Repression of IL-2 Promoter Activity by the Novel Basic Leucine Zipper p21 SNFT Protein This information is current as Milena Iacobelli, William Wachsman and Kathleen L. of September 29, 2021. McGuire J Immunol 2000; 165:860-868; ; doi: 10.4049/jimmunol.165.2.860 http://www.jimmunol.org/content/165/2/860 Downloaded from References This article cites 49 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/165/2/860.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 September 29, 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. Repression of IL-2 Promoter Activity by the Novel Basic Leucine Zipper p21SNFT Protein1,2 Milena Iacobelli,* William Wachsman,† and Kathleen L. McGuire3* IL-2 is the major autocrine and paracrine growth factor produced by T cells upon T cell stimulation. The inducible expression of IL-2 is highly regulated by multiple transcription factors, particularly AP-1, which coordinately activate the promoter. De- scribed here is the ability of the novel basic leucine zipper protein p21SNFT to repress AP-1 activity and IL-2 transcription. A detailed analysis of the repression by p21SNFT repression on the IL-2 promoter distal NF-AT/AP-1 site demonstrates that it can bind DNA with NF-AT and Jun, strongly suggesting that it represses NF-AT/AP-1 activity by competing with Fos proteins for Jun dimerization. The importance of this repression is that p21SNFT inhibits the trans-activation potential of protein complexes that contain Jun, thereby demonstrating an additional level of control for the highly regulated, ubiquitous AP-1 transcription factor and the IL-2 gene. The Journal of Immunology, 2000, 165: 860–868. Downloaded from ctivated Th cells of the immune system produce high adjacent AP-1 or AP-1-like sites for binding and/or functional ac- levels of the IL-2 cytokine when properly stimulated tivity. The significance of AP-1 for the activity of this promoter A through both the TCR and the CD28 costimulatory mol- has been demonstrated for the positive regulation of IL-2 expres- ecule (1, 2). IL-2 production is indicative of T cell activation and sion, as well as in anergic T cells, where a lack of AP-1 activity is is the major autocrine and paracrine growth factor for T cells. thought to be responsible for the inhibition of IL-2 production http://www.jimmunol.org/ While not produced by normal, quiescent T cells, the IL-2 gene (13–17). rapidly becomes transcriptionally active upon stimulation. IL-2 The AP-1 transcription factor consists of heterodimers between mRNA production is detectable within 40 min, peaks at 4–6 h, and the Fos and Jun families of proteins through their basic leucine returns to background levels by 24 h (2, 3). Although control of zipper (bZIP) domains. The expression and trans-activation po- IL-2 protein synthesis occurs at many levels, it is principally reg- tential of the AP-1 transcription factor are highly dependent on the ulated at the level of transcription. activation of stress and mitogenic signal transduction cascades Although there is evidence that sequences outside of the (18). Activation of the JNK and MEK MAP kinase pathways not ϳ300-bp human IL-2 promoter contribute to transcriptional con- only controls the overall levels of Fos and Jun proteins, but also trol in vivo, this region is primarily responsible for inducible ex- leads to the proper phosphorylation states of these proteins, which by guest on September 29, 2021 pression of the gene (4). The promoter contains a variety of bind- are required for transcriptional activity (18). Although c-Jun and ing sites for known activating transcription factors, such as NF- c-Fos dimers are considered to be the classical AP-1 transcription AT, NF-␬B, AP-1, and OCT,4 as well as the inhibitory protein factor, a variety of dimer combinations can exist among Fos, Jun, NIL-2 (2, 5). All four of the activating factors have been shown to and CREB/ATF family members, lending specificity of a particu- have multiple binding sites within the promoter. In addition, the lar dimer pair to an AP-1 or AP-1-like enhancer (TRE). Dimers of proximal OCT (6, 7), the CD28 response element (8–10), and the Jun and Fos or CREB/ATF have been shown to regulate the ex- distal NF-AT (11, 12) elements have all been shown to work with pression of a variety of immunologically