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• W2004381788 abstract "Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells. Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells. interleukin base pair(s) chloramphenicol acetyltransferase CCAAT-binding factor CP2-response element CP2 mutant lacking the Elf-1-homologous region of the DNA-binding domain electrophoretic mobility shift assay glyceraldehyde-3-phosphate dehydrogenase leader-binding protein nuclear factor of activated T cells recombinant CP2 signal transducer and activator of transcription T helper cytomegalovirus Interleukin (IL)1-4 is a pleiotropic cytokine that modulates the differentiation and the biologic activities of virtually all cells of hematopoietic origin (1Boulay J.-L. Paul W.E. J. Biol. Chem. 1992; 264: 20525-20528Abstract Full Text PDF Google Scholar). IL-4 is typically expressed in (and, at the same time, promotes the differentiation of) the T helper 2 (Th2) functional subset of CD4+ T cells, thereby playing a pivotal role in the regulation of humoral and allergic responses. Conversely, Th1 cell-associated cytokines, such as IL-2 and interferon-γ, are central to the development of cell-mediated, delayed type, and autoimmune responses (2O'Garra A. Immunity. 1998; 8: 275-283Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar). The biochemical and molecular mechanisms accounting for polarized expression of cytokine genes in T cells have been the focus of a number of studies over the past few years (3Fitch F.W. McKisic M.D. Lancki D.W. Gajewski T.F. Annu. Rev. Immunol. 1993; 11: 29-48Crossref PubMed Scopus (345) Google Scholar). Calcineurin-mediated activation of members of the nuclear factor of activated T cell (NFAT) family of transcription factors has been associated with antigen-dependent cytokine gene expression in both Th1 and Th2 cells (4Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2195) Google Scholar). However, NFAT-directed IL-4 transcription is preferentially induced in Th2 cells, presumably due to the involvement of Th2-restricted co-factors (5Li B. Tournier C. Davis R.J. Flavell R.A. EMBO J. 1999; 18: 420-432Crossref PubMed Scopus (294) Google Scholar, 6Ho I.-C. Hodge M.R. Rooney J.W. Glimcher L.H. Cell. 1996; 85: 973-983Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). It is likely that the commitment toward an IL-4-producing, Th2 phenotype is the outcome of a complex, dynamic interaction of multiple transcriptional regulators rather than the effect of a single protein. Irrespective of the cytokine milieu leading to preferential Th1 or Th2 differentiation (2O'Garra A. Immunity. 1998; 8: 275-283Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar), priming of T cells by antigen or mitogens is a requirement for IL-4 but not IL-2 production (7Weinberg A.D. English M. Swain S.L. J. Immunol. 1990; 144: 1800-1807PubMed Google Scholar). In fact, the IL-4 gene is only transcribed in actively dividing T cells, while IL-2 expression occurs independently of the cell cycle (8Bird J.J. Brown D.R. Mullen A.C. Moskowitz N.H. Mahowald M.A. Sider J.R. Gajewski T.F. Wang C.R. Reiner S.L. Immunity. 1998; 9: 229-237Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). T cell priming affects the expression and/or activation of a number of factors presumably involved in the regulation of cytokine genes. For example, primed T cells express higher levels of NFAT-1 and support greater NFAT-directed transcription than naive T cells (9Cron R.Q. Bort S.J. Wang Y. Brunvand M.W. Lewis D.B. J. Immunol. 1999; 162: 860-870PubMed Google Scholar). An earlier study also showed rapid and marked up-regulation of the DNA binding activity of the transcription factor CP2 following mitogenic stimulation of peripheral blood naive T cells, suggesting the involvement of this protein in the activation of immediate and/or early genes in these cells (10Volker J.L. Rameh L.E. Zhu Q. DeCaprio J. Hansen U. Genes Dev. 1997; 11: 1435-1446Crossref PubMed Scopus (51) Google Scholar). CP2, also known as leader-binding protein (LBP)-1c (11Yoon J.