Abstract

We have recently identified and clonedFoxp3, the gene defective in mice with thescurfy mutation. The immune dysregulation documented in these mice and in humans with mutations in the orthologous gene indicates that the foxp3 gene product, scurfin, is involved in the regulation of T cell activation and differentiation. The autoimmune state observed in these patients with the immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome, or X-linked autoimmunity-allergic dysregulation syndrome also points to a critical role for scurfin in the regulation of T cell homeostasis.FOXP3 encodes a novel member of the forkhead family of transcription factors. Here we demonstrate that this structural domain is required for nuclear localization and DNA binding. Scurfin, transiently expressed in heterologous cells, represses transcription of a reporter containing a multimeric forkhead binding site. Upon overexpression in CD4 T cells, scurfin attenuates activation-induced cytokine production and proliferation. We have identified FKH binding sequences adjacent to critical NFAT regulatory sites in the promoters of several cytokine genes whose expression is sensitive to changes in SFN abundance. Our findings indicate that the ability of scurfin to bind DNA, and presumably repress transcription, plays a paramount role in determining the amplitude of the response of CD4 T cells to activation. We have recently identified and clonedFoxp3, the gene defective in mice with thescurfy mutation. The immune dysregulation documented in these mice and in humans with mutations in the orthologous gene indicates that the foxp3 gene product, scurfin, is involved in the regulation of T cell activation and differentiation. The autoimmune state observed in these patients with the immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome, or X-linked autoimmunity-allergic dysregulation syndrome also points to a critical role for scurfin in the regulation of T cell homeostasis.FOXP3 encodes a novel member of the forkhead family of transcription factors. Here we demonstrate that this structural domain is required for nuclear localization and DNA binding. Scurfin, transiently expressed in heterologous cells, represses transcription of a reporter containing a multimeric forkhead binding site. Upon overexpression in CD4 T cells, scurfin attenuates activation-induced cytokine production and proliferation. We have identified FKH binding sequences adjacent to critical NFAT regulatory sites in the promoters of several cytokine genes whose expression is sensitive to changes in SFN abundance. Our findings indicate that the ability of scurfin to bind DNA, and presumably repress transcription, plays a paramount role in determining the amplitude of the response of CD4 T cells to activation. scurfy scurfin immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome X-linked autoimmunity-allergic dysregulation syndrome forkhead glutathione S-transferase V1 immunoglobulin heavy chain variable-region (VH) promoter fetal calf serum tetracycline-inducible green fluorescence protein anti electromobility shift assay transthyretin site nuclear factor of activated T cells granulocyte macrophage-colony stimulating factor interleukin tumor necrosis factor α phorbol myristate acetate signal transducer and activator of transcription base pair(s) scurfy(sf),1 a naturally occurring, X-linked, recessive mutation, has been described previously (1Lyon M. Peters J. Glenister P. Ball S. Wright E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2433-2437Crossref PubMed Scopus (151) Google Scholar, 2Godfrey V.L. Wilkinson J.E. Russell L.B. Am. J. Pathol. 1991; 138: 1379-1387PubMed Google Scholar, 3Godfrey, V. L., Wilkinson, J. E., Rinchik, E. M., and Russell, L. B. (1991) Proc. Natl. Acad. Sci. U. S. A.1991 88, 5528–5532Google Scholar, 4Godfrey V.L. Rouse B.T. Wilkinson J.E. Am. J. Pathol. 1994; 145: 281-286PubMed Google Scholar, 5Blair P.J. Carpenter D.A. Godfrey V.L. Russell L.B. Wilkinson J.E. Rinchik E.M. Mamm. Genome. 1994; 5: 652-654Crossref PubMed Scopus (25) Google Scholar, 6Blair P.J. Bultman S.J. Haas J.C. Rouse B.T. Wilkinson J.E. Godfrey V.L. J. Immunol. 