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• W2004720912 abstract "Antigen receptor engagement on T lymphocytes activates transcription factors important for stimulating cytokine gene expression. This is critical for clonal expansion of antigen-specific T cells and propagation of immune responses. Additionally, under some conditions antigen receptor stimulation initiates apoptosis of T lymphocytes through the induced expression of CD95 ligand and its receptor. Here we demonstrate that the transcription factor, NFAT, which is critical for the inducible expression of many cytokine genes, also plays a critical role in the regulation of T cell receptor-mediated CD95 ligand expression. Two sites within the CD95 ligand promoter, identified through DNase I footprinting, bind NFAT proteins from nuclear extracts of activated T cells. Although both sites appear important for optimal expression of CD95 ligand in activated T cells, mutational analysis suggests that the distal NFAT site plays a more significant role. Furthermore, these sites do not appear to be required for constitutive CD95 ligand expression in Sertoli cells. Antigen receptor engagement on T lymphocytes activates transcription factors important for stimulating cytokine gene expression. This is critical for clonal expansion of antigen-specific T cells and propagation of immune responses. Additionally, under some conditions antigen receptor stimulation initiates apoptosis of T lymphocytes through the induced expression of CD95 ligand and its receptor. Here we demonstrate that the transcription factor, NFAT, which is critical for the inducible expression of many cytokine genes, also plays a critical role in the regulation of T cell receptor-mediated CD95 ligand expression. Two sites within the CD95 ligand promoter, identified through DNase I footprinting, bind NFAT proteins from nuclear extracts of activated T cells. Although both sites appear important for optimal expression of CD95 ligand in activated T cells, mutational analysis suggests that the distal NFAT site plays a more significant role. Furthermore, these sites do not appear to be required for constitutive CD95 ligand expression in Sertoli cells. Adaptive immune responses require the activation of T lymphocytes through antigen-specific T cell receptor (TCR) 1The abbreviations used are: TCR, T cell receptor; mAb, monoclonal antibody; IL-2, interleukin 2; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PMA, phorbol myristate acetate; DTT, dithiothreitol; CsA, cyclosporin A; EMSA, electrophoretic mobility shift assays; bp, base pair(s). stimulation. Signaling events initiated by TCR ligation lead to the activation of transcription factors that regulate expression of cytokine genes, such as IL-2. This is important for the clonal expansion of antigen-specific T cells and propagation of immune responses (1Fraser J.D. Straus D. Weiss A. Immunol. Today. 1993; 14: 357-362Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 2Musci M.A. Latinis K.M. Koretzky G.A. Clin. Immunol. Immunopathol. 1997; 83: 205-222Crossref PubMed Scopus (27) Google Scholar). However, once the antigenic stimulus has been cleared, the expanded population of cells must be eliminated to prevent accumulation of excessive lymphocytes (2Musci M.A. Latinis K.M. Koretzky G.A. Clin. Immunol. Immunopathol. 1997; 83: 205-222Crossref PubMed Scopus (27) Google Scholar,3Osborne B.A. Curr. Opin. Immunol. 1996; 8: 245-254Crossref PubMed Scopus (166) Google Scholar). Recently, it has been proposed that one mechanism by which this occurs is through the induced expression of CD95 ligand (4Alderson M.R. Tough T.W. Davis-Smith T. Braddy S. Falk B. Schooley K.A. Goodwin R.G. Smith C.A. Ramsdell F. Lynch D.H. J. Exp. Med. 1995; 181: 71-77Crossref PubMed Scopus (868) Google Scholar, 5Brunner T. Mogil R.J. LaFace D. Yoo N.J. Mahboubi A. Echeverri F. Martin S.J. Force W.R. Lynch D.H. Ware C.F. Green D.R. Nature. 