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• W1963512312 abstract "Interleukin-4 (IL-4) is a multifunctional cytokine that plays an important role in immune and inflammatory responses. Expression of the IL-4 gene is tightly controlled at the level of gene transcription by both positive and negative regulatory elements in the IL-4 promoter. Several constitutive nuclear factors have been identified that can interact with IL-4 promoter elements in DNA binding assays. Here we report that the zinc-finger protein YY-1 (Yin-Yang 1) can bind to multiple elements within the human IL-4 promoter. Cotransfection of Jurkat T cells with different IL-4 promoter/reporter constructs together with expression vectors encoding antisense, wild-type, or zinc finger-deleted mutant YY-1 suggested that YY-1 enhanced IL-4 promoter activity in a DNA-binding domain-dependent manner. Site-directed mutagenesis revealed that a proximal YY-1-binding site, termed Y0 (−59TCATTTT−53), was essential for YY-1-driven IL-4 promoter activity. In addition, cotransfected YY-1 enhanced both IL-4 promoter activity and endogenous IL-4 gene expression in nontransformed peripheral blood T cells. Thus, YY-1 positively regulates IL-4 gene expression in lymphocytes. Interleukin-4 (IL-4) is a multifunctional cytokine that plays an important role in immune and inflammatory responses. Expression of the IL-4 gene is tightly controlled at the level of gene transcription by both positive and negative regulatory elements in the IL-4 promoter. Several constitutive nuclear factors have been identified that can interact with IL-4 promoter elements in DNA binding assays. Here we report that the zinc-finger protein YY-1 (Yin-Yang 1) can bind to multiple elements within the human IL-4 promoter. Cotransfection of Jurkat T cells with different IL-4 promoter/reporter constructs together with expression vectors encoding antisense, wild-type, or zinc finger-deleted mutant YY-1 suggested that YY-1 enhanced IL-4 promoter activity in a DNA-binding domain-dependent manner. Site-directed mutagenesis revealed that a proximal YY-1-binding site, termed Y0 (−59TCATTTT−53), was essential for YY-1-driven IL-4 promoter activity. In addition, cotransfected YY-1 enhanced both IL-4 promoter activity and endogenous IL-4 gene expression in nontransformed peripheral blood T cells. Thus, YY-1 positively regulates IL-4 gene expression in lymphocytes. interleukin-4 electrophoretic mobility shift assay interferon-γ Interleukin-4 (IL-4),1 a pleiotropic cytokine produced by activated T cells and basophils, plays a critical role in cellular and humoral immune responses (1Brown M.A. Hural J. Crit. Rev. Immunol. 1997; 17: 1-32Crossref PubMed Google Scholar). Dysregulated expression of IL-4 has been linked with autoimmune and allergic diseases (2Casolaro V. Georas S. Song Z. Ono S. Curr. Opin. Immunol. 1996; 8: 796-803Crossref PubMed Scopus (83) Google Scholar, 3Gallichan W.S. Balasa B. Davies J.D. Sarvetnick N. J. Immunol. 1999; 163: 1696-1703PubMed Google Scholar). In T cells, IL-4 gene expression is regulated at the transcriptional level by both ubiquitous and cell type-restricted factors (4Szabo S. Gold J. Murphy T. Murphy K. Mol. Cell. Biol. 1993; 13: 4793-4805Crossref PubMed Scopus (234) Google Scholar, 5Chuvpilo S. Schomberg C. Gerwig R. Heinfling A. Reeves R. Grummt F. Serfling E. Nucleic Acids Res. 1993; 21: 5694-5704Crossref PubMed Scopus (181) Google Scholar, 6Tara D. Weiss D. Brown M. J. Immunol. 1993; 151: 3617-3626PubMed Google Scholar, 7Li-Weber M. Krafft H. Krammer P. J. Immunol. 1993; 151: 1371-1382PubMed Google Scholar, 8Tara D. Weiss D. Brown M. J. Immunol. 1995; 154: 4592-4602PubMed Google Scholar, 9Burke T.F. Casolaro V. Georas S.N. Biochem. Biophys. Res. Commun. 