Interaction of NF-κB and NFAT with the Interferon-γ Promoter

Abstract

Interferon-γ (IFN-γ) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-γ gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-γ gene that binds the NF-κB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3′-half of the NF-κB consensus site. Sequence analysis of the IFN-γ promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-κB site (IFN-γ κB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-κB and NFAT family members in regulating IFN-γ transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-κB subunits bind specifically to the newly identified IFN-γ κB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-γ promoter through the cyclosporin-sensitive C3-3P site, whereas NF-κB proteins functionally interact with the C3-related sites. In addition, when located downstream to the β-galactosidase gene driven by the IFN-γ promoter, the intronic C3 site worked in concert with both the IFN-γ κB and the C3-3P site to enhance gene transcription.These results demonstrate that the coordinate activities of NFAT and NF-κB proteins are involved in the molecular mechanisms controlling IFN-γ gene transcription. Interferon-γ (IFN-γ) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-γ gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-γ gene that binds the NF-κB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3′-half of the NF-κB consensus site. Sequence analysis of the IFN-γ promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-κB site (IFN-γ κB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-κB and NFAT family members in regulating IFN-γ transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-κB subunits bind specifically to the newly identified IFN-γ κB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-γ promoter through the cyclosporin-sensitive C3-3P site, whereas NF-κB proteins functionally interact with the C3-related sites. In addition, when located downstream to the β-galactosidase gene driven by the IFN-γ promoter, the intronic C3 site worked in concert with both the IFN-γ κB and the C3-3P site to enhance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-κB proteins are involved in the molecular mechanisms controlling IFN-γ gene transcription. Interferon-γ (IFN-γ) 1The abbreviations used are: IFN-γ, interferon-γ; NF-κB, nuclear factor-κB; NFAT, nuclear factor of activated T cells; EMSA, electrophoretic mobility shift assay; PMA, phorbol 12-myristate 13-acetate; CsA, cyclosporin A, CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; IL, interleukin; PHA, phytohemagglutinin; RSV, Rous sarcoma virus; CMV, cytomegalovirus; TK, thymidine kinase; WT, wild type. 1The abbreviations used are: IFN-γ, interferon-γ; NF-κB, nuclear factor-κB; NFAT, nuclear factor of activated T cells; EMSA, electrophoretic mobility shift assay; PMA, phorbol 12-myristate 13-acetate; CsA, cyclosporin A, CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; IL, interleukin; PHA, phytohemagglutinin; RSV, Rous sarcoma virus; CMV, cytomegalovirus; TK, thymidine kinase; WT, wild type. is an important immunoregulatory protein responsible for several immunological effects (1Young H.A. Hardy K.J. Pharmacol. 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Immunol. 1997; 27: 1098-1107Crossref PubMed Scopus (28) Google Scholar). Interestingly, the IFN-γ gene belongs to a group of lymphokines whose expression is inhibited by the immunosuppressant cyclosporin A (1Young H.A. Hardy K.J. Pharmacol. Ther. 1990; 45: 137-151Crossref PubMed Scopus (104) Google Scholar, 19Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar), and in activated T cells, its transcription parallels that of other lymphokine genes (e.g. IL-2) whose promoter activity is enhanced by both NF-κB and NFAT proteins (19Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar). This laboratory identified a c-Rel binding site (C3) A. M. Ghosh P. Young H.A. S. A. PubMed Scopus Google Scholar), located in the of the IFN-γ that have a role in the of IFN-γ transcription. This site, which in a previously IFN-γ enhancer region (9Ciccarone V.C. Chrivia J. Hardy K.J. Young H.A. J. Immunol. 1990; 144: 725-730PubMed Google Scholar), a homology with the 3′-half of NF-κB consensus sequence and the NFAT binding sites. In this whereas NF-κB is a factor controlling the transcription of a of T. Annu. Rev. Immunol. 1994; PubMed Scopus Google Scholar, 1991; Google Scholar, D. PubMed Scopus Google Scholar, S. P. D. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar, M. V. J. P. A. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar), its role in the IFN-γ gene expression been A. M. Ghosh P. Young H.A. S. A. PubMed Scopus Google the of of NF-κB binding within its with NF-κB, a role for the cyclosporin A-sensitive NFAT proteins as of IFN-γ gene transcription been (19Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar, J.P. J.J. PubMed Scopus Google Scholar, PubMed Scopus Google and a potential NFAT binding site in the IFN-γ promoter been identified P.M. Pimm J. Ramassar V. Halloran P.F. 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Res. 1994; PubMed Scopus Google Scholar). mutagenesis analysis of the C3-3P site was by as A, and within the C3-3P was to the of the C3-3P These results that the two located in the 3′-half of the C3-3P site are for its binding In of this observation, when was used in the presence of a nuclear activated T binding activity was of the binding of the wild in a the of the C3-3P we the NF-κB and NFAT for the C3-3P complex we of the p65 NF-κB subunits and the protein in both the and to the of the C3-3P whereas a to and a of the complex the NF-κB we an a of the C3-3P complexes These results identified both NF-κB and NFAT proteins as the inducible binding factors with the C3-3P site. binding studies demonstrated the of NF-κB and NFAT proteins with the C3-3P site. the of we of the C3-3P site of the thymidine promoter in the shown in this was the T cell with expression for p65 NF-κB, p50 NF-κB, a of the PubMed Scopus Google Scholar). of the p65 NF-κB the expression in a of the activity the activities in the presence of the In cells the was activated by p65 and by whereas the p50 expression and the and In cells with the effects of the p65 and expression cyclosporin A the in promoter activity inhibited the to activity of the used as a was by we have identified a site within the IFN-γ promoter region that is to NF-κB p65 and to a that is activated by the presence of additional NF-κB binding an NF-κB activation of IFN-γ gene we the nuclear protein binding activity of the IFN-γ κB and sites. 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Res. 1994; PubMed Scopus Google Scholar), we investigated NFAT protein for the the IFN-γ region to shown in was with a the site and NFAT and to complex both the IFN-γ κB and to the NF-κB binding to the IFN-γ we analysis and analysis that both the IFN-γ κB and the specifically bind the inducible NF-κB In the and of Interestingly, the complex of region by as the NF-κB In addition, a complex was by the by the that this complex the p50 NF-κB A complex was the protein of this complex was These results the binding of NF-κB proteins NFAT proteins to both the IFN-γ κB and sites. of additional complexes that the its partial for other protein the of NF-κB protein binding to the IFN-γ promoter we of this region to of the promoter in the β-galactosidase This was in the T cell shown in both and with a p65 NF-κB expression to β-galactosidase activity in the presence of the IFN-γ promoter of in a In a p50 NF-κB expression was to β-galactosidase These results for a of NF-κB with the IFN-γ promoter Although both the C3-3P site and the IFN-γ promoter enhancer activity when of a promoter, we investigated role in the of the IFN-γ Based on mutagenesis studies II and we a of within the IFN-γ promoter region to that the binding activities of the IFN-γ κB to the β-galactosidase gene in the L. 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Transplantation. 1996; 61: 933-939Crossref PubMed Scopus (56) Google Scholar), that the induction of the IFN-γ promoter activity through the IFN-γ promoter the of the NFAT and NF-κB binding identified within the IFN-γ promoter and to with the intronic C3 enhancer two of the previously identified c-Rel intronic site C3 A. M. Ghosh P. Young H.A. S. A. PubMed Scopus Google downstream of the β-galactosidase gene in the WT, and the β-galactosidase activity by the two of the intronic C3 site the activities by the with the activity by the to which a of was the two of the intronic C3 site a of the β-galactosidase the C3-3P the IFN-γ κB binding activities in a of with the C3 whereas of binding both the C3-3P and IFN-γ κB binding to with the intronic C3 enhancer to activity of the IFN-γ A of have investigated the molecular mechanisms regulating the IFN-γ gene transcription. A consensus and an have been identified within the promoter to of the IFN-γ gene L. Weaver W.M. Pang Y. Young H.A. Wilson C.B. J. Exp. Med. 1993; 178: 1483-1496Crossref PubMed Scopus (178) Google Scholar), and the binding of and of transcription factors to this region of the promoter been L.A. Sweetser M.T. Weaver W.M. Hoeffler J.P. Kerppola T.K. Wilson C.B. J. Biol. Chem. 1996; 271: 31964-31972Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). This region of the promoter been two a to and a to L. Weaver W.M. Pang Y. Young H.A. Wilson C.B. J. Exp. Med. 1993; 178: 1483-1496Crossref PubMed Scopus (178) Google Scholar), and the of elements in regulating IFN-γ gene expression been demonstrated through the of by and T.M. Penix L.A. Rincon M.R. Flavell R.A. Mol. Cell. Biol. 1997; 17: 199-208Crossref PubMed Scopus (79) Google Scholar). In this the and elements shown to transcription in T cells in to through the T cell the the of T cells elements to for IFN-γ expression in cell and K. Büschenfeld K.