1995; 373: 441-444Crossref PubMed Scopus (1272) Google Scholar, 6Dhein J. Walczak H. Baumler C. Debatin K.M. Krammer P.H. Nature. 1995; 373: 438-441Crossref PubMed Scopus (1607) Google Scholar, 7Ju S.T. Panka D.J. Cui H. Ettinger R. el-Khatib M. Sherr D.H. Stanger B.Z. Marshak-Rothstein A. Nature. 1995; 373: 444-448Crossref PubMed Scopus (1453) Google Scholar). Once expressed, CD95 ligand engages its receptor, CD95, also expressed on the population of activated lymphocytes (8van Parijs L. Abbas A.K. Curr. Opin. Immunol. 1996; 8: 355-361Crossref PubMed Scopus (215) Google Scholar). In the absence of costimulatory signals that can delay apoptosis (9Boise L.H. Noel P.J. Thompson C.B. Curr. Opin. Immunol. 1995; 7: 620-625Crossref PubMed Scopus (134) Google Scholar, 10Boise L.H. Minn A.J. Noel P.J. June C.H. Accavitti M.A. Lindsten T. Thompson C.B. Immunity. 1995; 3: 87-98Abstract Full Text PDF PubMed Scopus (1078) Google Scholar, 11Radvanyi L.G. Shi Y. Homayoun V. Sharma A. Dhala R. Mills G.B. Miller R.G. J. Immunol. 1996; 156: 1788-1798PubMed Google Scholar, 12Noel P.J. Boise L.H. Green J.M. Thompson C.B. J. Immunol. 1996; 157: 636-642PubMed Google Scholar), CD95 ligation rapidly initiates the programmed cell death machinery thus efficiently eliminating excessive activated lymphocytes (8van Parijs L. Abbas A.K. Curr. Opin. Immunol. 1996; 8: 355-361Crossref PubMed Scopus (215) Google Scholar). Additionally, autoreactive T cells that are inappropriately activated in the periphery are believed to undergo apoptosis through a similar process (13Abbas A.K. Cell. 1996; 84: 655-657Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The significance of CD95 ligand expression in the process of activation-induced cell death has been highlighted by recent studies that demonstrate that blocking CD95/CD95 ligand interactions prevents apoptosis of TCR-stimulated lymphocytes (5Brunner T. Mogil R.J. LaFace D. Yoo N.J. Mahboubi A. Echeverri F. Martin S.J. Force W.R. Lynch D.H. Ware C.F. Green D.R. Nature. 1995; 373: 441-444Crossref PubMed Scopus (1272) Google Scholar, 7Ju S.T. Panka D.J. Cui H. Ettinger R. el-Khatib M. Sherr D.H. Stanger B.Z. Marshak-Rothstein A. Nature. 1995; 373: 444-448Crossref PubMed Scopus (1453) Google Scholar). Interestingly, in addition to its inducible expression on activated lymphocytes, CD95 ligand is constitutively expressed on epithelial cells within the eye and Sertoli cells within the testes (14Bellgrau D. Gold D. Selawry H. Moore J. Franzusoff A. Duke R.C. Nature. 1995; 377: 630-632Crossref PubMed Scopus (1101) Google Scholar, 15Griffith T.S. Brunner T. Fletcher S.M. Green D.R. Ferguson T.A. Science. 1995; 270: 1189-1192Crossref PubMed Scopus (1875) Google Scholar, 16Griffith T.S. Yu X. Herndon J.M. Green D.R. Ferguson T.A. Immunity. 1996; 5: 7-16Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Constitutive CD95 ligand expression participates in maintenance of the “immune privileged” status of these tissues by inducing apoptosis in infiltrating, CD95-bearing, activated lymphocytes (17Griffith T.S. Ferguson T.A. Immunol. Today. 1997; 18: 240-244Abstract Full Text PDF PubMed Scopus (251) Google Scholar). Despite improved understanding of the important physiological roles for CD95 ligand in immune privileged sites and in controlling T cell homeostasis, little is yet known about the regulation of CD95 ligand expression in these various cell types. In contrast, much is known about the signaling pathways that couple TCR ligation to expression of cytokine genes. Engagement of the TCR leads to rapid activation of protein tyrosine kinases of the Src and Syk families (18Chan A.C. Shaw A.S. Curr. Opin. Immunol. 