2000; 270: 1016-1023Crossref PubMed Scopus (31) Google Scholar, 10Georas S. Cumberland J. Burke T. Chen R. Park E. Ono S. Casolaro V. Leukemia (Baltimore). 2000; 14: 629-635Crossref PubMed Scopus (10) Google Scholar, 11Li-Weber M. Salgame P. Hu C. Davydov I.V. Laur O. Klevenz S. Krammer P.H. J. Immunol. 1998; 161: 1380-1389PubMed Google Scholar). These factors interact with a proximal promoter region composed of multiple regulatory elements and can both positively and negatively influence transcriptional activation (see Fig. 1). Other regions have been identified outside of the proximal IL-4 promoter that can regulate IL-4 gene expression, including the IL-4/IL-13 intergenic region (12Loots G.G. Locksley R.M. Blankespoor C.M. Wang Z.E. Miller W. Rubin E.M. Frazer K.A. Science. 2000; 288: 136-140Crossref PubMed Scopus (658) Google Scholar), the IL-4 second intron (13Henkel G. Weiss D. McCoy R. Deloughery T. Tara D. Brown M. J. Immunol. 1992; 149: 3239-3246PubMed Google Scholar), and downstream of the IL-4 gene (14Agarwal S. Avni O. Rao A. Immunity. 2000; 12: 643-652Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Some of these elements appear to coordinately regulate IL-4 gene expression at the chromatin level (15Lee R.L. Fields P.E. Flavell R.A. Immunity. 2001; 14: 447-459Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Major insights into the regulation of IL-4 gene expression came from studies using transgenic or knockout approaches to investigate the molecular basis of Th2 differentiation in mice (reviewed in Ref. 16Murphy K.M. Ouyang W. Farrar J.D. Yang J. Ranganath S. Asnagli H. Afkarian M. Murphy T.L. Annu. Rev. Immunol. 2000; 18: 451-494Crossref PubMed Scopus (540) Google Scholar). Using these approaches, several transcription factors have been identified that are critical for this process, including Stat6 (17Kaplan M.H. Schindler U. Smiley S.T. Grusby M.J. Immunity. 1996; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar), NFATc (18Ranger A. Hodge M. Gravallese E. Oukka M. Davidson L. Alt F. de la Brousse F. Hoey T. Grusby M. Glimcher L. Immunity. 1998; 8: 125-134Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), c-Maf (19Ho 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), GATA-3 (20Zhang D.-H. Cohn L. Ray P. Bottomly K. Ray A. J. Biol. Chem. 1997; 272: 21597-21603Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar), and JunB (21Li B. Tournier C. Davis R.J. Flavell R.A. EMBO J. 1999; 18: 420-432Crossref PubMed Scopus (294) Google Scholar). The mechanisms by which these factors influence T cell differentiation is under active investigation. To date, direct binding to and/or activation of the IL-4 promoter has been demonstrated for NFATc (22Chen R. Burke T. Cumberland J. Brummet M. Beck L. Casolaro V. Georas S. J. Immunol. 2000; 164: 825-832Crossref PubMed Scopus (56) Google Scholar), c-Maf (19Ho 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), and JunB (21Li B. Tournier C. Davis R.J. Flavell R.A. EMBO J. 1999; 18: 420-432Crossref PubMed Scopus (294) Google Scholar), but not for Stat6 (23Georas S. Cumberland J. Burke T. Chen R. Schindler U. Casolaro V. Blood. 1998; 92: 4529-4538Crossref PubMed Google Scholar) or GATA-3 (24Zhang D.-H. Yang L. Ray A. J. Immunol. 1998; 161: 3817-3821PubMed Google Scholar). A two-step model has recently been proposed to explain IL-4 gene expression in Th2 cells (25Agarwal S. Viola J.P. Rao A. J. Allergy Clin. Immunol. 1999; 103: 990-999Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Based on the appearance of DNase-hypersensitive sites in the IL-4 gene locus (26Agarwal S. Rao A. Immunity. 