-B. Li G. Roeder R.G. Mol. Cell. Biol. 1994; 14: 1776-1785Crossref PubMed Scopus (98) Google Scholar) or late simian virus 40 factor (12Shirra M.K. Zhu Q. Huang H.-C. Pallas D. Hansen U. Mol. Cell. Biol. 1994; 14: 5076-5087Crossref PubMed Scopus (51) Google Scholar), is the prototypical member of a novel family of mammalian proteins sharing a high degree of similarity to Elf-1, aDrosophila melanogaster tissue-specific factor encoded at the embryonic lethal locus Grainyhead (11Yoon J.-B. Li G. Roeder R.G. Mol. Cell. Biol. 1994; 14: 1776-1785Crossref PubMed Scopus (98) Google Scholar, 13Uv A.E. Thompson C.R.L. Bray S.J. Mol. Cell. Biol. 1994; 14: 4020-4031Crossref PubMed Google Scholar). By interacting with a hyphenated sequence composed of two directly repeated 4-base pair (bp) motifs separated by a 6-bp linker (CNRG-N6-CNR(G/C)) (11Yoon J.-B. Li G. Roeder R.G. Mol. Cell. Biol. 1994; 14: 1776-1785Crossref PubMed Scopus (98) Google Scholar, 14Lim L.C. Fang L. Swendeman S.L. Sheffery M. J. Biol. Chem. 1993; 268: 18008-18017Abstract Full Text PDF PubMed Google Scholar), CP2 has been reported to regulate transcription of a number of viral and cellular genes in response to developmental signals, including those encoding for rat γ-fibrinogen (15Chodosh L.A. Baldwin A.S. Carthew R.W. Sharp P.A. Cell. 1988; 53: 11-24Abstract Full Text PDF PubMed Scopus (434) Google Scholar), mouse α-globin (16Lim L.C. Swendeman S.L. Sheffery M. Mol. Cell. Biol. 1992; 12: 828-835Crossref PubMed Google Scholar), simian virus 40 (12), human immunodeficiency virus-1 (11Yoon J.-B. Li G. Roeder R.G. Mol. Cell. Biol. 1994; 14: 1776-1785Crossref PubMed Scopus (98) Google Scholar), herpes simplex virus-1 (17Dabrowski C.E. Carmillo P.J. Schaffer P.A. Mol. Cell. Biol. 1994; 14: 2545-2555Crossref PubMed Scopus (32) Google Scholar), γ- and ε-globin, and the human β-like globin gene cluster (18Cunningham J.M. Jane S.M. Blood. 1995; 86 Suppl. 1 (abstr.): 4Google Scholar), major histocompatibility complex class II Ea (19Bellorini M. Dantonel J.C. Yoon J.-B. Roeder R.G. Tora L. Mantovani R. Mol. Cell. Biol. 1996; 16: 503-512Crossref PubMed Scopus (30) Google Scholar), and human c-fos and mouse thymidylate synthase (20Shirra M.K. Hansen U. J. Biol. Chem. 1998; 273: 19260-19268Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). However, its contribution to cytokine gene expression in T cells has not been explored to date. In this study, we investigated the contribution of CP2 to the mechanisms regulating expression of the two pivotal T cell-restricted cytokines IL-2 and IL-4. A line of Jurkat T cells, which constitutively expresses IL-4 and produces IL-2 and interferon-γ following activation, was a gift by Dr. Jack L. Strominger (Harvard University). Cells were cultured in RPMI (Mediatech, Herndon, VA), containing 2 mml-glutamine, 10% heat-inactivated fetal bovine serum (Gemini Bio-Products, Calabasas, CA), and 50 μg/ml gentamicin (complete medium). Aliquots of cells frozen at early passages were recovered from liquid nitrogen and used for experiments between 1 and 6 weeks after thawing. The murine conalbumin-specific Th2 clone D10.G4.1 (D10; American Type Culture Collection, Manassas, VA) was maintained in culture by biweekly restimulation with antigen and irradiated (2,000 rads) syngeneic splenocytes as antigen-presenting cells in RPMI supplemented with 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 μm2-mercaptoethanol. IL-4 expression in these cultures was verified using a sensitive commercial enzyme-linked immunosorbent assay (Cytoscreen-US™, BIOSOURCE International, Camarillo, CA). The