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• W2050698668 abstract "We previously established that the class II transactivator CIITA binds GTP and disruption of the GTP binding ability of CIITA results in increased cytoplasmic CIITA, loss of nuclear CIITA, and thus diminished class II major histocompatibility complex transcription. Because of its role in facilitating nuclear localization, whether GTP binding is also required for CIITA-mediated transactivation of major histocompatibility class II genes remains unclear. We now show that recruitment of CIITA to the human leukocyte antigen (HLA)-DR promoter and activation of HLA-DR transcription is also GTP-dependent. After restoration of nuclear expression, CIITA mutants defective in GTP binding lack full transcriptional activation capacity. Although the availability of the activation domain of CIITA is unaltered, GTP mutants no longer cooperate with CREB-binding protein, p300, and pCAF and are defective in recruitment to the HLA-DR promoter. We previously established that the class II transactivator CIITA binds GTP and disruption of the GTP binding ability of CIITA results in increased cytoplasmic CIITA, loss of nuclear CIITA, and thus diminished class II major histocompatibility complex transcription. Because of its role in facilitating nuclear localization, whether GTP binding is also required for CIITA-mediated transactivation of major histocompatibility class II genes remains unclear. We now show that recruitment of CIITA to the human leukocyte antigen (HLA)-DR promoter and activation of HLA-DR transcription is also GTP-dependent. After restoration of nuclear expression, CIITA mutants defective in GTP binding lack full transcriptional activation capacity. Although the availability of the activation domain of CIITA is unaltered, GTP mutants no longer cooperate with CREB-binding protein, p300, and pCAF and are defective in recruitment to the HLA-DR promoter. Class II major histocompatibility complex (MHC II) 2The abbreviations used are: MHC IIclass II major histocompatibility complexCIITAclass II transactivatorHLA-DRhuman leukocyte antigen-DRNLSnuclear localization signal sequenceChIPchromatin immunoprecipitationCBPCREB-binding proteinCREBcAMP-response element-binding protein.2The abbreviations used are: MHC IIclass II major histocompatibility complexCIITAclass II transactivatorHLA-DRhuman leukocyte antigen-DRNLSnuclear localization signal sequenceChIPchromatin immunoprecipitationCBPCREB-binding proteinCREBcAMP-response element-binding protein. molecules are essential for T cell- and antibody-mediated immune responses through presentation of exogenous antigen-derived peptides to CD4+ T cells. Although professional antigen-presenting cells (B cells, monocytes, macrophages, and dendritic cells) express MHC II constitutively, interferon-γ will induce MHC II on most negative tissues/cells. Expression of both constitutive and inducible MHC II is regulated by a master transcriptional co-activator, CIITA (1Steimle V. Otten L.A. Zufferey M. Mach B. Cell. 1993; 75: 135-146Abstract Full Text PDF PubMed Scopus (759) Google Scholar). CIITA also regulates expression of MHC II-related genes such as HLA-DM and invariant chain through the W, X, and Y elements common to this family of promoters (2Chang C.H. Fontes J.D. Peterlin M. Flavell R.A. J. Exp. Med. 1994; 180: 1367-1374Crossref PubMed Scopus (299) Google Scholar, 3Kern I. Steimle V. Siegrist C.A. Mach B. Int. Immunol. 