1994; 153: 3764-3774PubMed Google Scholar, 7Derry J.M. Wiedemann P. Blair P. Wang Y. Kerns J.A. Lemahieu V. Godfrey V.L. Wilkinson J.E. Francke U. Genomics. 1995; 29: 471Crossref PubMed Scopus (39) Google Scholar, 8Kanangat S. Blair P. Reddy R. Deheshia J. Godfrey V. Rouse B.T. Wilkinson J.E. Eur. J. Immunol. 1996; 26: 161-165Crossref PubMed Scopus (106) Google Scholar, 9Clark L.B. Appleby M.W. Brunkow M.E. Wilkinson J.E. Ziegler S.F. Ramsdell F. J. Immunol. 1999; 162: 2546-2554PubMed Google Scholar). The disease observed in hemizygous mutant males features massive lymphoproliferation and subsequent infiltration of several organs, and results in death by ∼3 weeks of age (1Lyon M. Peters J. Glenister P. Ball S. Wright E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2433-2437Crossref PubMed Scopus (151) Google Scholar, 4Godfrey V.L. Rouse B.T. Wilkinson J.E. Am. J. Pathol. 1994; 145: 281-286PubMed Google Scholar). Both depletion and adoptive transfer experiments indicate that CD4 T cells are primarily responsible for the pathology observed in sfmice (2Godfrey V.L. Wilkinson J.E. Russell L.B. Am. J. Pathol. 1991; 138: 1379-1387PubMed Google Scholar, 6Blair P.J. Bultman S.J. Haas J.C. Rouse B.T. Wilkinson J.E. Godfrey V.L. J. Immunol. 1994; 153: 3764-3774PubMed Google Scholar). sf CD4 T cells are chronically activated, expressing up-regulated levels of several activation markers and secreting increased levels of several cytokines directly ex vivo (8Kanangat S. Blair P. Reddy R. Deheshia J. Godfrey V. Rouse B.T. Wilkinson J.E. Eur. J. Immunol. 1996; 26: 161-165Crossref PubMed Scopus (106) Google Scholar, 9Clark L.B. Appleby M.W. Brunkow M.E. Wilkinson J.E. Ziegler S.F. Ramsdell F. J. Immunol. 1999; 162: 2546-2554PubMed Google Scholar). sf-derived CD4 T cell effector function is also refractory to inhibition with several pharmacological reagents, particularly the immunosuppresants cyclosporin A and rapamycin (9Clark L.B. Appleby M.W. Brunkow M.E. Wilkinson J.E. Ziegler S.F. Ramsdell F. J. Immunol. 1999; 162: 2546-2554PubMed Google Scholar). The phenotype of sf mutant mice is strikingly similar to that observed in mice deficient in expression of CTLA-4, indicating that scurfin may also be an important regulator of the T cell activation program (10Waterhouse P. Penninger J.M. Timms E. Wakeham A. Shahinian A. Lee K.P. Thompson C.B. Griesser H. Mak T.W. Science. 1995; 270: 985-988Crossref PubMed Scopus (2376) Google Scholar, 11Tivol E.A. Borriello F. Schweitzer A.N. Lynch W.P. Bluestone J.A. Sharpe A.H. Immunity. 1995; 3: 541-547Abstract Full Text PDF PubMed Scopus (2393) Google Scholar, 12Chambers C.A. Sullivan T.J. Allison J.P. Immunity. 1997; 7: 885-895Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 13Walunas T.L. Lenschow D.J. Bakker C.Y. Linsley P.S. Freeman G.J. Green J.M. Thompson C.B. Bluestone J.A. Immunity. 1994; 1: 405-413Abstract Full Text PDF PubMed Scopus (1791) Google Scholar, 14Thompson C.B. Allison J.P. Immunity. 1997; 7: 445-450Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). Recently we have positionally cloned the sf gene (15Jeffery E.W. Hjerrild K.A. Paeper B. Clark L.B. Yasayko S.A. Wilkinson J.E. Galas D. Ziegler S.F. Ramsdell F. Brunkow M.E. Nat. Genet. 2001; 27: 68-73PubMed Google Scholar). The wild type sf gene (Foxp3) encodes a novel 48-kDa protein, scurfin (SFN). The protein is expressed at low levels, primarily in CD4 T cells (15Jeffery E.W. Hjerrild K.A. Paeper B. Clark L.B. Yasayko S.A. Wilkinson J.E. Galas D. Ziegler S.F. Ramsdell F. Brunkow M.E. Nat. Genet. 2001; 27: 68-73PubMed Google Scholar). Interestingly, SFN expression levels do not appear to be modulated following activation. The sfmutation is a 2-bp insertion that results in a premature stop codon. It is not known if the predicted truncated protein is stably expressed in mutants. However, overexpression of a transgene encoding the mutant form of the SFN gene in wild type mice yielded no phenotype (15Jeffery E.W. Hjerrild K.A. Paeper B. Clark L.B. Yasayko S.A. Wilkinson J.E. Galas D. Ziegler S.F. Ramsdell F. Brunkow M.E. Nat. Genet. 