1996; 8: 394-401Crossref PubMed Scopus (170) Google Scholar). These protein tyrosine kinases then couple to the activation of the Ras and phospholipase Cγ1 signaling intermediates (19Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1955) Google Scholar). Ras activation drives signals that ultimately lead to induction of members of the AP-1 family of transcription factors, important for regulation of the IL-2 gene promoter (20Rothenberg E.V. Ward S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9358-9365Crossref PubMed Scopus (169) Google Scholar, 21Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Crossref PubMed Scopus (595) Google Scholar). Stimulation of phospholipase Cγ1 leads to the calcium-dependent activation of the serine phosphatase, calcineurin (22Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1476) Google Scholar). Activated calcineurin then functions to dephosphorylate nuclear factor of activated T cell (NFAT) family members. Dephosphorylated NFAT proteins then enter the nucleus where they also serve an essential role in regulating the expression of many cytokine genes, including IL-2 (20Rothenberg E.V. Ward S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9358-9365Crossref PubMed Scopus (169) Google Scholar,23Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar). The immunosuppressant cyclosporin A (CsA) inhibits NFAT-dependent transcriptional events by binding calcineurin and blocking its enzymatic activity, thus preventing the redistribution of NFAT to the nucleus (24Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1968) Google Scholar). Previous studies demonstrated that treatment of lymphocytes with CsA also inhibits TCR-mediated CD95 ligand expression (6Dhein J. Walczak H. Baumler C. Debatin K.M. Krammer P.H. Nature. 1995; 373: 438-441Crossref PubMed Scopus (1607) Google Scholar, 25Anel A. Buferne M. Boyer C. Schmitt-Verhulst A.M. Golstein P. Eur. J. Immunol. 1994; 24: 2469-2476Crossref PubMed Scopus (214) Google Scholar, 26Brunner T. Nam J.Y. LaFace D. Ware C.F. Green D.R. Int. Immunol. 1996; 8: 1017-1026Crossref PubMed Scopus (103) Google Scholar). Additionally, lymphocytes from mice with targeted gene disruption of the transcription factor, NFATp, display a pronounced defect in activation-induced CD95 ligand expression (27Hodge M.R. Ranger A.M. de la Brouse F.C. Hoey T. Grusby M.J. Glimcher L.H. Immunity. 1996; 4: 397-405Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Furthermore, recent work from our laboratory (28Latinis K.M. Carr L.L. Peterson E.J. Norian L.A. Eliason S.L. Koretzky G.A. J. Immunol. 1997; 158: 4602-4611PubMed Google Scholar) utilizing a reporter construct driven by elements of the CD95 ligand promoter suggests that NFAT may play an important role in regulating CD95 ligand expression in activated T cells. In this study, we explore further the hypothesis that NFAT regulates CD95 ligand expression in activated lymphocytes. Using DNase I footprint analysis, we define two potential NFAT binding sites within the CD95 ligand promoter region. Both sites bind NFAT proteins independently, in an inducible and specific fashion. Yet, mutational analysis demonstrates that the distal NFAT site is more important than the proximal site for regulation of the CD95 ligand promoter in activated T cells. In contrast to the findings in T cells, experiments examining constitutively expressed CD95 ligand in Sertoli cells demonstrate that neither NFAT site is required for constitutive promoter activity in these cells. The following reagents were used in this study: sodium d-luciferin (Sigma), CsA (Sigma), phorbol myristate acetate (PMA, Sigma: used for stimulations at 50 ng/ml), ionomycin (Sigma: used for stimulations at 1 