IL-4 promoter fragments used in this study were generated by polymerase chain reaction using human genomic DNA as a template and cloned into the HindIII and XbaI sites of a Bluescript vector (Stratagene Cloning Systems, La Jolla, CA). An XbaI-tailed oligonucleotide, corresponding to bp +36 to +55 of the human IL-4 gene, and one of eightHindIII-tailed oligonucleotides, corresponding to bp −741 to −722, −311 to −292, −265 to −246, −225 to −206, −175 to −156, −145 to −126, −95 to −76, or −65 to −46, were used to introduce the appropriate restriction sites at the invariant 3′- and at the 5′-ends, respectively. After sequence verification, each fragment was inserted into the HindIII and XbaI sites of the chloramphenicol acetyltransferase (CAT)-expressing reporter vector, pCAT-Basic (Promega, Madison, WI) to construct the corresponding reporter plasmids IL-4.741, IL-4.311,IL-4.265, IL-4.225, IL-4.175,IL-4.145, IL-4.95, and IL-4.65 (21Georas S. Cumberland J. Burke T. Park E. Ono S. Casolaro V. Leukemia. 2000; 14: 629-635Crossref PubMed Scopus (10) Google Scholar). An IL-4.225 plasmid carrying two point mutations within the CP2-response element (CPRE) was generated by site-directed mutagenesis (QuikChange™; Stratagene). The following mutagenic primer (mutations are underlined) and its complement were synthesized (Genosys Biotechnologies, The Woodlands, TX): 5′-CATTTTCCTATTGGTATTATTTCACAGGAACATTTTACCTG-3′. The CP2 expression plasmid was generated by insertion of the murine CP2 cDNA into the HindIII and NotI sites of a pRc/CMV vector (Invitrogen, San Diego, CA) (14Lim L.C. Fang L. Swendeman S.L. Sheffery M. J. Biol. Chem. 1993; 268: 18008-18017Abstract Full Text PDF PubMed Google Scholar). A CP2 mutant lacking the Elf-1-homologous region of the DNA-binding domain (ΔElf-1) was generated from a full-length template as described (22Zhong F. Swendeman S.L. Popik W. Pitha P.M. Sheffery M. J. Biol. Chem. 1994; 269: 21269-21276Abstract Full Text PDF PubMed Google Scholar) and inserted into the same vector. TheIL-2.15ΔCX reporter plasmid, bearing bp −319 to +52 of the human IL-2 gene in pCAT-Basic (23Shaw J.-P. Utz P.J. Durand D.B. Toole J.J. Emmel E.A. Crabtree G.R. Science. 1988; 241: 202-205Crossref PubMed Scopus (10) Google Scholar), has been kindly donated by Dr. Gerald R. Crabtree (Stanford University, Stanford, CA). Jurkat cells (0.5–1 × 106/ml) were transfected with 1 μg of reporter plasmids and 2 μg of expression plasmids by 48-h culture in 5 ml of complete medium containing 5.2 mg/ml LipofectAMINE (Life Technologies, Inc.) as described (24Casolaro V. Georas S.N. Song Z. Zubkoff I.D. Abdulkadir S.A. Thanos D. Ono S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11623-11627Crossref PubMed Scopus (83) Google Scholar). Equal amounts of the corresponding noncoding vectors were added to control samples to yield a constant amount (3 μg) of DNA in each transfection. Where indicated, cells were stimulated 16–18 h before harvest. 40–80 μg of proteins extracted from each sample were analyzed for CAT concentration using a commercial enzyme-linked immunosorbent assay (Roche Molecular Biochemicals). In all experiments, CAT concentrations were normalized by total protein content, measured in cell lysates using the Bradford reagent (Bio-Rad). D10 cells (5 × 107 cells/ml) were separated from irradiated splenocytes by centrifugation onto a Ficoll-Isopaque gradient (Amersham Pharmacia Biotech) and then electroporated (960 microfarads, 270 V) with 45 μg of expression or control plasmids in 0.4 ml of serum-free RPMI using a Gene Pulser II System (Bio-Rad). Following 20-h recovery in complete medium, D10 cells were seeded on plates coated with 1 μg/ml anti-CD3 antibody (clone 145–2C11; BD PharMingen, San Diego, CA) and then incubated for 16 h prior to RNA extraction. Total RNA was extracted from