2001; 27: 68-73PubMed Google Scholar). This finding suggests that the sf phenotype most likely results from a loss of SFN function. The observations that females heterozygous for the sf mutation are phenotypically normal and display random X-inactivation also support this interpretation. Concurrent with the cloning of the sf gene, several reports of mutations in the human ortholog of the Foxp3 gene have been made (16Chatila T.A. Blaeser F. Ho N. Lederman H.M. Voulgaropoulos C. Helms C. Bowcock A.M. J. Clin. Invest. 2000; 106: R75-R81Crossref PubMed Scopus (737) Google Scholar, 17Wildin R.S. Ramsdell F. Peake J. Faravelli F. Casanova J.L. Buist N. Levy-Lahad E. Mazzella M. Goulet O. Perroni L. Dagna Bricarelli F. Byrne G. McEuen M. Proll S. Appleby M. Brunkow M.E. Wildin R.S. Nat. Genet. 2001; 27: 18-20Crossref PubMed Scopus (1481) Google Scholar, 18Bennett C.L. Christie J. Ramsdell F. Brunkow M.E. Ferguson P.J. Whitesell L. Kelly T.E. Saulsbury F.T. Chance P.F. Ochs H.D. Nat. Genet. 2001; 27: 20-21Crossref PubMed Scopus (2639) Google Scholar). Affected males present symptoms similar to those observed in sf mice and include a predilection to autoimmune diseases and allergy. The majority of the mutations in these patients also result in premature stop codons. Although the phenotype ofsf mice and of patients with the immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), or X-linked autoimmunity-allergic dysregulation syndrome (XLAAD) indicate a critical role for the foxp3 gene product in the negative regulation of T cell activation, it is not immediately evident how the protein exacts this effect. The most prominent structural feature of SFN is a forkhead/winged helix domain at the C-terminal end of the protein. Forkhead/winged helix domain-containing proteins are members of a rapidly growing family of DNA binding factors. Since the identification of the original forkhead (FKH) protein in Drosophila in the early 1990s, over 80 FKH family proteins, classified into 17 different subfamilies, have been described in a variety of species, including nematodes, yeast, and mammals (Ref. 19Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar and www.biology.pomona.edu/fox.html). Although both transcriptional activators and repressors have been identified in this family, FKH domain-containing proteins typically function in the regulation of lineage commitment and developmental differentiation (reviewed in Ref. 20Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (573) Google Scholar). SFN, with an FKH domain of only 84 amino acids, is an atypical winged helix family member. QRF-1, a previously described partial protein sequence (21Li C. Tucker P.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11583-11587Crossref PubMed Scopus (56) Google Scholar), contains a similarly truncated FKH domain. Li and Tucker (21Li C. Tucker P.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11583-11587Crossref PubMed Scopus (56) Google Scholar) have demonstrated that the FKH domain of QRF-1 is necessary and sufficient for binding to oligonucleotides containing DNA sequences described to bind FKH domain-containing proteins. The sequence identity between the FKH domain of QRF-1 and scurfin suggested that the latter would also function as a DNA binding protein. Another distinct feature of SFN is the location of the FKH domain at its C terminus. Generally, the FKH domain is located near the amino terminus of proteins in this family. This unique structural feature may be significant in the protein's function and help distinguish SFN from other FKH family proteins active in CD4 T cells. With the ultimate goal of identifying how SFN acts to regulate T cell function, we have carried out fundamental analyses of this protein. Here, we report that scurfin is a DNA binding protein that can repress transcription. The FKH domain is required for this activity. Complimentary to the observation of hyper-responsive T cells in mice carrying the inactivating sf mutation, and the description of T cell-mediated autoimmunity in humans with the IPEX or XLAAD syndrome, overexpression of scurfin attenuates the T cell activation response, as evidenced by reduced cytokine production in response to activating stimuli. The ability to bind DNA, i.e. inclusion of the FKH domain, is required for this effect. Although the exact mechanisms by which SFN regulates T cell activation remain to be elucidated, the findings presented here