μm). The following antibodies were used in this study: anti-Jurkat clonotypic TCR β chain mAb, C305 (29Weiss A. Stobo J.D. J. Exp. Med. 1984; 160: 1284-1299Crossref PubMed Scopus (385) Google Scholar); MOPC IgG2 (Organon Teknika Corp., West Chester, PA); anti-NFATp (mAb G1-D10) and anti-NFATc (mAb 7A6) were gifts of G. Crabtree. C305 ascites was immobilized on plastic culture dishes by incubating at 37 °C with 10 μg/ml C305 in phosphate-buffered saline (PBS) for 2 h. PDT102, a pGEX3X (Pharmacia Biotech Inc.) construct containing the NFATc Rel similarity domain (amino acids 415–591), was a gift of G. Crabtree. The following reporter constructs were used in this study: CMV-βgal; CD95L-486 (28Latinis K.M. Carr L.L. Peterson E.J. Norian L.A. Eliason S.L. Koretzky G.A. J. Immunol. 1997; 158: 4602-4611PubMed Google Scholar); Luc-link (28Latinis K.M. Carr L.L. Peterson E.J. Norian L.A. Eliason S.L. Koretzky G.A. J. Immunol. 1997; 158: 4602-4611PubMed Google Scholar), Distal Mut-486 (see below), Prox. Mut-486 (see below), and Double Mut-486 (see below). The Jurkat human leukemic T cell line was maintained in RPMI supplemented with 10% fetal calf serum, penicillin (1000 units/ml), streptomycin (1000 units/ml), and glutamine (20 mm). The TM4 Sertoli cell line (American Type Culture Collection, Rockville, MD) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (1000 units/ml), streptomycin (1000 units/ml), and glutamine (20 mm). For transient transfections, 15 × 106 Jurkat or 20 × 106 TM4 Sertoli cells were subjected to electroporation in 400 μl of cytomix intracellular buffer (120 mm KCl, 0.15 mm CaCl2, 10 mmK2HPO4/KH2PO4 (pH 7.6), 25 mm HEPES (pH 7.6), 2 mm EGTA (pH 7.6), 5 mm MgCl2: pH adjusted with KOH and freshly added 2 mm ATP (pH 7.6) and 5 mm glutathione) with a Gene Pulser (Bio-Rad) at 250 V/960 microfarads (Jurkats) or 320 V/960 microfarads (TM4 cells). Transfection efficiencies were normalized by co-transfecting 5 μg of a CMV-βgal reporter construct followed by quantification of β-galactosidase expression using the Galacto-Light assay kit (Tropix, Bedford, MA). The distal, proximal, and double NFAT binding mutants were created using overlap extension polymerase chain reaction with the forward (F) and reverse (R) end primers (F, 5′-GAACAAGCTTAATGTATAAAAAAGCATGCAATTATAATTC-3′; R, 5′-ACATAAGCTTGGCAGCTGGTGAGTCAGGCCA-3′) and the following primers to incorporate the NFAT binding site mutations: CD95 ligand Distal Mut (F, 5′-GTGGGAATCAACTTCCAGG-3′; R, 5′-CCTGGAAGTTGATTCCCAC-3′), CD95 ligand Prox Mut (F, 5′-TAGCTATTAGATCTCTATAA-3′; R, 5′-TTATAGAGATCTAATAGCTA-3′). Wild type CD95L-486 served as a template for creation of distal and proximal NFAT mutants. The distal mutant served as a template for creation of the double mutant. All polymerase chain reaction-derived constructs were confirmed by fluorescent automated sequencing (University of Iowa DNA facility, Iowa City, IA). The minimal IL-2 promoter luciferase construct was created by digesting the triplicated IL-2 distal NFAT reporter with Xho-1 to drop out the IL-2 NFAT sequences followed by religation of the Xho-1 ends. The triplicated distal and proximal CD95 ligand NFAT constructs were created by annealing the following oligonucleotides containing overhanging Xho-1-compatible sites followed by cloning into the Xho-1 site of the IL-2 minimal promoter construct: distal NFAT triplication (F, 5′-TCGAGGTGGGCGGAAACTTCCAGTGGGCGGAAACTTCCAGTGGGCGGAAACTTCCAC-3′) and (R, 5′-TCGAGTGGAAGTTTCCGCCCACTGGAAGTTTCCGCCCACTGGAAGTTTCCGCCCACC-3′); proximal NFAT triplication (F, 5′-TCGAGTTAGCTATGGAAACTCTTTAGCTATGGAAACTCTTTAGCTATGGAAACTCTC-3′) and (R, 5′-TCGAGAGAGTTTCCATAGCTAAAGAGTTTCCATAGCTAAAGAGTTTCCATAGCTAAC-3′). Transformants of the PDT102 vector were grown to OD 0.5 at 600 nm visible light and then