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• W1530082693 abstract "p21SNFT (21-kDa small nuclear factor isolated from T cells) is a novel human protein of the basic leucine zipper family. The overexpression of p21SNFTleads to the significant and specific repression of transcription from the interleukin-2 promoter as well as from several essential activator protein 1 (AP-1)-driven composite promoter elements. One example is the distal nuclear factor of activated T cells (NF-AT)/AP-1 element where the AP-1 (Fos/Jun) basic leucine zipper heterodimer interacts with members of the NF-AT family. p21SNFT has been shown to replace Fos in dimerization with Jun on a consensus AP-1 binding site (12-O-tetradecanolyphorbol-13-acetate response element (TRE)) and to interact with Jun and NF-AT at the distal NF-AT/AP-1 enhancer element. A detailed biochemical analysis presented here compares interactions involving p21SNFT with those involving Fos. The results demonstrate that a p21SNFT/Jun dimer binds a TRE similarly to AP-1 and like AP-1 binds cooperatively with NF-AT at the NF-AT/AP-1 composite element. However, Fos interacts significantly more efficiently than p21SNFT with Jun and NF-AT, and the replacement of Fos by p21SNFT in the trimolecular complex drastically alters protein-DNA contacts. The data suggest that p21SNFT may repress transcriptional activity by inducing a unique conformation in the transcription factor complex. p21SNFT (21-kDa small nuclear factor isolated from T cells) is a novel human protein of the basic leucine zipper family. The overexpression of p21SNFTleads to the significant and specific repression of transcription from the interleukin-2 promoter as well as from several essential activator protein 1 (AP-1)-driven composite promoter elements. One example is the distal nuclear factor of activated T cells (NF-AT)/AP-1 element where the AP-1 (Fos/Jun) basic leucine zipper heterodimer interacts with members of the NF-AT family. p21SNFT has been shown to replace Fos in dimerization with Jun on a consensus AP-1 binding site (12-O-tetradecanolyphorbol-13-acetate response element (TRE)) and to interact with Jun and NF-AT at the distal NF-AT/AP-1 enhancer element. A detailed biochemical analysis presented here compares interactions involving p21SNFT with those involving Fos. The results demonstrate that a p21SNFT/Jun dimer binds a TRE similarly to AP-1 and like AP-1 binds cooperatively with NF-AT at the NF-AT/AP-1 composite element. However, Fos interacts significantly more efficiently than p21SNFT with Jun and NF-AT, and the replacement of Fos by p21SNFT in the trimolecular complex drastically alters protein-DNA contacts. The data suggest that p21SNFT may repress transcriptional activity by inducing a unique conformation in the transcription factor complex. Combinatorial regulation is a powerful mechanism enabling transcription to be tightly controlled. The expression and induction levels of various transcription factors are cell and tissue type-dependent, leading to highly context-specific activities that are also coordinated by cooperativity between factors and sequence-specific DNA affinities (1Kohler J.J. Schepartz A. Biochemistry. 2001; 40: 130-142Crossref PubMed Scopus (99) Google Scholar, 2Kerppola T.K. Curran T. Cell. 1991; 66: 317-326Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 3Halazonetis T.D. Georgopoulos K. Greenberg M.E. Leder P. Cell. 1988; 55: 917-924Abstract Full Text PDF PubMed Scopus (769) Google Scholar, 4Chen L. Curr. Opin. Struct. Biol. 1999; 9: 48-55Crossref PubMed Scopus (49) Google Scholar, 5Wolberger C. Curr. Opin. Genet. Dev. 1998; 8: 552-559Crossref PubMed Scopus (47) Google Scholar). T lymphocytes represent a well characterized model for inducible combinatorial regulation, and activation of several cytokines and other essential genes in T cells is controlled by such transcription factor interactions (6Bert A.G. Burrows J. Hawwari A. Vadas M.A. Cockerill P.N. J. Immunol. 2000; 165: 5646-5655Crossref PubMed Scopus (35) Google Scholar, 7Crabtree G.R. Science. 1989; 243: 355Crossref PubMed Scopus (913) Google Scholar, 8Jain J. Loh C. Rao A. Curr. Opin. Immunol. 1995; 7: 333-342Crossref PubMed Scopus (501) Google Scholar, 9Foletta V.C. Segal D.H. Cohen D.R. J. Leukocyte Biol. 1998; 63: 139-152Crossref PubMed Scopus (315) Google Scholar, 10Lenardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1259) Google Scholar, 11Bassuk A.G. Leiden J.M. Immunity. 