suggest that the ability of scurfin to act as a negative regulator of cytokine production by CD4 T cells may involve direct repression of NFAT-mediated transcription. cDNA encoding full-length human scurfin (amino acids 1–431) or a fragment lacking the FKH domain (amino acids 1–327) was inserted into the expression vector pIRES2-EGFP (CLONTECH, Palo Alto, CA), the GST fusion vector pGEX-4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ), and the tetracycline-responsive vector pREV-TRE (CLONTECH). A multimeric FKH binding site construct was created by annealing complementary oligonucleotides containing three tandem repeats of the FKH binding site V1P (sequence given below). Restriction sites were included at the ends of the oligonucleotides to facilitate subcloning of the multimer into the SV40 promoter-driven luciferase reporter, pGL-3 Promoter (Promega, Madison, WI) to create 3xFKH-luc. Sequencing confirmed all constructs to be correct and free of mutation. HEK 293T cells and COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS, penicillin, and streptomycin. The Jurkat subline D4 was maintained in RPMI 1640 supplemented with 10% FCS, penicillin, and streptomycin. Tetracycline-inducible (Teti) Jurkat clones were generated following neomycin selection of D4 cells transfected with pREV-Tet-On (CLONTECH). Co-expression of a Tet-responsive GFP construct allowed selection of a clone yielding minimal promoter activity in the absence of treatment with the tetracycline analog, doxycycline (CLONTECH). This clone, hereafter referred to as TO, was used to create Tetiexpressers of empty vector (TO.TRE), full-length scurfin (TO.SFN), or scurfin lacking the FKH domain (TO.ΔFKH). The full-length (SFN), deletion mutant (ΔFKH), or empty pIRES2-EGFP construct was transiently expressed in HEK 293T cells using a calcium phosphate transfection kit (5 Prime → 3 Prime, Inc., Boulder, CO). After 24 h of culture, ∼5000 cells/well were plated on poly-lysine-coated microscope slides and allowed to adhere overnight at 37 °C. 48 h post-transfection, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% saponin. Cells were stained with pre-immune sera (Pre) or polyclonal antisera raised against full-length human scurfin (αSFN), followed by goat α-rabbit IgG Alexa-568, the actin stain phalloidin-Alexa 488, and the DNA intercalating dye Toto-3 (all from Molecular Probes, Eugene, OR). After several phosphate-buffered saline washes, mount medium (50% glycerol in phosphate-buffered saline) was added to each well. A coverslip was then applied and sealed. The Laser Scanning Confocal Imaging System, MRC 1024 (Bio-Rad, Hercules, CA), was used to visualize subcellular localization of full-length and ΔFKH scurfin. Cytoplasmic and nuclear protein was isolated from HEK 293T transient transfectants as previously described (22Penix L. Weaver W.M. Pang Y. Young H.A. Wilson C.B. J. Exp. Med. 1993; 178: 1483-1496Crossref PubMed Scopus (178) Google Scholar). Protein concentration was determined by Bradford assay. Equal quantities of cytoplasmic and nuclear protein were resolved by polyacrylamide gel electrophoresis and transferred to nitrocellulose. The subcellular localization of scurfin was assessed by immunoblotting with αSFN, followed by detection with a chemiluminescent reagent. Single-stranded oligonucleotides containing the consensus FKH binding sites (21 and references therein) TTR-S (5′-TCG AGT TGA CTA AGT CAA TAA TCA GAA TCA G) and V1P (5′-TCA AAA ATA TTG AAG TGT TAT CAC ATA CAC), an irrelevant sequence (5′-ATG AAT ATG CAA ATC AGG TG), or putative FKH/NFAT composite binding sites in the human GM-CSF enhancer (5′-TCT GCC CTG CCA CAA CCC CAT CGG AGC CCC TGA GTC AGC ATG G), the human IL-2 promoter (5′-ATC AGA AGA GGA AAA ATG AAG GTA ATG TTT TTT CAG ACT GGT AA), or the NFAT multimeric reporter construct (5′-AAG AGG AAA ATT TGT TTC ATA CAG AAG GCG) were annealed with their complementary strand and used as cold competitors or radiolabeled probe in gel shift assays with Jurkat-derived nuclear protein. A dimer of the TTR-S oligonucleotide (5′-CTG AAT TCT GAT TAT TGA CTT AGT CAA CAT TCT GAT TAT TGA CTT AGT CAA C) and a different irrelevant competitor (5′-GAT TTA AAA GTG TGT CCC AGC AGC CCT GGT CCA GCC CTC T) were