1995; 7: 1295-1299Crossref PubMed Scopus (95) Google Scholar). Defects in CIITA (as in some MHC II deficiency patients or CIITA knock-out mice) nearly abolish all MHC II expression and are accompanied by a near complete lack of CD4+ T cells, causing a severe and lethal combined type immunodeficiency (4Ting J.P. Trowsdale J. Cell. 2002; 109: S21-S33Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Class II MHC expression levels, both constitutive and cytokine-induced, are in large part regulated via transcriptional control of CIITA through multiple promoters, each with particular tissue restriction and induction patterns (5Lennon A.M. Ottone C. Rigaud G. Deaven L.L. Longmire J. Fellous M. Bono R. Alcaide-Loridan C. Immunogenetics. 1997; 45: 266-273Crossref PubMed Scopus (75) Google Scholar, 6Muhlethaler-Mottet A. Otten L.A. Steimle V. Mach B. EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (432) Google Scholar, 7Piskurich J.F. Linhoff M.W. Wang Y. Ting J.P. Mol. Cell. Biol. 1999; 19: 431-440Crossref PubMed Google Scholar). However, post-translational modification of CIITA function, either directly via signaling events (e.g. phosphorylation and ubiquitination) or indirectly through histone deacetylase association during infection also influences MHC II expression, suggesting that CIITA is highly regulated (8Li G. Harton J.A. Zhu X. Ting J.P. Mol. Cell. Biol. 2001; 21: 4626-4635Crossref PubMed Scopus (56) Google Scholar, 9Sisk T.J. Nickerson K. Kwok R.P. Chang C.H. Int. Immunol. 2003; 15: 1195-1205Crossref PubMed Scopus (37) Google Scholar, 10Greer S.F. Harton J.A. Linhoff M.W. Janczak C.A. Ting J.P. Cressman D.E. J. Immunol. 2004; 173: 376-383Crossref PubMed Scopus (27) Google Scholar, 11Greer S.F. Zika E. Conti B. Zhu X.S. Ting J.P. Nat. Immunol. 2003; 4: 1074-1082Crossref PubMed Scopus (83) Google Scholar, 12Schnappauf F. Hake S.B. Camacho Carvajal M.M. Bontron S. Lisowska-Grospierre B. Steimle V. Eur. J. Immunol. 2003; 33: 2337-2347Crossref PubMed Scopus (36) Google Scholar, 13Wang Y. Curry H.M. Zwilling B.S. Lafuse W.P. J. Immunol. 2005; 174: 5687-5694Crossref PubMed Scopus (101) Google Scholar).CIITA likely shuttles between the cytoplasm and the nucleus, a process regulated in part by at least three nuclear localization sequences, export sequences, GTP binding, and C-terminal leucine-rich repeats of CIITA (14Cressman D.E. Chin K.C. Taxman D.J. Ting J.P. Immunity. 1999; 10: 163-171Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 15Hake S.B. Masternak K. Kammerbauer C. Janzen C. Reith W. Steimle V. Mol. Cell. Biol. 2000; 20: 7716-7725Crossref PubMed Scopus (96) Google Scholar, 16Spilianakis C. Papamatheakis J. Kretsovali A. Mol. Cell. Biol. 2000; 20: 8489-8498Crossref PubMed Scopus (134) Google Scholar, 17Harton J.A. Zika E. Ting J.P. J. Biol. Chem. 2001; 276: 38715-38720Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 18Raval A. Weissman J.D. Howcroft T.K. Singer D.S. J. Immunol. 2003; 170: 922-930Crossref PubMed Scopus (28) Google Scholar). CIITA can self-associate and likely forms larger complexes mediated by homotypic and heterotypic interactions between the GTP-binding domain and the leucine-rich repeats (19Linhoff M.W. Harton J.A. Cressman D.E. Martin B.K. Ting J.P. Mol. Cell. Biol. 2001; 21: 3001-3011Crossref PubMed Scopus (82) Google Scholar, 20Sisk T.J. Roys S. Chang C.H. Mol. Cell. Biol. 2001; 21: 4919-4928Crossref PubMed Scopus (43) Google Scholar). These interactions may be important for transcription activation, but it remains unclear whether they are critical for nuclear import. Once in the nucleus, CIITA participates in chromatin remodeling and activates MHC II transcription. Although a non-DNA-binding protein, CIITA directs MHC II transcription by forming a scaffold or enhanceosome at the promoter through interactions with RFX, CREB, NF-Y, and components of the basal transcription machinery (21Zhu X.S. Linhoff M.W. Li G. Chin K.C. Maity S.N. Ting J.P. Mol. Cell Biol. 2000; 20: 6051-6061Crossref PubMed Scopus (168) Google Scholar, 22Moreno C.S. Beresford G.W. Louis-Plence P. Morris A.C. Boss J.M. Immunity. 