-H.M. Neurath M.E. Eur. J. Immunol. 1997; 27: 1098-1107Crossref PubMed Scopus (28) Google have used in to a site to that binds a complex in T cells and T cells. These have a induction of the proteins to this region of the promoter is involved in the transcription of the IFN-γ gene in to Although the promoter in are for the transcription of the IFN-γ is that additional J. Ghosh P. M. J. Hardy K.J. Ortaldo J.R. Young H.A. J. Biol. Chem. 1994; Full Text PDF PubMed Google and D.A. Nelson F.B. Reinherz E.L. Diamond D.J. Eur. J. Immunol. 1991; 21: 1879-1885Crossref PubMed Scopus (51) Google elements a role in the of IFN-γ and that for IFN-γ gene expression the We have previously identified a c-Rel binding site (C3) A. M. Ghosh P. Young H.A. S. A. PubMed Scopus Google that is located within an IFN-γ intronic enhancer region and that sequence homology with NFAT NF-κB binding In this we have the binding activity and properties of two newly identified C3-related (C3-1P and and a NF-κB (IFN-γ κB) site located in the promoter region and for a newly identified and the intronic c-Rel site demonstrate the within the IFN-γ promoter, of for the NF-κB binding of in regulating IFN-γ expression the that IFN-γ We have previously demonstrated J. Ortaldo J.R. K. Young H.A. J. Biol. 1995; PubMed Scopus Google that the nuclear in the of NF-κB proteins in a NK cell and the of the NF-κB proteins within the of the IFN-γ promoter to enhance the transcription of the Although the the activation of IFN-γ expression have been a S. A. M. Int. Immunol. 1997; Scopus Google as an important for the induction of IFN-γ expression through the T cell in cells. been demonstrated to important for activation G. 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Transplantation. 1996; 61: 933-939Crossref PubMed Scopus (56) Google the region as a potential NFAT binding site. In both and NFAT to of the C3-3P that NF-κB and NFAT proteins bind the C3-3P site. of both NF-κB and NFAT proteins to bind the site is the that the is a of the proteins Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, J.P. L. D.J. L.A. A. 1994; PubMed Scopus Google Scholar). In addition, a the binding of an factor to a κB site J. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with the site A. S. Res. 1994; PubMed Scopus Google Scholar), the of NF-κB and NFAT proteins to functionally interact with the C3-3P site the that NF-κB and NFAT through transcriptional activity through enhancer These results IFN-γ to the family of genes that are in by the NF-κB and NFAT of transcription We have previously A. M. Ghosh P. Young H.A. S. A. PubMed Scopus Google that a p50 NF-κB protein bind to the intronic C3 we binding of the p50 the C3-3P site, its binding by the is that the NF-κB p50 binds the κB 1991; Google Scholar, Mol. Cell. Biol. PubMed Google Scholar), the binding of the p50 to the of the is that of the IFN-γ κB site by the the of the p50 NF-κB the of the site. that p50 NF-κB the activity of the IFN-γ DNA as as the IFN-γ promoter activity is in with the that p50 is of an activation and that p65 is for transcriptional activity M. V. J. P. A. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar). the role of the in regulating IFN-γ gene expression is of as and T. 1996; PubMed Scopus Google have identified binding the intronic C3 the enhanced promoter activity when the C3 site was downstream of the gene was when the IFN-γ κB the C3-3P binding activity by transcription of the IFN-γ gene results promoter and intronic that and NF-κB IFN-γ transcription. This analysis of in the of the IFN-γ DNA In this for the the NF-κB and NFAT family of proteins with of the IFN-γ promoter and the C3-3P site and the IFN-γ as enhancer elements for IFN-γ gene transcription. the IFN-γ gene as an additional transcriptional the NF-κB and NFAT complexes a role in transcriptional We and for Ortaldo and for of the and and for and We the of the p65 NF-κB expression of for the NF-κB expression and A. and for and of for the of expression and the