1998; 9: 765-775Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, 27Takemoto N. Koyano-Nakagawa N. Yokota T. Arai N. Miyatake S. Arai K. Int. Immunol. 1998; 10: 1981-1985Crossref PubMed Scopus (123) Google Scholar), this is thought to involve an initial chromatin remodeling step followed by cytokine gene transcription in response to T cell receptor-activated transcription factors. Although chromatin remodeling is likely an important regulatory step during Th2 differentiation, the proximal IL-4 promoter confers a high degree of tissue specificity when linked to reporter genes in transgenic mice (15Lee R.L. Fields P.E. Flavell R.A. Immunity. 2001; 14: 447-459Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 28Todd M. Grusby M. Lederer J. Lacy E. Lichtman A. Glimcher L. J. Exp. Med. 1993; 177: 1663-1674Crossref PubMed Scopus (104) Google Scholar, 29Wenner C.A. Szabo S.J. Murphy K.M. J. Immunol. 1997; 158: 765-773PubMed Google Scholar). Thus, an analysis of the factors that regulate the activity of the proximal IL-4 promoter will enhance our understanding of IL-4 gene expression. We recently performed a detailed deletional analysis of the human proximal IL-4 promoter and discovered novel binding sites for several transcription factors, including NFAT (nuclearfactor of activated T cells) (9Burke T.F. Casolaro V. Georas S.N. Biochem. Biophys. Res. Commun. 2000; 270: 1016-1023Crossref PubMed Scopus (31) Google Scholar), CP-2 (30Casolaro V. Keane-Myers A.M. Swendeman S.L. Steindler C. Zhong F. Sheffery M. Georas S.N. Ono S.J. J. Biol. Chem. 2000; 275: 36605-36611Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and an uncharacterized repressor factor (10Georas S. Cumberland J. Burke T. Chen R. Park E. Ono S. Casolaro V. Leukemia (Baltimore). 2000; 14: 629-635Crossref PubMed Scopus (10) Google Scholar). This analysis also uncovered binding sites for several other constitutive nuclear factors, in keeping with previous studies on the IL-4 promoter in other systems (4Szabo S. Gold J. Murphy T. Murphy K. Mol. Cell. Biol. 1993; 13: 4793-4805Crossref PubMed Scopus (234) Google Scholar, 5Chuvpilo S. Schomberg C. Gerwig R. Heinfling A. Reeves R. Grummt F. Serfling E. Nucleic Acids Res. 1993; 21: 5694-5704Crossref PubMed Scopus (181) Google Scholar, 8Tara D. Weiss D. Brown M. J. Immunol. 1995; 154: 4592-4602PubMed Google Scholar, 31Li-Weber M. Davydov I. Krafft H. Krammer P. J. Immunol. 1994; 153: 4122-4133PubMed Google Scholar). Unlike the inducible IL-4 promoter-binding factors that have been intensively studied, only a few of these constitutive factors have been well characterized. Here we report that YY-1 (Yin-Yang 1), a constitutive nuclear member of the GLI-Krüppel family of zinc-finger transcription factors, can interact with four binding sites in the human proximal IL-4 promoter. We use site-directed mutagenesis together with cotransfection assays to define the role of YY-1 in regulating the transcriptional activation of the IL-4 promoter. A luciferase-based human IL-4 promoter construct containing 270 bp upstream from the transcription start site and ending at +65 (termed 270luc) was synthesized as described (9Burke T.F. Casolaro V. Georas S.N. Biochem. Biophys. Res. Commun. 