D10 cells using RNAzol B (Tel-Test, Inc., Friendswood, TX). Specific antisense DNA templates for murine IL-4 and for the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased (BD PharMingen). These were used to synthesize the α-[32P]UTP (3,000 Ci/mmol, 10 mCi/ml; Amersham Pharmacia Biotech)-labeled probes in the presence of a GACU pool using a T7 RNA polymerase (Promega). Riboprobes (500,000 cpm) were hybridized with 5–15 μg of target RNA at 45 °C, followed by digestion with RNase A (Promega) for 30 min at 30 °C as described (25Song Z. Krishna S. Thanos D. Strominger J.L. Ono S.J. J. Exp. Med. 1994; 180: 1763-1774Crossref PubMed Scopus (79) Google Scholar). The samples were loaded on an acrylamide-urea sequencing gel next to labeled DNA molecular weight markers and next to the labeled probes and run at 50 watts in 45 mm Tris, pH 8.2, 45 mm boric acid, and 1 mm EDTA. The gel was adsorbed to filter paper, dried under vacuum, and exposed to film (X-OmatTM AR; Eastman Kodak Co.) at −70 °C with an intensifying screen. Autoradiographs were quantified by computerized densitometry using a Kodak Digital Science Electrophoresis Documentation and Analysis System. Results are shown as the ratio of IL-4/GAPDH densitometric units. Nuclear extracts were prepared by a modification of a described protocol (26Li Y. Ross J. Scheppler J.A. Franza Jr., B.R. Mol. Cell. Biol. 1991; 11: 1883-1893Crossref PubMed Scopus (76) Google Scholar). Jurkat cells (2–5 × 107) were allowed to swell in 10 mm Hepes, pH 7.9, 30 mm KCl, 1 mmdithiothreitol, 0.2 mm EDTA, 0.5 mmphenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, and 1 μg/ml aprotinin and then lysed (5 min, 4 °C) by the addition of 0.075% Nonidet P-40. Nuclei were isolated by centrifugation (4 min, 3,000 rpm) and then extracted (40 min, 4 °C) in 20 mm Hepes, pH 7.9, 420 mm KCl, 1 mm dithiothreitol, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 1 μg/ml aprotinin, and 20% glycerol. Extracts were separated from cellular debris and membranes (10 min, 14,000 rpm), aliquoted, quick-frozen in liquid nitrogen, and then stored at −80 °C. Protein concentrations in all extracts were measured by the Bradford protocol (Bio-Rad). Recombinant CP2 (rCP2) was prepared and enriched as described previously (14Lim L.C. Fang L. Swendeman S.L. Sheffery M. J. Biol. Chem. 1993; 268: 18008-18017Abstract Full Text PDF PubMed Google Scholar). The following oligonucleotides and their complements were synthesized (Genosys Biotechnologies): 5′-TGCTGAAACTTTGTAGTTAATTTTTTAAAAAGGTTTCATTTTCCTATTGG-3′ (225), 5′-AGGTTTCATTTTCCTATTGGTCTGATTTCACAGGAACATTTTACCTGTTT-3′ (195), 5′-TCTGATTTCACAGGAACATTTTACCTGTTT-3′ (175), 5′-TTGGTCTGATTTCACAGGAACAT-3′ (IL-4 CPRE), 5′-TTGGTATTATTTCACAGGAACAT-3′ (IL-4 CPRE MUT), and 5′-TAGAGCAAGCACAAACCAGGCCAA-3′ (α-globin CPRE). These were either 5′-end-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [γ-32P]ATP or labeled by random hexamer priming using the Klenow fragment (Roche Molecular Biochemicals) and [α-32P]dCTP (Amersham Pharmacia Biotech). In both cases, radiolabeled probes were purified by spin column chromatography (ProbeQuant; Amersham Pharmacia Biotech) and then by electroelution from 4% nondenaturing polyacrylamide gels. For EMSA, probes (10,000–30,000 cpm, corresponding to 5–20 fmol) were incubated (20–30 min, 25 °C) with 1–5 μg of nuclear extracts or 10–100 ng of rCP2 in 15 μl of 12 mm Hepes, pH 7.9, 50 mm KCl, 0.5 mm MgCl2, 0.12 mm EDTA, 0.12 mm EGTA, 4 mmdithiothreitol, 0.1% Nonidet P-40, 12% glycerol, 0.1 mg/ml bovine serum albumin, and 30 μg/ml (10 μg/ml in the case of rCP2) poly(dI-dC) (Amersham Pharmacia Biotech). Where indicated, 20 min after the addition of the probe, the binding reactions were incubated (30 min, 4 °C) with rabbit antisera specific for the transcription factors CP2 (16Lim L.C. Swendeman S.L. Sheffery