1999; 10: 143-151Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 23Fontes J.D. Jiang B. Peterlin B.M. Nucleic Acids Res. 1997; 25: 2522-2528Crossref PubMed Scopus (116) Google Scholar, 24Mahanta S.K. Scholl T. Yang F.C. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6324-6329Crossref PubMed Scopus (108) Google Scholar). The histone acetyltransferases CBP (CREB-binding protein), p300, and pCAF cooperatively activate CIITA-driven gene expression, and CIITA may have its own acetyltransferase activity (25Raval A. Howcroft T.K. Weissman J.D. Kirshner S. Zhu X.S. Yokoyama K. Ting J. Singer D.S. Mol. Cell. 2001; 7: 105-115Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Moreover, CBP and pCAF acetylation of CIITA enhances nuclear import (16Spilianakis C. Papamatheakis J. Kretsovali A. Mol. Cell. Biol. 2000; 20: 8489-8498Crossref PubMed Scopus (134) Google Scholar). Conversely, CIITA can be negatively regulated by phosphorylation at a number of different serine residues (8Li G. Harton J.A. Zhu X. Ting J.P. Mol. Cell. Biol. 2001; 21: 4626-4635Crossref PubMed Scopus (56) Google Scholar, 9Sisk T.J. Nickerson K. Kwok R.P. Chang C.H. Int. Immunol. 2003; 15: 1195-1205Crossref PubMed Scopus (37) Google Scholar, 10Greer S.F. Harton J.A. Linhoff M.W. Janczak C.A. Ting J.P. Cressman D.E. J. Immunol. 2004; 173: 376-383Crossref PubMed Scopus (27) Google Scholar, 26Spilianakis C. Kretsovali A. Agalioti T. Makatounakis T. Thanos D. Papamatheakis J. EMBO J. 2003; 22: 5125-5136Crossref PubMed Scopus (72) Google Scholar).Although binding GTP, CIITA does not appear to be a GTPase in vitro (27Harton J.A. Cressman D.E. Chin K.C. Der C.J. Ting J.P. Science. 1999; 285: 1402-1405Crossref PubMed Scopus (85) Google Scholar). A point mutation that confers GTPase activity noticeably diminishes CIITA function. Mutants with defective GTP binding have been used to demonstrate the importance of the GTP-binding domain of CIITA in nuclear import and export, dimerization, and intrinsic acetyltransferase activity (18Raval A. Weissman J.D. Howcroft T.K. Singer D.S. J. Immunol. 2003; 170: 922-930Crossref PubMed Scopus (28) Google Scholar, 19Linhoff M.W. Harton J.A. Cressman D.E. Martin B.K. Ting J.P. Mol. Cell. Biol. 2001; 21: 3001-3011Crossref PubMed Scopus (82) Google Scholar, 25Raval A. Howcroft T.K. Weissman J.D. Kirshner S. Zhu X.S. Yokoyama K. Ting J. Singer D.S. Mol. Cell. 2001; 7: 105-115Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 27Harton J.A. Cressman D.E. Chin K.C. Der C.J. Ting J.P. Science. 1999; 285: 1402-1405Crossref PubMed Scopus (85) Google Scholar). Because GTP-binding domain mutations render CIITA largely cytoplasmic, whether GTP binding is actually required for CIITA transcription activation potential remains unclear. In this report we explore the GTP dependence of CIITA-mediated MHC II transcription activation using CIITA constructs containing the strong SV40 nuclear localization signal sequence (NLS). Under conditions of enforced nuclear expression, activation of MHC II transcription by CIITA appears to be largely GTP-dependent. Further GTP binding appears important for recruitment to the MHC II promoter and may mediate CIITA interactions with promoter-bound transcription factors.EXPERIMENTAL PROCEDURESTissue Culture Cells and Conditions—COS-7 and HeLa cells were maintained in Dulbecco's modified Eagle's medium (with 4.5 g/liter glucose) supplemented with 10% fetal calf serum, 5 mm l-glutamine, and 0.1% streptomycin-penicillin. All cells were grown at 37 °C with 5% CO2.Plasmid Constructs—pcDNA3 (Invitrogen) and FLAG-tagged versions of wild-type CIITA and the CIITA GTP binding mutants GTP1, GTP2, GTP3, C2RASG1, and C2RG1L465Q have been described previously (27Harton J.A. Cressman D.E. Chin K.C. Der C.J. Ting J.P. Science. 