2000; 270: 1016-1023Crossref PubMed Scopus (31) Google Scholar). A construct containing 235 bp upstream from the transcription start site was amplified by PCR from 270luc and ligated into the pCR-2.1-TOPO vector (Invitrogen), followed by ligation into the XhoI site of pGL3-Basic (Promega). A construct containing 145 bp upstream from the transcription start site was amplified by PCR from genomic DNA and ligated into the KpnI and SacI sites of pGL3. Mutations were introduced into the Y0 YY-1-binding site within 270luc using site-directed mutagenesis with the QuikChange kit (Stratagene) to generate construct 270lucY0mut. The following mutations specifically disrupted the Y0 site (see Fig. 4): wild-type 270luc,−59TCATTTT−53; to 270lucY0mut,−59TgtaTTT−53 (mutations in lowercase). All products were sequenced to confirm accurate replication. An SV40 promoter-driven YY-1 expression vector was synthesized by ligating theEcoRI insert from pcDNA1-YY-1 (described in Refs. 34Jain J. Burgeon E. Badalian T. Hogan P. Rao A. J. Biol. Chem. 1995; 270: 4138-4145Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar and35Schreiber E. Matthias P. Müller M. Schaffner W. Nucleic Acids Res. 1989; 176419Crossref PubMed Scopus (3903) Google Scholar) into the EcoRI sites of pSG5 (Stratagene). An expression vector encoding a YY-1 mutant with a deletion of amino acids 333–408 within the zinc-finger domain (ZFDmut) was synthesized by restricting pSG5-YY-1 with HindIII and BamHI and religation using the following linkers: HindIII, 5′-AGCTTCCAACAACCAGTGAG-3′; and BamHI, 5′-GATCCTCACTGGTTGTTGGA-3′. The antisense YY-1 vector was a kind gift of Dr. Michael Atchison (University of Pennsylvania) and has been described (32Breslin M.B. Vedeckis W.V. J. Steroid Biochem. Mol. Biol. 1998; 67: 369-381Crossref PubMed Scopus (57) Google Scholar). Expression vectors encoding full-length NFATp (pREP4-NFATp) and NFATc (pSH107-NFATc) were gifts of Dr. Timothy Hoey (Tularik Inc.) and Dr. Gerald Crabtree (Stanford University), respectively. Jurkat T cells (courtesy of Dr. Jack Strohminger, Harvard University) were maintained and transfected using 2.5 × 106 cells, 1 μg of reporter, and 3 μl of Superfect® (QIAGEN Inc.) per μg of plasmid DNA as previously reported (22Chen R. Burke T. Cumberland J. Brummet M. Beck L. Casolaro V. Georas S. J. Immunol. 2000; 164: 825-832Crossref PubMed Scopus (56) Google Scholar). COS-7 cells were obtained from American Type Culture Collection (Manassas, VA). Jurkat cells and a Jurkat subline stably transfected with the SV40 large T antigen (JTAg cells, kindly provided by Dr. Ron Wange, NIA, National Institutes of Health) were transfected using electroporation as follows. Cells were resuspended at a concentration of 8 × 106 cells/300 μl of Opti-MEM (Invitrogen) together with the indicated amounts of reporter plasmid and then electroporated at 300 V and 960 microfarads in cuvettes with a 0.4-cm gap width (Bio-Rad Gene-Pulser II). Transfection efficiencies of standard Jurkat and JTAg cells using electroporation averaged 40 and 85%, respectively, as determined by analyzing green fluorescence of cells cotransfected with a GFP expression vector (data not shown). Eighteen hours after transfection, cells were lysed and analyzed by luminometry using a Monolight 3010C luminometer and luciferase assay kit (Analytical Bioluminescence, Gaithersburg, MD). In some experiments, cells were incubated with the calcium ionophore A23187 (Calbiochem) for 18 h prior to analysis of reporter gene expression as indicated. Two strategies were taken to control for transfection efficiency. First, at least three different reporter plasmid and expression vector preparations were used, and cells of similar passage number were transfected under identical reaction conditions. In cotransfection experiments, cells were also transfected with empty vector to keep the total amount of DNA constant in a given condition. Second, the efficiency of transfection was monitored in some experiments by cotransfecting a second internal control plasmid. In these experiments, cells were cotransfected with pSEAP2-Control (1 μg; CLONTECH), and firefly luciferase activity was normalized to secreted alkaline phosphatase measured by