M. Mol. Cell. Biol. 1992; 12: 828-835Crossref PubMed Google Scholar), NFAT-1 (Upstate Biotechnology, Inc., Lake Placid, NY), CCAAT-binding factor (CBF)-A (Accurate Chemical & Scientific, Westbury, NY), or signal transducer and activator of transcription 6 (STAT6) (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA). This led, under the experimental conditions described, to ablation of the immunoreactive complexes, with almost no detectable “supershift” (14Lim L.C. Fang L. Swendeman S.L. Sheffery M. J. Biol. Chem. 1993; 268: 18008-18017Abstract Full Text PDF PubMed Google Scholar). Free probes and DNA-protein complexes were resolved by electrophoresis on 4% native polyacrylamide gels in 45 mmTris, pH 8.2, 45 mm boric acid, 1 mm EDTA, and 1% glycerol and then visualized by autoradiography of fixed and dried gels. Footprinting experiments were performed as described (27Sigman D.S. Biochemistry. 1990; 29: 9097-9105Crossref PubMed Scopus (292) Google Scholar). To identify discrete nucleotide contacts for rCP2 within the CP2-responsive IL-4 promoter region, a 195–146 oligonucleotide was 5′-end-labeled on the coding or noncoding strand and then incubated (30 min, 25 °C) with 100 ng of rCP2 in 25 μl of EMSA buffer (described above). Samples were electrophoresed on a 4% native polyacrylamide gel, which was then immersed into a solution containing 10 mm Tris, pH 8.0, 0.2 mm1,10-phenanthroline, and 0.045 mm CuSO4(Sigma). The chemical nuclease reaction was started by the addition of mercaptopropionic acid (Sigma) to 0.05%, allowed to proceed for 12 min at 25 °C, and then quenched in 2 mm2,9-dimethyl-1,10-phenanthroline (Sigma). Free DNA and DNA-protein complexes, visualized by autoradiography, were eluted from the gel matrix (18 h at 37 °C) in 0.5 m ammonium acetate, pH 7.5, 1 mm EDTA, and 0.1% sodium dodecyl sulfate. Equivalent amounts of DNA from each sample and of a Maxam-Gilbert G+A ladder of the same probe (27Sigman D.S. Biochemistry. 1990; 29: 9097-9105Crossref PubMed Scopus (292) Google Scholar) were resolved by electrophoresis on an 8% acrylamide, 7 m urea gel. Several potential CP2 elements are located within the proximal 300 bp of the human IL-4 promoter. We analyzed the effect of transient CP2 overexpression on CAT gene expression driven by an IL-4 promoter region included between bp −311 and +55 in the human T cell line Jurkat. In agreement with previous studies (24Casolaro V. Georas S.N. Song Z. Zubkoff I.D. Abdulkadir S.A. Thanos D. Ono S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11623-11627Crossref PubMed Scopus (83) Google Scholar, 28Arai N. Nomura D. Villaret D. DeWaal-Malefijt R. Seiki M. Yoshida M. Minoshima S. Fukuyama R. Maekawa M. Kudoh J. Shimizu N. Yokota K. Abe E. Yokota T. Takebe Y. Arai K. J. Immunol. 1989; 142: 274-282PubMed Google Scholar, 29Li-Weber M. Eder A. Krafft-Czepa H. Krammer P.H. J. Immunol. 1992; 148: 1913-1918PubMed Google Scholar), we found that these cells are in a preactivated state with respect to IL-4 expression, characterized by constitutively elevated IL-4 mRNA accumulation (data not shown) and promoter activity (Fig.1). Constitutive IL-4 promoter activity was enhanced up to 5-fold in Jurkat cells transiently transfected with a CP2 expression plasmid (Fig. 1). CP2 overexpression resulted in less noticeable increase of IL-4 promoter activity in cells stimulated with PMA (10 ng/ml) and the Ca2+ ionophore A23187 (1 μm), presumably due to the contribution of endogenous CP2 to IL-4 activation in these cells (see below). In striking contrast, PMA- and A23187-induced IL-2 promoter activity was reduced by up to 80% in CP2-overexpressing cells (Fig. 1). To map the IL-4 promoter element(s) necessary for transactivation by CP2, we generated a panel of deletional mutants for use in reporter studies (Fig.2 A) (21Georas S. Cumberland J. Burke T. Park E. Ono S. Casolaro V. Leukemia. 