annealed with their complementary strand, filled in with Klenow, and used in EMSA assays performed with recombinant proteins. GST fusion proteins were affinity purified from isopropyl-1-thio-β-d-galactopyranoside-induced bacterial lysates in the presence of protease inhibitors using pre-swelled glutathione-agarose beads (Sigma Chemical Co., St. Louis, MO). Nuclear extracts were prepared from D4 cells as previously described (22Penix L. Weaver W.M. Pang Y. Young H.A. Wilson C.B. J. Exp. Med. 1993; 178: 1483-1496Crossref PubMed Scopus (178) Google Scholar). Nuclear protein (4 μg/lane) or a comparable amount of purified GST fusion protein was preincubated at room temperature with binding buffer supplemented with 500 ng of poly(dI-dC) (Roche Molecular Biochemicals, Indianapolis, IN) and a 50- to 100-fold molar excess of cold oligonucleotide competitor for 10 min (23Schubert L.A. King G. Cron R.Q. Lewis D.B. Aruffo A. Hollenbaugh D. J. Biol. Chem. 1995; 270: 29624-29627Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Probe (2 × 104 cpm, end-labeled with [γ-32P]ATP) was then added to a final volume of 25 μl, and the incubation continued for an additional 15 min at room temperature. In some reactions, rabbit pre-immune sera or antisera raised against full-length SFN was incubated with nuclear protein for 30 min on ice prior to addition of the probe. All reactions were analyzed by polyacrylamide gel electrophoresis using 4% gels in 0.027 m Tris borate, 0.6 mm EDTA buffer. The activity of the 3xFKH-luc or the pGL-3 Promoter luciferase reporter construct was assessed in COS-7 cells. Calcium phosphate transfection was used to transiently express the β-galactosidase control vector, pSV-β-gal (Promega), the luciferase reporter pGL-3 Promoter or 3xFKH-luc, and the SFN, ΔFKH, or empty pIRES2-EGFP expression construct in COS-7 cells. 48 h post-transfection, cells were harvested and lysates were prepared in 1× Reporter lysis buffer according to the manufacturer's specifications (Promega). Luciferase activity was measured in cellular lysates using the Luciferase assay system (Promega) and the EG & G Berthold Lumat 9507 luminometer (PerkinElmer Life Sciences, Boston, MA) according to the manufacturers' specifications. The β-galactosidase activity in duplicates of each lysate was determined using the β-galactosidase enzyme assay system (Promega) and used to normalize measured luciferase activity. The Jurkat subline D4 was transiently transfected with the expression construct encoding full-length human SFN or the parent vector as described previously (23Schubert L.A. King G. Cron R.Q. Lewis D.B. Aruffo A. Hollenbaugh D. J. Biol. Chem. 1995; 270: 29624-29627Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). After 24 h of culture in the absence or presence of 1 or 10 μg/ml CD3 antibody (OKT3), culture supernatants were collected. IL-2 secretion was evaluated by bioassay using the IL-2-dependent murine cell line HT-2 (9Clark L.B. Appleby M.W. Brunkow M.E. Wilkinson J.E. Ziegler S.F. Ramsdell F. J. Immunol. 1999; 162: 2546-2554PubMed Google Scholar). The activity of a luciferase reporter containing a multimer of a regulatory NFAT site (−280) from the murine IL-2 gene (NFAT-luc) (24Petrak D. Memon S.A. Birrer M.J. Ashwell J.D. Zacharchuk C.M. J. Immunol. 1994; 153: 2046-2051PubMed Google Scholar) or the SV40 promoter-driven luciferase reporter, pGL-3 Promoter, was assessed in the TO.TRE, TO.SFN, and TO.ΔFKH cell lines. Cells were cultured in 1 μg/ml doxycycline for 24 h prior to transfection. Equal concentrations (107 cells/0.4 ml) of each clone were electroporated with 10 μg of the control vector pSV-β-gal and either NFAT-luc or pGL-3 Promoter as previously described (23Schubert L.A. King G. Cron R.Q. Lewis D.B. Aruffo A. Hollenbaugh D. J. Biol. Chem. 1995; 270: 29624-29627Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Cells were rested for 2 h at 37 °C. Cell viability was determined by trypan blue exclusion, and 106 cells per well were cultured in the absence or presence of ionomycin (1.5 μm) and PMA (25 ng/ml) for 6 h. Cells were harvested, and lysates were prepared in 1× reporter lysis buffer. Luciferase activity was measured and normalized based on the β-galactosidase activity