1999; 285: 1402-1405Crossref PubMed Scopus (85) Google Scholar, 28Chin K.C. Li G.G. Ting J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2501-2506Crossref PubMed Scopus (91) Google Scholar). To generate the chimera C2RAS, an Eco47III site at nucleotides 1279–1284 and a ClaI site at nucleotides 1707–1712 of wild-type CIITA were generated using QuikChange mutagenesis (Stratagene). Following digestion, an amplified Eco47III/ClaI-flanked fragment of the GTPase-inactive RasQ61L oncogene was inserted. All the NLS-tagged forms of these mutants were generated by transferring the AocI/SacII fragment containing the entire nucleotide-binding domain into AocI/SacII-digested NLS-CIITA (14Cressman D.E. Chin K.C. Taxman D.J. Ting J.P. Immunity. 1999; 10: 163-171Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The sequences of all DNA amplification-generated constructs were confirmed by fluorescent dye terminator sequencing.Luciferase Assays—Transient cotransfection was performed using FuGENE 6 (Roche Applied Science). Briefly, 5 × 104 cells/well were plated in 24-well plates, cultured overnight, and transfected with DRA300Luc (a luciferase reporter driven by the MHC class II HLA-DR promoter) and either pcDNA3 (vector control), FLAG-tagged NLS-CIITA, or NLS-CIITA mutant plasmids (14Cressman D.E. Chin K.C. Taxman D.J. Ting J.P. Immunity. 1999; 10: 163-171Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The transfected cells were lysed 16–18 h posttransfection in 1× reporter lysis buffer (Promega), and luciferase assays were performed as previously described (29Piskurich J.F. Wang Y. Linhoff M.W. White L.C. Ting J.P. J. Immunol. 1998; 160: 233-240PubMed Google Scholar). Each transfection was performed in triplicate, and luciferase data were normalized to total protein.Western Blotting—Following transfection of cells with 1 μg of DNA, cell were lysed in 1× reporter lysis buffer (Promega) containing 1× Complete™ (Roche Applied Science) protease inhibitors. Protein concentrations were normalized, and 5 μgof protein was subjected to SDS-PAGE (8%) and transferred to nitrocellulose. Blotting for the FLAG epitope was performed as described (19Linhoff M.W. Harton J.A. Cressman D.E. Martin B.K. Ting J.P. Mol. Cell. Biol. 2001; 21: 3001-3011Crossref PubMed Scopus (82) Google Scholar).Immunofluorescence Microscopy—Immunofluorescent staining of transiently transfected COS-7 cells was performed as previously described (14Cressman D.E. Chin K.C. Taxman D.J. Ting J.P. Immunity. 1999; 10: 163-171Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Briefly, 5 to 8 × 104 cells were plated on two-well chamber slides (Nunc), incubated overnight at 37 °C, and transfected with 1 to 1.5 μg of plasmid DNA using FuGENE 6. After a second overnight incubation, the cells were washed with 1× phosphate-buffered saline, blocked in 1× phosphate-buffered saline, 1% bovine serum albumin, 10% goat serum, and stained with anti-FLAG(M5) (Sigma) and goat anti-mouse IgG-fluorescein isothiocyanate (Southern Biotech). To ensure representative data in these experiments, prior to taking representative photomicrographs for each experiment the entire slide was examined under 400–500× magnification to observe the pattern of nuclear and cytoplasmic expression.ChIP Assay—HeLa cells were plated (1 × 106 cells/100-mm plate) 24 