stimulated with 0.005m isopropyl-1-thio-β-d-galactopyranoside for 1 h at room temperature with shaking. Bacteria were pelleted, resuspended in 3 ml of cold PBS, and sonicated to disrupt cell walls. After addition of 330 μl of 10% Triton (in PBS), cellular debris was pelleted. The supernatant was tumbled with glutathioneS-transferase-conjugated agarose beads for 10 min at room temperature and washed three times in 1% Triton followed by PBS. Glutathione S-transferase-NFAT protein was then eluted by tumbling beads with 1 ml of elution buffer (20 mmglutathione in 100 mm Tris (pH 8.0), 120 mmNaCl) for 10 min at 4 °C. The eluate was then dialyzed for 2 h in DB buffer (see DNase footprinting) without bovine serum albumin but supplemented with 0.3% Nonidet P-40 and 0.5 mm PMSF. Protein was quantified by the Bio-Rad protein assay. 5 × 107Jurkat cells were left unstimulated, stimulated with immobilized C305 mAb, 50 ng/ml PMA plus 1 μm ionomycin, or with PMA plus ionomycin in the presence of 200 ng/ml CsA and then lysed in 500 μl of TLB buffer (40 mm KCl, 10 mm HEPES (pH 7.0), 3 mm MgCl2, 5% glycerol, 0.2% Nonidet P-40, 2 μg/ml leupeptin, 8 μg/ml aprotinin, 0.5 mm PMSF, and 1.0 mm DTT). Samples were centrifuged immediately at 4 °C for 2 min at 14,000 rpm. The supernatant was removed and nuclei lysed in 300 μl of NB buffer (0.42 m KCl, 20 mm HEPES (pH 7.9), 1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol, 2 μg/ml leupeptin, 8 μg/ml aprotinin, 0.5 mm PMSF, and 0.5 mm DTT) by tumbling for 30 min at 4 °C. Lysates were then dialyzed twice for 2 h in dialysis buffer (0.1 m KCl, 20 mmHEPES (pH 7.9), 0.2 mm EDTA, 20 glycerol, 0.5 mm PMSF, and 0.5 mm DTT). Protein concentrations were determined by the Bio-Rad protein assay. CD95 ligand constructs were subcloned into the HindIII site of pBluescript (Stratagene, La Jolla, CA) to allow digestion with ClaI andEcoRV to generate a 5′ overhang at the −1 end of the promoter and a blunt site at the −486 end. 25 μg of DNA was digested and gel-purified followed by incubation with 50 μCi of [α-32P]dCTP and [α-32P]dGTP (Amersham Corp.) and 5 units of Klenow enzyme for 25 min at room temperature. Probes were then purified with Stratagene push columns. DNase footprint analysis was performed by incubating 20,000 cpm of probe with indicated amounts of recombinant protein and 1.0 μg of poly(dI-dC) (Pharmacia) in 20 μl of DB buffer (10 mm Tris (pH 8.0), 15 mm HEPES (pH 7.9), 50 mm NaCl, 5 mmMgCl2, 1 mm DTT, 1 mg/ml bovine serum albumin, 5% glycerol) for 30 min on ice. Reactions were then digested with 0.75 units of DNase I diluted in DD buffer (50 mmCaCl2, 20 mm HEPES (pH 7.9)) for 5 min. Digestion was stopped by addition of 200 μl of DNase stop solution (2.5 m NH4 acetate, 25 mg/ml sheared salmon sperm DNA) and 500 μl of 100% EtOH. DNA was precipitated in a dry ice/EtOH bath for 15 min followed by centrifugation for 10 min at 4 °C. Pellets were washed in 70% EtOH, resuspended in 5 μl of formamide loading buffer, and heated at 90 °C for 5 min. Reactions were resolved on a 5% sequencing gel. Electrophoretic mobility shift assays were performed with 4 μg of nuclear extract protein or 5 ng of recombinant protein in 50 mm KCl, 10 mm Tris (pH 7.5), 10 mm HEPES, 1.25 mm DTT, 1.1 mm EDTA, and 15% (v/v) glycerol in a volume of 20 μl. The binding reactions were incubated for 30 min at room temperature with 20,000 cpm (0.1–0.5 ng) double-stranded oligonucleotides end-labeled with [γ-32P]ATP (Amersham Corp.) using T4 polynucleotide kinase. A 1000-fold excess of unlabeled specific and nonspecific oligonucleotides were used as competitors where indicated. Supershifts were performed by addition of 1 μl of both α-NFATp and α-NFATc antibodies in the binding reactions or 1 μl MOPC as a negative control. In addition to