2000; 14: 629-635Crossref PubMed Scopus (10) Google Scholar). Transiently overexpressed CP2 was more effective on promoter constructs truncated at bp −265 or −225 than on IL-4.311 or IL-4.741 (Fig. 2 B). However, removal of the region spanning bp −225 to −176 decreased transcriptional activation in CP2-overexpressing cells by almost 4-fold, while constructs truncated at bp −145 through −65 were unresponsive to the factor (Fig. 2 B). This indicated that a CPRE is located between bp −175 and −146, although additional nucleotide contacts upstream of bp −175 might be required for full promoter inducibility by CP2. The IL-4 promoter region included between bp −225 and −146, shown in Fig. 2 C, harbors a number of elements contributing to a varying degree to IL-4 transcriptional activation (21Georas S. Cumberland J. Burke T. Park E. Ono S. Casolaro V. Leukemia. 2000; 14: 629-635Crossref PubMed Scopus (10) Google Scholar, 30Takemoto N. Koyano-Nakagawa N. Arai N. Arai K. Yokota T. Int. Immunol. 1997; 9: 1329-1338Crossref PubMed Scopus (29) Google Scholar, 31Li-Weber M. Davydov I.V. Krafft H. Krammer P.H. J. Immunol. 1994; 153: 4122-4133PubMed Google Scholar). To verify whether CP2 can directly bind to elements located within this IL-4 promoter region, we generated three oligonucleotides spanning partially overlapping sequences for use in EMSA (Fig. 2 C). rCP2, used at a concentration (100 ng/15 μl) sufficient for saturation of an α-globin consensus oligonucleotide (Fig.2 D, lane 1), did not bind to oligonucleotides 175–146 (lane 2) and225–176 (lane 4). Since nucleotide contacts in both subregions might be required for CP2 binding, we also used as a probe an oligonucleotide (195–146) including the proximal 20 bp of the 225–176 segment in addition to175–146 (Fig. 2C). This resulted in consistent formation of a CP2 complex of apparently lower affinity than that formed on the α-globin probe (Fig. 2 D, lane 3). A complex of faster mobility also formed on the195-146 oligonucleotide, which was accounted for by binding of monomeric CP2, as assessed by coincubation with CP2-specific antibodies (data not shown). To define the nucleotide contacts necessary for CP2 binding to this region of the IL-4 promoter, we analyzed the pattern of protection from chemical cleavage of a 195–146 oligonucleotide following EMSA with rCP2. In experiments using copper-phenanthroline as the cleaving agent (27Sigman D.S. Biochemistry. 1990; 29: 9097-9105Crossref PubMed Scopus (292) Google Scholar), rCP2 (100 ng) protected a sequence extending from bp −177 to −158 (Fig. 2 E). A CPRE (−174CTGATTTCACAGG−162) is recognizable within this sequence. Its homology to CP2 elements identified within other cellular and viral promoters is shown in Fig. 2 F. The IL-4 CPRE displays a conserved (−165CAGG−162) and an imperfect CNRG motif (−174CTGA−171) separated by a 5-bp linker. A constitutive CP2-immunoreactive complex was formed in EMSA using a consensus α-globin CP2 oligonucleotide and nuclear extracts from Jurkat cells (Fig. 3, A and B). CP2 nuclear expression and/or DNA binding activity in these cells was not affected by stimulation with PMA and A23187 (Fig. 3 A,lanes 2 and 3). Four complexes were detectable in EMSA with Jurkat nuclear extracts and a similar length oligonucleotide probe centered on the IL-4 promoter CPRE (lanes 4–6). Differently from the α-globin complex, Ca2+-mediated stimulation did affect formation of the slower mobility complexes I and II, with consistently increased formation of complex II and diminished complex I formation (lane 5). Both complexes appeared to contain immunoreactive CP2, as indicated by the neutralizing effect of rabbit anti-CP2 antibodies (Fig. 3 B, lane 6), while neither complex reacted to CBF-A-, NFAT-1-, or STAT6-specific antibodies (lanes 7 and 8 and