chemiluminescence (Great EscAPe SEAP kit,CLONTECH). pSEAP2 was chosen because, compared with other standard internal control vectors, its expression was affected the least by cell stimulation, and it did not interfere with YY-1 overexpression in pilot cotransfection experiments (data not shown). In experiments with the antisense YY-1 vector, cell growth was analyzed by (i) counting viable cells using trypan blue exclusion and (ii) comparing total protein content in lysates of control and antisense-transfected cells. Highly enriched peripheral blood T cells were obtained by countercurrent elutriation of leukapheresis packs (Johns Hopkins Oncology Center). This procedure yielded ∼70% pure CD3+ cells as assessed by flow cytometry (data not shown). Cells were transfected by electroporation according to the method of Cron et al. (33Cron R.Q. Schubert L.A. Lewis D.B. Hughes C.C. J. Immunol. Methods. 1997; 205: 145-150Crossref PubMed Scopus (35) Google Scholar) as follows. Cells were first incubated for 18 h in the presence of 1 μg/ml phytohemagglutinin (Calbiochem). Subsequently, 10 × 106 cells were resuspended in a volume of 300 μl Opti-MEM with 10 μg of reporter together with the indicated amounts of expression vector or empty vector to keep total DNA constant. Cells were electroporated using a 0.4-cm gap width cuvette and a 1-s duration pulse at 300 V and 960 microfarads (Bio-Rad Gene-Pulser II). These settings were determined in pilot experiments to yield maximal luciferase expression (data not shown). Omission of phytohemagglutinin from the overnight culture significantly reduced transfection efficiency (data not shown). Cells were stimulated with calcium ionophore (1 μmA23187) plus phorbol 12-myristate 13-acetate (20 ng/ml) or Me2SO control for 18 h prior to analysis of reporter gene activity by luminometry as described above. IL-4 protein secretion was measured in supernatants using a sensitive enzyme-linked immunosorbent assay (Ultrasensitive IL-4 kit, detection limit of 0.27 pg/ml, BIOSOURCE) according to the manufacturer's instructions. All measured values fell within the standard curve. Whole cell, cytoplasmic, and nuclear lysates were obtained from Jurkat cells. Nuclear extracts from mouse Th1 (AE7) and Th2 (D10) clones were kindly provided by Dr. Anuradha Ray (Yale University). Cell extracts were separated by 6% SDS-PAGE and then transferred to Trans-Blot transfer medium polyvinylidene difluoride membrane (Bio-Rad). After blocking in phosphate-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 for 1 h, membranes were probed with mouse anti-YY-1 monoclonal antibody H-10 (1:500 dilution; Santa Cruz Biotechnology) for 10 h at 4 °C, followed by incubation for 1 h with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:3000 dilution; Amersham Biosciences, Inc.). After a final washing step, immunoreactive bands were visualized by enhanced chemiluminescence and autoradiography using the ECL Western blotting detection kit (Amersham Biosciences, Inc.) according to the manufacturer's directions. The following 30-bp oligonucleotides and their complements were synthesized: 5′-ATTGCTGAAACCGAGGGAAAATGAGTTTACATTG-3′ (P0, −36 to −69), 5′-TGAGTTTACATTGGAAATTTTCGTTACACCAGATTG-3′ (P1, −57 to −92), 5′-TCTGATTTCACAGGAACATTTTACCTGTTT-3′ (P2, −175 to −146), 5′-AATCAGACCAATAGGAAAATGAAACCTTTTTAA-3′ (P3, −169 to −201), 5′-AGTTTCAGCATAGGAAATTACACCATAATTTGC-3′ (P4, −216 to −248), and 5′-GCAGTCCTCCTGG GGAAA GATAGAGTAATATCA-3′ (P5, −340 to −372). Mutations were introduced into the P0 and P2 consensus YY-1-binding sites (underlined) as indicated in Figs. 2 and 4. A recombinant fragment of murine NFATp (including 298 amino acids of the DNA-binding domain, highly conserved among