the CD95 ligand promoter probes described in Fig. 1 B, the following double-stranded oligonucleotide probes were used in these experiments: nonspecific, 5′-TGTCGAATGCAAATCACTAGAA-3′; IL-2 dist WT, 5′-GGTCAGAAAGGAGGAAAACTGTTTCATA-3′. Transfected cells were treated as indicated, followed by lysis in 100 μl of harvest buffer (100 mm KPO4 (pH 7.8), 1.0 mm DTT, 1% Triton X-100). Lysates were then mixed with 100 μl of assay buffer (200 mm KPO4 (pH 7.8), 10 mm ATP, 20 mm MgCl2) followed by addition of 100 μl of 1.0 mm luciferin. Luciferase activity, expressed in arbitrary light units, was determined in triplicate for each experimental condition using a luminometer (Monolight 2010; Analytical Luminescence Laboratory, San Diego, CA). We have shown previously that a CD95 ligand reporter comprised of 486 base pairs of DNA immediately 5′ of the translational start site of the human CD95 ligand gene contains critical promoter elements for TCR-induced transcriptional activation in the Jurkat T cell line (28Latinis K.M. Carr L.L. Peterson E.J. Norian L.A. Eliason S.L. Koretzky G.A. J. Immunol. 1997; 158: 4602-4611PubMed Google Scholar). Additionally, this reporter reflects constitutive expression of CD95 ligand in the TM4 Sertoli cell line. This previous study also suggests that NFAT may play a role in the induced but not constitutive expression of CD95 ligand. To explore the role of NFAT in CD95 ligand expression further, we first attempted to map NFAT binding sites within the CD95 ligand promoter. As shown in Fig. 1 A (lanes 3 and 4), DNase I footprint analysis reveals two regions within the 486-base pair probe that are protected from enzymatic digestion through interactions with recombinant NFAT protein. We designated these regions as putative NFAT distal and NFAT proximal sites based on their location relative to the predicted TATA box.Lane 1 demonstrates that in the absence of recombinant protein both NFAT sites are DNase I-sensitive. The two sites appear to bind recombinant protein with grossly equivalent affinities as each site is partially protected from DNase I cleavage with 50 ng of protein and protected more with 500 ng. Additionally, NFAT appears to bind both DNA strands, as labeling either strand individually provides NFAT-mediated DNase I-protected sites at similar regions of the promoter (data not shown). Each protected site corresponds to approximately 20 base pairs within the CD95 ligand promoter. Sequence analysis reveals that both regions contain an identical GGAAA sequence that differs sufficiently from previously defined NFAT binding sites to fail recognition by a GCG computer search using the data bases described in Refs. 30Wingender E. J. Biotech. 1994; 35: 273-280Crossref PubMed Scopus (59) Google Scholar and 31Ghosh D. Nucleic Acids Res. 1993; 21: 3117-3118Crossref PubMed Scopus (119) Google Scholar. Many of the previously described NFAT sites within cytokine promoters bind NFAT family members cooperatively with other transcription factors (23Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar). Interestingly, there are no consensus AP-1, ATF-2, Oct-1, or other recognizable transcription factor binding sites surrounding the CD95 ligand promoter NFAT elements. This suggests that the putative NFAT sites we have identified in the CD95 ligand promoter might bind NFAT proteins in the absence of these other transcription factors. In support of this, DNase footprint analysis using recombinant AP-1 protein failed to demonstrate AP-1 binding either independently or in cooperation with recombinant NFAT protein (data not shown). It is possible, of course, that other unidentified transcription factors may interact with NFAT proteins at these sites. To address further the specificity of NFAT binding to the two sites, each