h prior to transfection. Cells were transfected with 3 μg of the indicated plasmids using FuGENE 6 (Roche) according to the manufacturer's recommendations. Chromatin immunoprecipitations (ChIP) were performed in accordance with the ChIP assay kit (Upstate Biotechnology). Briefly, 18–24 h post-transfection DNA-protein cross-linking was accomplished at room temperature with 0.37% formaldehyde for 10 min and stopped by addition of 0.125 m glycine for 5 min. Cells were lysed on ice for 15 min with SDS cell lysis buffer (50 mm Tris, pH 8.1, 10 mm EDTA, 1% sodium dodecyl sulfate) supplemented with Complete EDTA-free protease inhibitors (Roche). After lysis, chromatin was sheared to an average size of 200–1200 bp by sonication. Lysates were diluted 1:2 with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-HCL, pH 8.1, 167 mm NaCl) and precleared with Protein G Plus A-agarose beads (Santa Cruz). Immunoprecipitation was carried out with 10 μg of anti-FLAG M5 (Sigma). DNA-protein-antibody-bead complexes were then washed at room temperature using the following: TSE-500 wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCL, pH 8.1), LiCl wash buffer (500 mm LiCl, 1% Nonidet P-40, 1% deoxycholate, 100 mm Tris-HCL, pH 8.1) and 1× TE buffer (10 mm Tris-HCL, pH 7.5, 1 mm EDTA). In addition, no-antibody control immunoprecipitations were performed. Elution of the DNA-protein complexes was accomplished with a 1% SDS and 50 mm NaHCO3 buffer solution. Cross-links were reversed overnight at 65 °C. Samples were then incubated with 1.0 μgof Proteinase K for 1 h, and DNA was purified using phenol-chloroform extraction. Recovered HLA-DR promoter DNA was assayed by real-time PCR using 15% of the recovered DNA.Real-time PCR—Real-time PCR was performed using a MyIQ Thermocycler (Bio-Rad) with the following MHC II promoter primers: sense primer, 5′-GAT CTC TTG TGT CCT GGA CCC TTT GCA A-3′; antisense primer, 5′-CCC AAT TAC TCT TTG GCC AAT CAG AAA A-3′. Quantification of the HLA-DR promoter was conducted in a total volume of 25 μl with 2× Sybr Green (Bio-Rad). Thermal cycler parameters were 3 min at 95 °C and 40 cycles of denaturation at 95 °C for 15 s, annealing/extension at 55 °C for 1 min.RESULTSThe GTP-dependent functions of CIITA are not fully understood. Previously we demonstrated that CIITA mutants with decreased abilities to bind GTP remain principally cytoplasmic and thus unable to effectively activate MHC II transcription (Table 1) (27Harton J.A. Cressman D.E. Chin K.C. Der C.J. Ting J.P. Science. 1999; 285: 1402-1405Crossref PubMed Scopus (85) Google Scholar). Interestingly, these observations may be due to increased nuclear export as other studies suggest enhanced binding of the same mutants to CREM1, the major protein responsible for nuclear export (18Raval A. Weissman J.D. Howcroft T.K. Singer D.S. J. Immunol. 2003; 170: 922-930Crossref PubMed Scopus (28) Google Scholar). Diminished self-association of certain CIITA-GTP mutants suggests that molecular interactions between CIITA molecules may be disrupted or non-productive if GTP is not bound (19Linhoff M.W. Harton J.A. Cressman D.E. Martin B.K. Ting J.P. Mol. Cell. Biol. 2001; 21: 3001-3011Crossref PubMed Scopus (82) Google Scholar, 20Sisk T.J. Roys S. Chang C.H. Mol. Cell. Biol. 2001; 21: 4919-4928Crossref PubMed Scopus (43) Google Scholar). Thus it remains unclear what role, if any, GTP binding actually plays in transcriptional activation. To directly test whether CIITA requires GTP binding to activate transcription of MHC II, we generated NLS-tagged versions of several GTP binding mutants and examined their ability to activate transcription from the HLA-DRA promoter (Fig. 