detected in duplicate lysates. Distinct sequences present in the FKH domain have been described to mediate nuclear localization and DNA binding by proteins containing this structural feature (25Hellqvist M. Mahlapuu M. Blixt A. Enerback S. Carlsson P. J. Biol. Chem. 1998; 273: 23335-23343Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To analyze scurfin localization, we first assessed the ability of transiently expressed scurfin to localize to the nucleus. A construct directing expression of scurfin (SFN), a fragment lacking the FKH domain (ΔFKH), or the parent vector pIRES2-EGFP (293) was transiently transfected into HEK 293T cells as described under “Experimental Procedures.” Scurfin localization was assessed at both the single cell level by confocal microscopy (Fig.1, A–D) and at the level of the transfected population by fractionation of total cellular protein into nuclear and cytoplasmic components and subsequent Western blotting (Fig. 1E). Scurfin (in red) predominantly localizes to the nucleus of HEK 293T cells (Fig. 1C). As predicted, removal of the FKH domain results in the exclusion of the majority of scurfin from the nucleus (Fig. 1D). The residual red staining observed in the nucleus of ΔFKH-transfected cells could reflect staining of scurfin endogenously expressed in HEK 293T cells or, more likely, cross-reactivity of the antisera with other FKH domain-containing proteins expressed in these cells. A similar, low level of reactivity with the SFN antisera is observed in the nucleus of cells transfected with vector alone (Fig. 1B). Finally, staining with the scurfin antisera is specific, because no detectable signal is observed when pre-immune sera is used for staining (Fig.1A). To demonstrate that the results observed by confocal microscopy were representative of the entire transfected population, cytoplasmic and nuclear protein were concomitantly isolated from transfectants. Equal quantities of cytoplasmic (c) and nuclear (n) protein were immunoblotted with scurfin antisera (Fig. 1E). As observed with confocal microscopy, the majority of scurfin (∼48 kDa) localizes to the nuclear protein fraction, and removal of the FKH domain (∼34-kDa band) results in its retention in the cytoplasm. These findings indicate that scurfin can localize to the nucleus and that the FKH domain is required for this to occur. Confocal microscopy and Western blotting of nuclear protein isolated from Jurkat cells both indicate that endogenously expressed SFN also localizes to the nucleus (data not shown). Based on the sequence identity between the FKH domain of the previously described partial protein QRF-1 and scurfin, we predicted that scurfin would form a complex with the same DNA sequences (21Li C. Tucker P.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11583-11587Crossref PubMed Scopus (56) Google Scholar). We tested the ability of both recombinant GST fusions with scurfin and endogenously expressed scurfin to bind to oligonucleotides containing these sequences. As shown in Fig.2A, a fusion of GST with full-length scurfin binds to a probe containing a dimer of the FKH consensus binding site from the TTR-S gene (Fig. 2A,lane 3). Addition of a 50-fold molar excess of cold competitor of the same sequence eliminates formation of the lowest band (Fig. 2A, lane 4), but inclusion of the same amount of an irrelevant DNA sequence does not (Fig. 2A,lane 5). Addition of polyclonal antisera raised against SFN in the gel shift reaction results in retardation of the lowest band (Fig. 2A, lane 6). This indicates that SFN is a primary component of the lowest DNA-protein complex and that the two higher bands may represent nonspecific binding activity. As expected, a fusion of GST with a deletion mutant of scurfin, which lacks the FKH domain, fails to form a complex with the probe (Fig. 2A,lane 2). Finally a fusion between GST and c-Jun, a protein known to bind a distinct DNA sequence, fails to form a complex with the TTR-S probe (Fig. 2A, lane 1). These results suggest that scurfin is a DNA binding protein that specifically complexes with a consensus FKH binding site and that the FKH domain is required for this activity. The FKH domain-dependent ability of recombinant scurfin to bind to DNA