guest on October 2, 2021 members; NF-AT1 and NF-AT2 are negative and positive reg- ulators of the IL-4 gene, respectively (29, 36–42). In this study we showed that IL-4 gene transcription is synergistically enhanced by the interaction of CBP/p300 with NF-AT1 or NF- AT2 by transfection experiments. The transactivation mediated by CBP/p300 and NF-AT is reduced by CIITA activity. How- ever, CIITA does not affect the DNA binding ability of NF-AT, as demonstrated by EMSA in vitro. In addition, CIITA does not bind to NF-AT (T. J. Sisk and C.-H. Chang, unpublished ob- FIGURE 5. CIITA mutants that can interact with p300 are able to re- servations). Instead, our data imply that the repression effect of press the IL-4 promoter. A, A schematic diagram of CIITA domains. CIITA is probably due to the reduced availability of CBP/p300 Acidic, region rich in acidic amino acids; P/S/T, region rich in proline, to interact with NF-AT on the IL-4 promoter. Because both serine, and threonine residues; GBD, GTP binding domain; LRR, leucine- CIITA and NF-AT bind to the same domains of CBP/p300, it is rich repeats. Numbers indicate amino acid positions (4). B, Comparison of not surprising to observe the competition between the two. CIITA mutants for activation and repression functions. 293T cells were CBP/p300 seems to behave as a bridging factor to assemble a transfected with 1 ␮g of the MHC class II E␣ (left panel) or the 748-bp competent transcriptional machinery rather than act as a acetyl- IL-4 promoter (middle panel)-driven luciferase reporter construct with 1 ␮g of different forms of CIITA. The IL-4 promoter activity in 68-41 T transferase on the exogenous IL-4 promoter. A CBP mutant lack- cells is shown in the right panel. For MHC class II, the relative activity was ing the enzymatic activity was able to transactivate the IL-2 pro- normalized using the luciferase value transfected with reporter alone as 1. moter (26), and the same mutant activated the IL-4 promoter For repression of IL-4, the IL-4 luciferase value cotransfected with the (S. Roys and C.-H. Chang, unpublished observations). Unfortu- NF-AT2 and the wild-type p300 is considered as 1. AS-WT, Wild-type nately, CBP or p300 homozygous knockout mice are embryonic p Ͻ 0.05 vs control (n ϭ 3). C, The GTP lethal (12), and thus the role of CBP/p300 in transcription of the ,ء .CIITA in antisense orientation binding domain of CIITA interacts with N-terminus p300. CIITA mutants IL-4 gene in vivo remains unclear. tagged with FLAG were cotransfected with p300. The immunoprecipita- None of the CIITA mutants tested activated the MHC class II tion was performed using an anti-p300 Ab followed by Western blotting promoter, indicating that the integrity of CIITA domains is critical using the anti-FLAG Ab. D, Interaction of the wild-type CIITA and p300. FLAG-tagged wild-type CIITA was transfected into 293T cells with the for the activation function on the MHC class II promoter. On the contrary, CIITA mutants showed different degrees of IL-4 inhibi- full-length p3001–2414 or N-terminus p3001–347. The immunoprecipitation was performed as described in C. Cell lysate is 1/20th of the total cell lysate tion. Mutants capable of interacting with CBP/p300 can repress used for immunoprecipitation. IL-4. This further supports the idea that the inhibition observed in 2516 INHIBITION OF IL-4 GENE TRANSCRIPTION BY CIITA

IL-4 gene expression is due to the competition between transcrip- 9. Mahanta, S. K., T. Scholl, F.-C. Yang, and J. L. Strominger. 1997. Transactiva- tion factors. It would be ideal to show the interaction of CBP/p300 tion by CIITA, the type II bare lymphocyte syndrome-associated factor, requires participation of multiple regions of the TATA box binding protein. Proc. Natl. with NF-AT or CIITA present in T cells. However, due to low Acad. Sci. USA 94:6324. levels of CBP/p300 in T cells, we were unable to detect immuno- 10. Shikama, N., J. Lyon, and N. La Thangue. 1997. The p300/CBP family: inte- precipitation product by CBP/p300 under a variety of conditions. grating signals with transcription factors and chromatin. Trends Cell Biol. 7:230. Therefore, like others, we relied on overexpression systems 11. Yao, T.-P., S. P. Oh, M. Fuchs, N.-D. Zhou, L.-E. Ch’ng, D. Newsome, (25, 26). R. T. Bronson, E. Li, D. M. Livingston, and R. Eckner. 