1). All of the NLS-tagged constructs display nuclear localization comparable with that seen with wild-type NLS-CIITA (Fig. 1A). Consistent with our previous results where we observed no apparent nuclear localization of the untagged mutants, the phosphate-binding domain (GTP1) and guanine ring-binding domain (GTP3) mutants essentially lack the ability to activate MHC II transcription (Fig. 1B). Surprisingly, however, the GTP2 mutation that eliminates the consensus magnesium ion coordination site (DAYG) retains substantial ability to activate transcription from the HLA-DRA promoter. We have previously observed that reducing the amount of transfected DNA can reveal the effects of nuclear import-related differences in activation potential relative to wild-type controls (17Harton J.A. Zika E. Ting J.P. J. Biol. Chem. 2001; 276: 38715-38720Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 30Harton J.A. O'Connor W. Conti B.J. Linhoff M.W. Ting J.P. Hum. Immunol. 2002; 63: 588-601Crossref PubMed Scopus (23) Google Scholar). However, transfections of cells with a tenth of the DNA yielded essentially the same pattern, suggesting that NLS-GTP2 is functionally impaired, retaining only ∼50% activity relative to wild-type, and that the reduced activity is not a consequence of import differences. These data strongly suggest that CIITA requires GTP for the full activation of HLA-DRA transcription.TABLE 1Summary of transactivation potential and nuclear localization for CIITA GTP-binding domain mutantsConstructG1G3G4GTPase activityRelative activationNuclear localization%CIITAGKAGQGKSDAYG LSKAD-100C/NGTP1–AGQGKSDAYG LSKADND38CGTP2GKAGQGKS—- LSKADND10CGTP3GKAGQGKSDAYG L—-ND21CC2RASG1GAGVGGKSDAYG LSKAD-95C/NC2RASG1L465QGAGVGGKSDAYG QSKAD+30C Open table in a new tab Mutation of the phosphate-binding domain of CIITA to that of Ras (C2RasG1) does not diminish activation of HLA-DRA transcription; however, the addition of the L465Q point mutation to either C2RASG1 (C2RG1L465Q) or to wild-type CIITA imparted intrinsic GTPase activity to CIITA (27Harton J.A. Cressman D.E. Chin K.C. Der C.J. Ting J.P. Science. 1999; 285: 1402-1405Crossref PubMed Scopus (85) Google Scholar). We previously demonstrated that CIITA GTPase activity correlated with diminished transcriptional activation ability and an apparent loss of nuclear translocation. Presumably, limited nuclear import of C2RG1L465Q is sufficient to partially activate transcription but insufficient for successful immunofluorescent staining. If the diminished capacity of this mutant is due principally to the import defect, restoration of nuclear localization would be expected to completely restore function. The NLS-tagged forms of both C2RASG1 and C2RASG1L465Q display nuclear localization comparable with wild-type NLS-CIITA (Fig. 2A), but the GTPase active form of CIITA has ∼50% of the activity of the parent construct (Fig. 2B). Thus, even with detectable and apparently normal nuclear localization, GTPase-active CIITA is impaired in its ability to activate transcription. This observation further supports the hypothesis that the GTP-bound state of CIITA influences its transcriptional activity.FIGURE 2The GTPase-active CIITA mutant C2RG1L465Q decreases CIITA-mediated transactivation. COS-7 cells were co-transfected with either 1 μg (A) or 100 ng (B) of the indicated constructs and the HLA-DR luciferase reporter. Transactivation ability was normalized to that of NLS-C2RASG1 (100%; relative activation), the functional, non-GTPase parent construct. Data shown represent the mean ± S.E. for three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)How GTP binding imparts transactivation capacity