important genes involved in T cell activation and cytokine expression (19). *Department of Biology and Molecular Biology Institute, San Diego State University, Additional proteins have been described that are capable of San Diego, CA 92182; and †Medical and Research Services, San Diego Veterans dimerizing with Jun but that do not belong to either the Fos or Affairs Medical Center, Division of Hematology/Oncology, and Cancer Center, Uni- CREB/ATF family. These rat proteins are called Jun dimerization versity of California-San Diego, La Jolla, CA 92093 proteins 1 and 2 (JDP1 and JDP-2) and are capable of repressing Received for publication January 10, 2000. Accepted for publication April 25, 2000. the expression of an AP-1-regulated reporter construct (20). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance JDP-1, like the closely related human B-ATF protein, can bind with 18 U.S.C. Section 1734 solely to indicate this fact. with Jun to a TRE sequence (20, 21). JDP-1 and B-ATF are also 1 This work was supported by Grants CRP CA99-00542V-10051 (to K.L.M.), closely related to the Merek’s disease virus EcoQ protein (MEQ), CA53382 (to K.L.M.), and CA59713 (to W.W.) from the National Cancer Institute another bZIP protein capable of Jun dimerization but whose inter- (Bethesda, MD) and by a Veterens Affairs Career Development Award (to W.W.). actions with Jun lead to viral gene transcription (22). It currently 2 SNFT The GenBank accession number for the p21 coding sequence is AF255346. is not clear what the physiological relevance is of Jun interactions 3 Address correspondence to Dr. Kathleen L. McGuire, Department of Biology, San with proteins such as B-ATF, JDP1, and JDP-2, because their abil- Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4614. E-mail address: [email protected] ity to dimerize with Jun has not been shown to influence the tran- 4 Abbreviations used in this paper: OCT, octomer binding protein; NIL-2, negative scription of any cellular gene. regulator of IL-2; bZIP, basic leucine zipper; JNK, Jun N-terminal kinase; MEK, Recently, a novel human protein named p21SNFT (for 21-kDa mitogen-activated kinase-extracellular regulated kinase kinase; MEKK, MEK kinase; small nuclear factor isolated from T cells) was identified that CREB, cAMP response element binding protein; ATF, activating transcription factor; p21SNFT, 21-kDa small nuclear factor isolated from T cells; TRE, 12-O-tetradecano- dimerizes with Jun proteins and represses AP-1 activity (W. ate-13-acetate response element; JDP, Jun dimerization protein; MEQ, Merek’s dis- Klump and W. Wachsman, manuscript in preparation). The bio- ease virus EcoQ protein; HTLV-I, human T cell leukemia virus type I; LTR, long SNFT terminal repeat; RSV, Rous sarcoma virus; FRK, Fos-regulating kinase; PKA, protein chemical characteristics of p21 are similar to the known ac- kinase A; CAT, chloramphenicol acetyltransferase. tivities of the JDP proteins, and it shares high amino acid identity Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 861 (69%) to rat JDP-1, suggesting that p21SNFT may be its human STAT-60 (Tel-Test, Friendswood, TX) and was probed with a radioac- homologue. Due to the properties of p21SNFT and the function of tively labeled IL-2 probe. The filter was later probed with a TCR␤ probe AP-1 in IL-2 gene expression, the involvement of this factor in the to control for RNA loading. transcriptional regulation of IL-2 was investigated. Cytokine expression The work shows that p21SNFT significantly and specifically The J-CI-1, J-CI-2, J-SNFT-1, and J-SNFT-2 stable cell lines were resus- down-regulates IL-2 promoter activity and endogenous IL-2 pro- pended at 2.5 ϫ 105 cells/ml in culture medium and stimulated with 10 SNFT duction by Jurkat cells. The ability of p21 to repress the IL-2 ng/ml PMA and 1.5 ␮M ionomycin with or without mAb 9.3 at a 1/2500 promoter occurs though multiple IL-2 enhancers that functionally dilution (CD28 stimulation). Cells were incubated for 4 days, and culture require AP-1. p21SNFT is shown to bind TRE sequences with Jun, supernatants were harvested and tested for IL-2 and GM-CSF levels by to the exclusion of Fos, and to bind to the IL-2 promoter distal ELISA (BioSource International, Camarillo, CA) according to the manu- facturer’s directions.