1998. Gene dosage- dependent embryonic development and proliferation defects in mice lacking the Our study also indicates that the interaction between CIITA and transcriptional integrator p300. Cell 93:361. CBP/p300 is bivalent. A similar bivalent interaction was demon- 12. Eckner, R., M. E. Ewen, D. Newsome, M. Gerdes, J. A. DeCaprio, strated between NF-␬B and CBP/p300; interestingly, this interac- J. B. Lawrence, and D. M. Livingston. 1994. Molecular cloning and functional tion is regulated by the conformational change in NF-␬B upon analysis of the adenovirus E1A-associated 300-KD protein (p300) reveals a protein with properties of a transcriptional adaptor. Genes Dev. 8:869. phosphorylation (43). CIITA activity as a repressor also depends 13. Kwok, R. P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, on its conformational change, which results in the alteration of its R. G. Brennan, S. G. E. Robers, M. R. Green, and R. H. Goodman. 1994. Nuclear potential to interact with CBP/p300 (T. J. Sisk and C.-H. Chang, protein CBP is a coactivator for the transcription factor CREB. Nature 370:223. unpublished observations). We observed that the CIITA mutant 14. Arany, Z., D. Newsome, E. Oldread, D. M. Livingston, and R. Eckner. 1995. A containing the acidic and P/S/T domain inhibited the IL-4 pro- family of transcriptional adapter proteins targeted by the E1A oncoprotein. Na- ture 374:81. moter more efficiently than the full-length CIITA even at a lower 15. Zhang, J. J., U. Vinkemeier, W. Gu, D. Chakravarti, C. M. Horvath, and J. E . concentration (S. Roys and C.-H. Chang, unpublished observa- J. Darnell. 1996. Two contact regions between Stat1 and CBP/p300 in interferon tions). This may be due to a different conformation of the CIITA ␥ signaling. Proc. Natl. Acad. Sci. USA 93:15092. Downloaded from mutant that could have better accessibility to CBP/p300 or a higher 16. Nakajima, T., C. Uchida, S.F. Anderson, J. D. Parvin, and M. Montminy. 1997. affinity to CBP/p300. CIITA nearly abolishes IL-4 promoter Analysis of a cAMP-responsive activator reveals a two-component mechanism 1–331 for transcriptional induction via signal-dependent factors. Genes Dev. 11:738. activity, whereas CIITA leads to only a modest reduction in 408–857 17. Horvai, A., L. Xu, E. Korzus, G. Brard, D. Kalafus, T. M. Mullen, D. W. Rose, promoter activity in 68-41 T cells (Fig. 5B). This suggests that the M. G. Rosenfeld, and C. K. Glass. 1997. Nuclear integration of JAK/STAT and acidic domain has a higher affinity for CBP/p300 than does Ras/AP-1 signaling by CBP and p300. Proc. Natl. Acad. Sci. USA 94:1074.

CIITA408–805; however, experiments that will determine the rela- 18. Puri, P. L., V. Sartorelli, X.-J. Yang, Y. Hamamori, V. V. Ogryzko, tive affinities of these specific protein-protein interactions remain B. H. Howard, L. Kedes, J. Y. J. Wang, A. Graessmann, Y. Nakatani, et al. 1997. http://www.jimmunol.org/ Differential roles of p300 and PCAF acetyltransferases in muscle differentiation. to be performed. Mol. Cell 1:35. It should be noted that CIITA is not a global inhibitor of genes 19. Ogryzko, V., R. L. Schiltz, V. Russanova, B. H. Howard, and Y. Nakatani. 1996. that can be activated by CBP/p300. For example, IFN-␥ gene ex- The transcriptional coactivators p300 and CBP are histone acetyltransferases. pression was not altered in Th1 cells derived from CIITA-deficient Cell 87:953. mice, although CBP/p300 interaction with STAT1 induces the 20. Boyes, J., P. Byfield, Y. Nakatani, and V. Ogryzko. 1998. Regulation of activity of the transcription factor GATA-1 by acetylation. Nature 396:594. IFN-␥ gene transcription (16, 18). The subtle variance in the rec- 21. Gu, W., and R. G. Roeder. 1997. Activation of p53 sequence-specific DNA bind- ognition of CBP/p300 domain by a different transcription factor in ing by acetylation of the p53 C-terminal domain. Cell 90:595. the context of promoter elements may explain the final conse-