to CIITA is unclear. One possibility is that GTP binding confers a transactivation-permissive conformation. To test this, wild-type CIITA and GTP mutants were fused to the DNA-binding domain of the yeast transcription factor GAL4 and tested for their ability to activate a GAL4-luciferase promoter/reporter construct (Fig. 3). From previous studies, it is known that phosphorylation of CIITA by cAMP-dependent protein kinase inhibits the ability of GAL4-CIITA to activate a GAL4-luciferase promoter (8Li G. Harton J.A. Zhu X. Ting J.P. Mol. Cell. Biol. 2001; 21: 4626-4635Crossref PubMed Scopus (56) Google Scholar). However, deletion of critical GTP binding elements in GAL4-CIITA had no effect on activation of GAL4-luciferase, suggesting that GTP binding defects do not impair the availability of the activation domain. Interestingly, GAL4-C2RASG1 has an increased ability to activate transcription whereas that of GAL4-C2RG1L465Q is decreased relative to its parent construct (C2RASG1). Although some correlation with GTP binding may account for the increased activity of C2RasG1, the lack of differences between wild type and deletion or GTPase-active mutants strongly suggests that GTP binding differences do not alter transactivation domain availability.FIGURE 3GTP domain mutations do not adversely affect CIITA activation domain function. COS-7 cells were transfected with DNA coding for the GAL4 DNA-binding domain (pSG424), GAL4 fused to wild-type CIITA (GAL4-CIITA), or the indicated GTP-binding domain mutants and a Gal4 luciferase reporter with five copies of the Gal4 DNA binding motif. Luciferase activity was normalized to the parent construct, GAL4-CIITA (100%; relative activation). Luciferase activity in triplicate transfections is shown as the mean ± S.E. and is representative of results from one of three similar experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Our data suggested that GTP does not alter availability of the distal N-terminal transactivation domain; however, GTP-dependent conformational changes affecting residues near the motifs mediating GTP binding might be important. To explore this idea, the GTP-binding domain of Ras61L (GTPase-deficient) was cloned into CIITA to generate C2RAS (Fig. 4A). C2RAS retains the phosphate binding loop (G1) of CIITA but contains the nucleotide-binding domain of the oncogenic, GTPase-defective Ras61L mutant from residue 18 through 127 and displays GTP binding comparable with Ras in an in vitro exchange assay (not shown). C2RAS fails to activate HLA-DRA transcription (not shown) and has defective nuclear import (Fig. 4B). To rule out failed nuclear import as a reason for lack of transactivation ability, C2RAS was tagged with the SV40 NLS. NLS-C2RAS enters the nucleus but still fails to activate transcription (Fig. 4, B and C). These observations suggest that residues within the domain, but not directly involved in nucleotide binding, are required for activation, even in the presence of an intact GTP-binding domain.FIGURE 4CIITA nuclear localization and HLA-DRA transactivation require the CIITA GTP-binding domain.A, schematic of the C2RAS construct that contains the GTP-binding domain of the Ras Q61L, non-GTPase-active oncogene. The wild-type phosphate-binding site (G1) from CIITA is maintained and the G2-G5 motifs derive from RasQ61L. B, anti-FLAG immunofluorescence staining is shown for COS-7 cells expressing transfected wild-type CIITA and C2RAS, without and with the addition of an N-terminal NLS sequence. C, HLA-DR luciferase reporter activation by NLS-CIITA versus NLS-C2RAS. Data are the mean ± S.E. of three independent experiments. GTP binding by C2RAS in in vitro exchange assays was similar to that of the GTPase-impaired RasV12 (not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Various studies support the contention that CIITA drives both opening of the promoter and activation of transcription (25Raval A. Howcroft T.K. Weissman J.D. Kirshner S. Zhu X.S. Yokoyama K. Ting J. Singer D.S. Mol. Cell. 2001; 7: 105-115Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 31Steimle V. Siegrist C.A. Mottet A. Lisowska-Grospierre B. Mach B. Science. 