data not shown). Complexes III and IV apparently consisted of unrelated constitutive nuclear protein(s) of unclear sequence specificity. To elucidate the contribution of endogenously expressed CP2 to constitutive and inducible activity of the IL-4 promoter, we analyzed CAT expression in Jurkat cells transiently transfected with plasmids carrying point mutations within the IL-4 CPRE that would selectively affect the formation of the CP2-containing complexes. Disruption of the distal CNRG motif (−174ATTATTTCACAGG−162) was sufficient to markedly decrease CP2 binding in EMSA using Jurkat nuclear extracts (Fig. 3 C) or rCP2 (not shown). This was accompanied by an almost 50% decrease in constitutive and induced CAT expression in Jurkat cells transiently transfected with anIL-4.225 plasmid carrying the same mutation within the CPRE (Fig. 3 D). To further assess the contribution of endogenous CP2 to IL-4 promoter activity, we generated, for use in transient transfection experiments, a plasmid encoding a CP2 polypeptide lacking the Elf-1 homology region of the DNA-binding domain (13Uv A.E. Thompson C.R.L. Bray S.J. Mol. Cell. Biol. 1994; 14: 4020-4031Crossref PubMed Google Scholar) and referred to as ΔElf-1 mutant. In a previous study, this mutant did not bind to DNA and specifically inhibited binding of full-length CP2 in a dominant negative fashion (22Zhong F. Swendeman S.L. Popik W. Pitha P.M. Sheffery M. J. Biol. Chem. 1994; 269: 21269-21276Abstract Full Text PDF PubMed Google Scholar). Its overexpression inhibited by >75% constitutive IL-4 promoter activity in Jurkat cells (Fig. 4 A). A similar degree of IL-4 promoter inhibition was seen in cells stimulated with A23187, while IL-2 promoter activity was fundamentally unaffected (data not shown). Up-regulation of IL-4 promoter activity in CP2-overexpressing Jurkat cells was often paralleled by markedly increased IL-4 secretion, suggesting direct activation of the endogenous IL-4 gene (data not shown). However, due to low level expression of IL-4 and the poor transfection efficiency in these cells, the effect of overexpressed ΔElf-1 CP2 was far less apparent (not shown). To elucidate the role of CP2 in transcriptional activation of the endogenous IL-4 gene, we therefore assessed the effect of overexpressed CP2 polypeptides on IL-4 transcript accumulation in the murine Th2 cell line D10. As shown in Fig. 4, B and C, D10 cells transfected to higher than 50% efficiency with a full-length CP2-encoding plasmid expressed, upon CD3 ligation, markedly higher levels of IL-4 mRNA than cells transfected with the corresponding noncoding plasmid. In contrast, and in clear agreement with our findings shown in Fig. 4 A, overexpression of the dominant negative ΔElf-1 mutant consistently interfered with CD3-dependent activation of the IL-4 gene in D10 cells. In this study, we provide the first evidence of the involvement of CP2, the prototypical member of a novel family of transcription factors related to the Drosophila developmental protein Elf-1, in the regulation of cytokine genes expressed in T cells. CP2 has been involved in gene regulation in a variety of cell lineages, where it can act as both a transcriptional activator and a repressor (14Lim L.C. Fang L. Swendeman S.L. Sheffery M. J. Biol. Chem. 1993; 268: 18008-18017Abstract Full Text PDF PubMed Google Scholar, 22Zhong F. Swendeman S.L. Popik W. Pitha P.M. Sheffery M. J. Biol. Chem. 1994; 269: 21269-21276Abstract Full Text PDF PubMed Google Scholar, 32Parada C.A. Yoon J.B. Roeder R.G. J. Biol. Chem. 1995; 270: 2274-2283Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar,33Cunningham J.M. Vanin E.F. Tran N. Valentine M. Jane S.M. Genomics. 1995; 30: 398-399PubMed Google Scholar). We confirm here the dual nature of this factor, a" @default.
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