was not surprising. However, this finding did not prove that scurfin binds DNA under more physiologically relevant circumstances. Because CD4 T cells express scurfin and are most affected by the inactivating sf mutation, we next studied the DNA binding ability of endogenously expressed scurfin. Nuclear protein was extracted from Jurkat cells activated with αCD3 for 2 h and used in gel shift assays. As observed with recombinant full-length protein, endogenously expressed scurfin forms a complex with a probe that contains a FKH protein consensus DNA binding site from the V1P promoter (Fig. 2B, lanes 1 and5). A 50-fold molar excess of cold oligonucleotide competitor of the same DNA sequence as the probe (Fig. 2B,lane 2) or the FKH binding site from the TTR-S promoter (Fig. 2B, lane 3) competes away the protein/probe complex, indicating that a specific complex is formed. Furthermore, addition of an irrelevant DNA sequence does not disrupt complex formation (Fig. 2B, lane 4). The same findings were attained when the FKH binding site from the TTR-S promoter was used as the probe (data not shown). Inclusion of antisera raised against scurfin (Fig. 2B, lane 6), but not pre-immune sera (Fig. 2B, lane 7), results in retardation of the migration of the complex, demonstrating that scurfin is a major component of the protein-DNA complex. These findings confirm that scurfin is present in the nucleus of CD4 T cells and is capable of binding DNA. The same DNA binding activity is observed in nuclear protein isolated from unstimulated Jurkat cells (data not shown). Next we investigated the functional significance of the ability of scurfin to bind DNA. The FKH family of DNA binding factors contains transcriptional activators as well as repressors of transcription. The documented transactivation domains of other FKH family proteins are located C-terminal of the FKH domain (26Mahlapuu M. Pelto-Huikko M. Aitola M. Enerback S. Carlsson P. Dev. Biol. 1998; 202: 183-195Crossref PubMed Scopus (72) Google Scholar, 27Tang E.D. Nunez G. Barr F.G. Guan K., L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 28Schuddekopf K. Schorpp M. Boehm T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9661-9664Crossref PubMed Scopus (78) Google Scholar). As previously mentioned, the scurfin FKH domain is unique in that it ends only 11 amino acids from the C terminus of the protein. We hypothesized that SFN might lack a transactivation domain and possibly inhibit transcription. To address this question, we examined the effect of SFN on the expression of the SV40 minimal promoter reporter flanked by multiple FKH binding sites. COS-7 cells were transiently transfected with an expression construct directing the production of scurfin (SFN), a mutant lacking the FKH domain (ΔFKH), or the parent vector pIRES2-EGFP (Control). The control vector pSV-β-gal and either 3xFKH-luc (Fig. 3,speckled bars) or the parent luciferase reporter pGL-3 Promoter (Fig. 3, solid bars) were co-transfected with each expression construct. Lysates were harvested and luciferase and β-galactosidase activity measured 48 h later. Comparable levels of luciferase activity are detected in cells transfected with the pGL-3 Promoter, irrespective of the expression construct co-transfected (Fig.3, solid bars). Activity of the 3xFKH-lucconstruct in COS-7 cells transfected with the parent vector (Fig. 3,Control, speckled bar) and SFN lacking the DNA binding domain (Fig. 3, ΔFKH, speckled bar) is comparable. However, overexpression of full-length scurfin consistently results in more than a 50% reduction of transcription of the reporter gene (Fig. 3, SFN, speckled bar), suggesting that SFN can repress transcription. The observation that SFN overexpression does not affect promoter activity in the absence of FKH binding sites (Fig. 3, SFN, solid bar) and that removal of the DNA binding domain eliminates the reduction in expression of the 3xFKH-luc reporter gene (Fig. 3, ΔFKH,speckled bar) both indicate that this repression is specific and most likely mediated by the FKH domain. Although Fig. 3 shows an apparent reduction in expression of the 3xFKH-luc in ΔFKH-transfected cells, relative to that observed in control transfected cells, this trend has not been