was mutated independently in the context of the 486-base pair promoter (Fig. 1 B). Both mutant probes were then assessed for their ability to bind recombinant NFAT protein in the DNase I cleavage assay. As shown in lanes 7 and 8 of Fig.1 A (representing an experiment performed with duplicate samples), mutation of the distal NFAT site disrupts the ability of NFAT protein to protect this site from DNase I cleavage. However, the non-mutated proximal site is still protected from DNase I cleavage in the presence of NFAT protein. Similarly, as shown in lanes 11 and 12, mutation of the proximal NFAT site prevents NFAT protein-mediated protection from DNase I cleavage, whereas the intact distal site still binds protein and is efficiently protected from digestion. Note that DNase I enzymatic activity is primarily targeted to purine residues, and hence both mutations alter the DNase I cleavage patterns slightly. To confirm the specificity of NFAT protein binding to these two sites we designed 19-mer double-stranded oligonucleotide probes corresponding to either the distal or proximal NFAT binding sites (Fig.1 B) for use in electrophoretic mobility shift assays (EMSA). Each radiolabeled probe was incubated with recombinant NFAT protein in the absence or presence of specific or nonspecific DNA competitors, and complex formation was resolved by polyacrylamide gel electrophoresis. As shown in Fig. 1 C, the wild type probe containing the distal NFAT site forms a complex with NFAT protein (lane 1) which is competed with an excess of unlabeled specific probe (lane 2) but not with an excess of a nonspecific probe (lane 3). Furthermore, a probe with a mutation in the predicted distal NFAT binding site fails completely to bind protein (lanes 4-6). Similarly, a probe containing the wild type proximal NFAT site sequence forms a complex with NFAT protein (lane 7) which is competed with excess unlabeled specific probe (lane 8) but not excess unlabeled nonspecific probe (lane 9). A mutation in the predicted proximal NFAT binding site again abolishes all protein binding (lanes 10–12). As a positive control, a probe containing a previously defined IL-2 NFAT site binds NFAT protein (lane 13) and is competed with excess unlabeled specific probe (lane 14) but not excess unlabeled nonspecific probe (lane 15). Overall these data indicate that there are at least two NFAT binding elements in the 486-bp CD95 ligand promoter region which can bind NFAT protein in a sequence-specific fashion. Next, to address whether NFAT proteins from activated lymphocytes can bind the putative CD95 ligand NFAT sites, distal and proximal NFAT site probes were used in EMSAs with nuclear extracts from Jurkat T cells. Nuclear extracts were prepared from cells left unstimulated or activated with immobilized anti-TCR mAb or PMA plus ionomycin (agents that bypass TCR-mediated protein tyrosine kinase events to initiate signals leading to NFAT activation). In addition, nuclear extracts were prepared from cells stimulated with PMA plus ionomycin in the presence of CsA, blocking signaling events leading to NFAT nuclear translocation. The EMSAs reveal similar results using either the distal (Fig.2, A and B) or proximal (Fig. 2, C and D) NFAT probes. Neither probe forms a specific complex with nuclear proteins from unstimulated extracts (lane 1, Fig. 2, A and C). However, activation via the TCR or with PMA plus ionomycin induces complex formation with both the distal and proximal probes (lanes 2 and 3, Fig. 2, A and C). Treatment with CsA inhibits complex formation with both distal and proximal probes (lane 4, Fig. 2, A andC). To address the specificity of binding in these experiments both probes were mixed with nuclear extracts from PMA and ionomycin-activated Jurkat cells in the presence