22. Fontes, J. D., S. Kanazawa, D. Jean, and B. M. Peterlin. 1999. Interactions be- by guest on October 2, 2021 quence. It remains to be demonstrated that CIITA controls the tween the class II transactivator and CREB binding protein increase transcription binding of NF-AT to CBP/p300 during Th1 cell differentiation to of major histocompatibility complex class II genes. Mol. Cell. Biol. 19:941. prevent IL-4 gene transcription. 23. Kretsovali, A., T. Agalioti, C. Spilianakis, E. Tzortzakaki, M. Merika, and J. Papamatheakis. 1998. Involvement of CREB binding protein in expression of major histocompatibility complex class II genes via interaction with the class II Acknowledgments transactivator. Mol. Cell. Biol. 18:6777. 24. Garcia-Rodriguez, C., and A. Rao. 1998. Nuclear factor of activated T cells We thank Drs. Roland Kwok, Mike Imperiale, Gary Nabel, Ken Murphy, (NFAT)-dependent transactivation regulated by the coactivators p300/CREB- Mercedes Rincon, Jenny Ting, and Gerald Crabtree for proving reagents. binding protein (CBP). J. Exp. Med. 187:2031. We are very much indebted to Dr. Wes Dunnick for valuable suggestions 25. Avots, A., M. Buttmann, S. Chuvpilo, C. Escher, U. Smola, A. J. Bannister, and the critical reading of the manuscript. U. R. Rapp, T. Kouzarides, and E. Serfling. 1999. CBP/p300 integrates Raf/Rac- signaling pathways in the transcriptional induction of NF-ATc during T cell activation. Immunity 10:515. References 26. Kubo, M., J. Ransom, D. Webb, Y. Hashimoto, T. Tada, and T. Nakayama. 1997. 1. Chin, K.-C., C. Mao, C. Skinner, J. L. Riley, K. L. Wright, C. S. Moreno, T-cell subset-specific expression of the IL-4 gene is regulated by a silencer ele- G. R. Stark, J. M. Boss, and J. P.-Y. Ting. 1994. Molecular analysis of G1B and ment and STAT6. EMBO J. 16:4007. G3A IFN-␥ mutants reveals that defects in CIITA or RFX result in defective class II MHC and Ii gene induction. Immunity 2:533. 27. Chin, K.-C., G. G.-X. Li, and J. P.-Y. Ting. 1997. Importance of acidic, proline/ 2. Chang, C.-H., J. Fontes, M. Peterlin, and R. A. Flavell. 1994. Class II transac- serine/threonine-rich, and GTP-binding regions in the major histocompatibility tivator (CIITA) is sufficient for the inducible expression of major histocompati- complex class II transactivator: generation of transdominant-negative mutants. bility complex class II genes. J. Exp. Med. 180:1367. Proc. Natl. Acad. Sci. USA 94:2501. 3. Chang, C.-H., and R. A. Flavell. 1995. Class II transactivator regulates the ex- 28. Timmerman, L. A., J. I. Healy, S. N. Ho, L. Chen, C. C. Goodnow, and pression of multiple genes involved in antigen presentation. J. Exp. Med. 181: G. R. Crabtree. 1997. Redundant expression but selective utilization of nuclear 765. factor of activated T cells family members. J. Immunol. 159:2735. 4. Steimle, V., L. A. Otten, M. Zufferey, and B. Mach. 1993. Complementation 29. Perkins, D. N., L. K. Felzien, J. C. Betts, K. Leung, D. H. Beach, and G. J. Nabel. cloning of an MHC class II transactivator mutated in hereditary MHC class II 1977. Regulation of NF-␬B by cyclin-dependent kinases associated with the p300 deficiency (or bare lymphocyte syndrome). Cell 75:135. coactivator. Science 275:523. 5. Chang, C.-H., S. Guerder, S.-C. Hong, W. van Ewijk, and R. A. Flavell. 1996. Mice lacking the MHC class II transactivator (CIITA) show tissue-specific im- 30. Moran, B., and B. Zerler. 1988. Interactions between cell growth-regulating do- pairment of MHC class II expression. Immunity 4:167. mains in the products of the adenovirus E1A oncogene. Mol. Cell. Biol. 8:1756. 6. Gourley, T., S. Roys, N. W. Lukacs, S. L. Kunkel, R. A. Flavell, and C.-H. 31. Wenner, C. A., S. J. Szabo, and K. M. Murphy. 1997. Identification of IL-4 Chang. 1999. A novel role for the major histocompatibility complex class II promoter elements conferring Th2-restricted expression during T helper cell sub- transactivator CIITA in the repression of IL-4 production. Immunity 10:377. set development. J. Immunol. 158:765. 7. Fontes, J. D., N. Jabrane-Ferrat, C. R. Toth, and B. M. Peterlin. 1996. Binding 32. Rincon, M., and R. A. Flavell. 1994. AP-1 transcriptional activity requires both and cooperative interactions between two B cell-specific transcriptional coactivators. J. Exp. Med. 183:2517. T-cell receptor-mediated and co-stimulatory signals in primary T lymphocytes. 8. Scholl, T., S. K. Mahanta, and J. L. Strominger. 1997. Specific complex forma- EMBO J. 13:4370. tion between the type II bare lymphocyte syndrome-associated transactivators 33. Cunningham, K., H. Ackerly, F. Alt, and W. Dunnick. 1998. Potential regulatory CIITA and RFX5. Proc. Natl. Acad. Sci. USA 94:6330. elements for germline transcription in or near murine Sg1. Int. Immunol. 10:527. The Journal of Immunology 2517

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