different NFAT family members) was expressed as a hexahistidine-tagged protein and extracted as described (34Jain J. Burgeon E. Badalian T. Hogan P. Rao A. J. Biol. Chem. 1995; 270: 4138-4145Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Recombinant YY-1 was extracted from bacteria transformed with a histidine-tagged YY-1 expression vector. Nuclear extracts were obtained from 5 × 106 Jurkat cells using the method of Schreiber et al. (35Schreiber E. Matthias P. Müller M. Schaffner W. Nucleic Acids Res. 1989; 176419Crossref PubMed Scopus (3903) Google Scholar). EMSAs were performed using 5 μg of nuclear protein and γ-32P-end-labeled probe with 0.8 μg of poly(dG·dC) (Amersham Biosciences, Inc.) in a final volume of 10 μl. Experiments with recombinant proteins were performed with 1-μl aliquots of the recombinant NFATp DNA-binding domain with or without 1 μl of YY-1, and the final buffer composition in all samples was adjusted to contain 84 mm KCl, 34 mm NaCl, 7% glycerol, 20 mm HEPES (pH 7.5), 1 mmdithiothreitol, and 0.1% Nonidet P-40. Free probes and protein-DNA complexes were resolved by 5% PAGE with 0.5× Tris borate/EDTA. In some experiments, extracts were incubated with 1 μl of the following antisera for 30 min at 4 °C prior to addition of probe: rabbit anti-NFATp polyclonal antibody (Upstate Biotechnology, Inc.), rabbit anti-YY-1 polyclonal antibody C-20 (Santa Cruz Biotechnology), rabbit anti-Stat6 polyclonal antibody S-20 (Santa Cruz Biotechnology), and isotype- and species-matched control IgG (Sigma). The IL-4 promoter contains multiple binding sites for members of the NFAT family of transcription factors, termed the P elements P0–P5 (4Szabo S. Gold J. Murphy T. Murphy K. Mol. Cell. Biol. 1993; 13: 4793-4805Crossref PubMed Scopus (234) Google Scholar, 5Chuvpilo S. Schomberg C. Gerwig R. Heinfling A. Reeves R. Grummt F. Serfling E. Nucleic Acids Res. 1993; 21: 5694-5704Crossref PubMed Scopus (181) Google Scholar, 9Burke T.F. Casolaro V. Georas S.N. Biochem. Biophys. Res. Commun. 2000; 270: 1016-1023Crossref PubMed Scopus (31) Google Scholar) (Fig. 1). During a detailed deletional analysis of the human IL-4 promoter, we identified a factor (termed complex IV) that bound downstream of NFAT to the P2 element (10Georas S. Cumberland J. Burke T. Chen R. Park E. Ono S. Casolaro V. Leukemia (Baltimore). 2000; 14: 629-635Crossref PubMed Scopus (10) Google Scholar). As shown in Fig. 2 (A–C), multiple nuclear factors from Jurkat cells recognized this element in EMSAs. These included a slowly migrating factor with apparent repressor properties (termed Rep-1 (10Georas S. Cumberland J. Burke T. Chen R. Park E. Ono S. Casolaro V. Leukemia (Baltimore). 2000; 14: 629-635Crossref PubMed Scopus (10) Google Scholar)), a Ca2+-induced factor containing NFATp, and the constitutive complexes III–V. We previously reported that complex IV binds in a sequence-specific manner and that its formation is not competed for by a panel of consensus oligonucleotides, including those containing high affinity NFAT and AP-1 sites (10Georas S. Cumberland J. Burke T. Chen R. Park E. Ono S. Casolaro V. Leukemia (Baltimore). 2000; 14: 629-635Crossref PubMed Scopus (10) Google Scholar). Mutational analysis revealed that the formation of complex IV required sequences located just downstream of the NFAT consensus sequence −163GGAACA−158(Fig. 2, A and B). Inspection of this region revealed a sequence (−160ACATTTT−154) highly homologous to a consensus YY-1-binding site, which we termed Y2 (Fig.2B). The most conserved nucleotide in the YY-1 consensus sequence is the second cytosine (5′-CCATNTT-3′), which appears to be required for high affinity DNA binding (36Shrivastava A. Calame K. Nucleic