of various DNA competitors. An excess of unlabeled nonspecific DNA probe does not inhibit complex formation (lane 5, Fig. 2, A and C andlane 11, Fig. 2, B and D) nor do probes that incorporate mutations in the NFAT binding sites (lane 7, Fig. 2, A and C). Yet excess unlabeled probes specific for either the distal or the proximal NFAT sites compete complex formation with the labeled distal or proximal NFAT probes, respectively (lane 6, Fig. 2, A andC and lane 12, Fig. 2, B andD). To begin to address whether the composition of the complexes associated with the distal and proximal NFAT sites are similar, a cross competition experiment was performed. Excess unlabeled proximal probe competes complex formation with the labeled distal probe (lane 8, Fig. 2 A) and unlabeled distal probe competes complex formation with the labeled proximal probe (lane 8, Fig.2 C). This suggests that the proteins from activated nuclear extracts that form complexes with either probe are likely to have similar binding characteristics. To assess further whether the putative NFAT sites bind to NFAT proteins from activated Jurkat cell nuclear extracts, supershift experiments were performed with NFAT-specific antibodies. Antibodies against NFATp and NFATc (which constitute the T cell-specific isoforms of NFAT (32McCaffrey P.G. Luo C. Kerppola T.K. Jain J. Badalian T.M. Ho A.M. Burgeon E. Lane W.S. Lambert J.N. Curran T. Verdine G.L. Rao A. Hogan P.G. Science. 1993; 262: 750-754Crossref PubMed Scopus (379) Google Scholar,33Ho S. Timmerman L. Northrop J. Crabtree G.R. Adv. Exp. Med. Biol. 1994; 365: 167-173Crossref PubMed Scopus (10) Google Scholar)) were incubated with nuclear extracts from PMA and ionomycin-activated Jurkat cells in the presence of labeled distal and proximal NFAT probes. In both instances the specific complexes are supershifted (lane 9, Fig. 2, A andC), indicating that each probe binds NFAT proteins from activated nuclear extracts. A nonspecific isotype control antibody does not induce a supershift (lane 10, Fig. 2, A andC). Interestingly, in contrast to what has been shown for the IL-2 NFAT sites, antibodies specific for AP-1 transcription factor components fail to supershift complexes formed with distal and proximal CD95 ligand NFAT probes, whereas supershift formation is detected with a control probe containing an AP-1 site (data not shown). Finally, we examined whether the specificities of NFAT complex formation with the CD95 ligand NFAT sites are similar to that of an NFAT site from the IL-2 promoter. Complex formation with either labeled distal or proximal CD95 ligand NFAT probe is competed efficiently with a probe containing a canonical NFAT binding site derived from the IL-2 gene promoter (lane 13, Fig. 2, B andD). This suggests that NFAT protein binding specificities for each site are similar. Collectively, these results indicate that both the distal and proximal CD95 ligand promoter NFAT sites are capable of binding nuclear NFAT proteins from T cells in an inducible and specific fashion. Having determined that both the distal and proximal CD95 ligand promoter NFAT sites are capable of binding NFAT proteins from activated lymphocytes, experiments were designed to test whether these sites are capa" @default.
• W2004720912 created "2016-06-24" @default.
• W2004720912 creator A5004594443 @default.
• W2004720912 creator A5039755136 @default.
• W2004720912 creator A5072888364 @default.
• W2004720912 creator A5076944041 @default.
• W2004720912 date "1997-12-01" @default.
• W2004720912 modified "2023-10-16" @default.
• W2004720912 title "Two NFAT Transcription Factor Binding Sites Participate in the Regulation of CD95 (Fas) Ligand Expression in Activated Human T Cells" @default.
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