1994; 265: 106-109Crossref PubMed Scopus (684) Google Scholar, 32Rigaud G. De Lerma Barbaro A. Nicolis M. Cestari T. Ramarli D. Riviera A.P. Accolla R.S. J. Immunol. 1996; 156: 4254-4258PubMed Google Scholar, 33Wright K.L. Chin K.C. Linhoff M. Skinner C. Brown J.A. Boss J.M. Stark G.R. Ting J.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6267-6272Crossref PubMed Scopus (63) Google Scholar, 34Beresford G.W. Boss J.M. Nat. Immunol. 2001; 2: 652-657Crossref PubMed Scopus (155) Google Scholar). This led us to consider whether the defect was in recruitment of the GTP-binding domain mutants to the promoter or in the ability of the mutants to transactivate. CIITA interacts cooperatively with the histone acetyltransferases CBP, pCAF, and p300 leading to increased transactivation and requires the N terminus of CIITA (16Spilianakis C. Papamatheakis J. Kretsovali A. Mol. Cell. Biol. 2000; 20: 8489-8498Crossref PubMed Scopus (134) Google Scholar, 17Harton J.A. Zika E. Ting J.P. J. Biol. Chem. 2001; 276: 38715-38720Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 35Fontes J.D. Kanazawa S. Jean D. Peterlin B.M. Mol. Cell. Biol. 1999; 19: 941-947Crossref PubMed Scopus (137) Google Scholar, 36Kretsovali A. Agalioti T. Spilianakis C. Tzortzakaki E. Merika M. Papamatheakis J. Mol. Cell. Biol. 1998; 18: 6777-6783Crossref PubMed Scopus (154) Google Scholar, 37Sisk T.J. Gourley T. Roys S. Chang C.H. J. Immunol. 2001; 165: 2511-2517Crossref Scopus (96) Google Scholar). We reasoned that if CIITA mutants were recruited to the promoter but unable to activate transcription, their intact N-terminal domain should still interact with these coactivators (in excess), resulting in increased transcriptional activity. Expression of the NLS-tagged CIITA constructs is comparable with wild type in transfected cells (Fig. 5A), with the exception of NLS-GTP2. NLS-GTP2, like the parent GTP2 construct, consistently displays reduced expression relative to other constructs (27Harton J.A. Cressman D.E. Chin K.C. Der C.J. Ting J.P. Science. 1999; 285: 1402-1405Crossref PubMed Scopus (85) Google Scholar, 28Chin K.C. Li G.G. Ting J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2501-2506Crossref PubMed Scopus (91) Google Scholar). Although no cooperation was observed between wild-type NLS-CIITA and CBP, pCAF and p300 both substantially enhanced the ability of NLS-CIITA to activate transcription (Fig. 5B). Of the mutants, only NLS-GTP2 displayed cooperation with both pCAF and p300, despite its lower expression. This confirms the initial observation that NLS-GTP2 is partially active and supports our conclusion that NLS-GTP2 is recruited to the promoter. Neither GTP1 nor GTP3 displayed any cooperativity with pCAF or p300, suggesting that they are unlikely to be recruited to the promoter. Curiously, C2RAS, although unable to appreciably activate HLA-DRA transcription, displayed some cooperation with p300. Although small, cooperation between C2RAS and p300 was consi" @default.
• W2050698668 created "2016-06-24" @default.
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• W2050698668 date "2007-09-01" @default.
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