Acids Res. 1994; 22: 5151-5155Crossref PubMed Scopus (278) Google Scholar). When we mutated this nucleotide within the P2 element, we did not detect complex IV binding in EMSA using Jurkat nuclear extracts (data not shown). Using anti-transcription factor antibodies and nuclear extracts from Jurkat T cells in EMSA, we found that complex IV indeed contained immunoreactive YY-1 (Fig. 2C, lanes 3 and6). Fig. 2D also shows that factors previously shown to bind to the P2 region, such as NFATp and Stat6 (23Georas S. Cumberland J. Burke T. Chen R. Schindler U. Casolaro V. Blood. 1998; 92: 4529-4538Crossref PubMed Google Scholar), did not affect complex IV. YY-1 appeared to bind in a competitive manner with complex III, which bound just upstream of the NFAT site (Fig.2A, compare lanes 1 and 7). The identities of the constitutive complexes III and V are currently unknown. Because the IL-4 promoter P elements are homologous even outside of the NFAT core consensus sequence, we speculated that YY-1 would interact with additional sequences within the IL-4 promoter. Fig.3 shows that a YY-1-immunoreactive complex formed on oligonucleotide probes encompassing the P3 and P4 (but not P1 and P5) elements using Jurkat nuclear extracts in EMSA. Importantly, sequence inspection revealed five of six base pair matches for the YY-1 consensus sequence (including the critical second cytosine) within the P3 and P4 (but not P1 and P5) oligonucleotides (Fig. 3). The most proximal IL-4 NFAT site (termed P0) has been shown to bind both constitutive and inducible nuclear factors and to contribute to promoter activity in T cell lines and Th2 cells (29Wenner C.A. Szabo S.J. Murphy K.M. J. Immunol. 1997; 158: 765-773PubMed Google Scholar, 37Hodge M.R. Rooney J.W. Glimcher L.H. J. Immunol. 1995; 154: 6397-6405PubMed Google Scholar). Sequence inspection revealed a potential YY-1-binding site located immediately adjacent to the P0 NFAT consensus sequence (Fig.4). Fig. 4A shows that a YY-1-immunoreactive complex formed on the P0 element using Jurkat nuclear extracts in EMSA. To precisely map the YY-1-binding site in this region, we introduced mutations into the YY-1 consensus sequence and studied the ability of recombinant YY-1 to bind to wild-type and mutant oligonucleotides. As shown in Fig. 4B, mutation of two nucleotides within the YY-1 consensus sequence (including the second cytosine, C−59) drastically impaired the ability of YY-1 to bind to this region (compare lanes 4 and6). As a control, we also found that recombinant YY-1 readily bound to the P2 oligonucleotide. These experiments mapped the proximal IL-4 promoter YY-1-binding site to sequence−59TCATTTT−53, which we termed Y0. The introduced mutations also inhibited the binding of native YY-1 to the Y0 element (Fig. 4C). Note that a constitutive complex that was partially inhibited by the anti-YY-1 antibody (Fig.4A, asterisk) bound equally well to the mutant probe (Fig. 4C). The identity of this complex, which does not represent sequence-specific binding by YY-1, is currently unknown. We did not detect NFATp binding to the P0 element in these experiments (data not shown), in keeping with prior studies showing that the P0 element binds NFAT with lower affinity than other P elements (37Hodge M.R. Rooney J.W. Glimcher L.H. J. Immunol. 1995; 154: 6397-6405PubMed Google Scholar). Our biochemical data showed that YY-1 can interact with four binding sites in the proximal IL-4 promoter. Sequence inspection and computer analysis did not detect additional YY-1-binding sites within ∼1000 bp surrounding the IL-4 transcription start site. To test the functional significance of YY-1 in regulating IL-4 promoter activity, we next studied the effect of cotransfecting an antisense YY-1 expression vector with different promoter constructs into Jurkat T cells. These experiments were prompted by the observations that (i) the IL-4 promoter is constitutively active in transiently transfected Jurkat cells (30Casolaro V. Keane-Myers A.M. Swendeman S.L. Steindler C. Zhong F. Sheffery M. Georas S.N. Ono S.J. J. Biol. Chem. 2000; 275: 36605-36611Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and (ii) YY-1 is a constitutive nuclear protein in these cells (Fig. 2). We used both a full-length promoter construct (270luc) and a minimal construct containing only the Y0 site (145luc; see “Experimental Procedures”) together with an antisense YY-1 vector that has been shown to inhibit glucocorticoid receptor expression by ∼50% in other systems (32Breslin M.B. Vedeckis W.V. J. Steroid Biochem. Mol. Biol. 1998; 67: 369-381Crossref PubMed Scopus (57) Google Scholar). Interestingly, cotransfection of antisense YY-1 inhibited transcription driven by the full-length IL-4 promoter by ∼80% (Fig.5). The activity of the minimal construct 145luc was also reduced by antisense YY-1, although this result did not reach statistical significance (p = 0.06). Transfection of antisense YY-1 did not appear to affect cell growth (see “Experimental Procedures”). These results suggest that YY-1 contributes to constitutive IL-4 promoter activity in Jurkat cells. We next overexpressed YY-1 and reasoned that increasing the cellular concentration of this factor would further enhance IL-4 promoter activity. In these experiments, Jurkat cells were cotransfected by electroporation with a series of IL-4 promoter constructs and a wild-type YY-1 expression vector. Cotransfection of YY-1 resulted in a concentration-dependent enhancement of transcription driven by the full-length IL-4 promoter (data not shown) as well as of transcription driven by 145luc (Fig.6A). Western blot analysis confirmed that total cellular YY-1 content was also increased in a concentration-dependent manner in these cells (Fig.6A). Overexpressed YY-1 also enhanced IL-4 promoter activity in transiently transfected JTAg cells, a subline of Jurkat cells stably transfected with the SV40 T antigen (Fig. 6B; see “Experimental Procedures”). In both JTAg and standard Jurkat cells, the expression of YY-1 was confined largely to the cell nucleus, indicating that the subcellular localization of overexpressed YY-1 was faithfully regulated (Fig. 6B and data not shown). The detection of some YY-1 within the cytoplasmic fraction likely reflects ongoing expression of YY-1 from the transfected construct. In parallel experiments, we examined the effects of overexpressing YY-1 on IL-4 promoter activity in non-lymphoid COS-7 cells. IL-4 145luc was constitutively active in these cells, as was reported for the human IL-4 promoter in non-lymphoid HeLa cells (38Li-Weber M. Eder A. Krafft-Czepa H. Krammer P. J. Immunol. 1992; 148: 1913-1918PubMed Google Scholar). Interestingly, cotransfection of YY-1 sufficient to increase total cellular YY-1 expression as determined by Western blotting down-regulated the IL-4 promoter in COS-7 cells (∼70% inhibition; n = 4) (data not shown). Thus, IL-4 promoter enhancement by YY-1 appears to be cell type-specific. The observation that 145luc, which contains only the Y0 element, can be transactivated by overexpressed YY-1 suggests that the Y0 element is critical for YY-1-dependent IL-4 promoter activity. To test this hypothesis, we mutated Y0 in the context of the full-length promoter to generate the construct 270lucY0mut and studied promoter activity under a variety of experimental conditions." @default.
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