1995; 3: 223-237Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 12Giese K. Kingsley C. Kirshner J.R. Grosschedl R. Genes Dev. 1995; 9: 995-1008Crossref PubMed Scopus (485) Google Scholar). Differential stimulation through the T cell receptor and various co-receptors leads to stimulus-dependent activation of factors, which selectively converge at promoter and enhancer elements to modulate transcription (13Paul W.E. Seder R.A. Cell. 1994; 76: 241-251Abstract Full Text PDF PubMed Scopus (1700) Google Scholar, 14Hughes C.C.W. Pober J.S. J. Biol. Chem. 1996; 271: 5369-5377Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Interactions between NF-AT 1The abbreviations used are: NF-AT, nuclear factor of activated T cells; IL, interleukin; SNFT, small nuclear factor isolated from T cells; AP, activator protein; Ds, specific unbound DNA; PDs, specific bound DNA; Dn, nonspecific sites; bZIP, basic leucine zipper; TRE, 12-O-tetradecanolyphorbol 13-acetate response element; GFP, green fluorescent protein; EMSA, electrophoretic mobility shift assay; MMP-1, matrix metalloproteinase 1, MPE, methedium propyl EDTA; GST, glutathioneS-transferase. and AP-1 family members at composite NF-AT/AP-1 binding elements are among the most common and highly studied examples of combinatorial regulation in T cells (15Diebold R.J. Rajaram N. Leonard D.A. Kerppola T.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7915-7920Crossref PubMed Scopus (36) Google Scholar, 16Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (681) Google Scholar, 17Chen L. Glover J.N.M. Hogan P.G. Rao A. Harrison S.C. Nature. 1998; 392: 42-48Crossref PubMed Scopus (414) Google Scholar, 18Ramirez-Carrozi V.R. Kerppola T.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4893-4898Crossref PubMed Scopus (33) Google Scholar, 19Macian F. Lopez-Rodriguez C. Rao A. Oncogene. 2001; 20: 2476-2489Crossref PubMed Scopus (623) Google Scholar). NF-AT/AP-1 interactions, which are often cooperative, strongly influence the production of many cytokines required in T cells including IL-2, IL-3, IL-4, and granulocyte macrophage colony-stimulating factor (20Rooney J.W. Hoey T. Glimcher L.H. Immunity. 1995; 2: 473-483Abstract Full Text PDF PubMed Scopus (238) Google Scholar, 21Cockerill P.N. Bert A.G. Jenkins F. Ryan G.R. Shannon M.F. Vadas M.A. Mol. Cell. Biol. 1995; 15: 2071-2079Crossref PubMed Scopus (116) Google Scholar, 22Macian F. Garcia-Rodriguez C. Rao A. EMBO J. 2000; 19: 4783-4795Crossref PubMed Scopus (267) Google Scholar). IL-2 is the major mitogenic cytokine produced in T lymphocytes in response to antigenic stimulation through the T cell receptor and coreceptor CD28. This activation is an essential event in the T cell-mediated immune response, leading to both clonal expansion of the T cell and activation of other cell types involved in the response (7Crabtree G.R. Science. 1989; 243: 355Crossref PubMed Scopus (913) Google Scholar,23Cantrell D.A. Smith K.A. Science. 1984; 224: 1312-1316Crossref PubMed Scopus (719) Google Scholar, 24Robb R.J. Immunol. Today. 1984; 5: 203-209Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 25Smith K.A. Immunol. Rev. 1980; 51: 337-357Crossref PubMed Scopus (632) Google Scholar). IL-2 activity is largely controlled at the level of transcription through convergence of transcription factors at the proximal 300-bp region of the IL-2 promoter (7Crabtree G.R. Science. 1989; 243: 355Crossref PubMed Scopus (913) Google Scholar, 8Jain J. Loh C. Rao A. Curr. Opin. Immunol. 1995; 7: 333-342Crossref PubMed Scopus (501) Google Scholar, 23Cantrell D.A. Smith K.A. Science. 1984; 224: 1312-1316Crossref PubMed Scopus (719) Google Scholar, 24Robb R.J. Immunol. Today. 1984; 5: 203-209Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 25Smith K.A. Immunol. Rev. 1980; 51: 337-357Crossref PubMed Scopus (632) Google Scholar, 26Garrity P.A. Chen D. Rothenberg E.V. Wold B.J. Mol. Cell. Biol. 1994; 14: 2159-2169Crossref PubMed Scopus (129) Google Scholar). This region contains several AP-1 composite elements at which AP-1 proteins interact with transcription factors from other families (8Jain J. Loh C. Rao A. Curr. Opin. Immunol. 1995; 7: 333-342Crossref PubMed Scopus (501) Google Scholar, 27Siebenlist U. Durand D.B. Bressler P. Holbrook N.J. Norris C.A. Kamoun M. Kant J.A. Crabtree G.R. Mol. Cell. Biol. 1986; 6: 3042-3049Crossref PubMed Scopus (121) Google Scholar). The distal NF-AT/AP-1 enhancer element of the human promoter is regulated by highly cooperative interactions between AP-1 and NF-AT proteins (6Bert A.G. Burrows J. Hawwari A. Vadas M.A. Cockerill P.N. J. Immunol. 2000; 165: 5646-5655Crossref PubMed Scopus (35) Google Scholar,8Jain J. Loh C. Rao A. Curr. Opin. Immunol. 1995; 7: 333-342Crossref PubMed Scopus (501) Google Scholar, 15Diebold R.J. Rajaram N. Leonard D.A. Kerppola T.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7915-7920Crossref PubMed Scopus (36) Google Scholar, 22Macian F. Garcia-Rodriguez C. Rao A. EMBO J. 2000; 19: 4783-4795Crossref PubMed Scopus (267) Google Scholar, 28Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 29Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar, 30Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar). Although NF-AT is able to bind this element alone, the presence of AP-1 greatly stabilizes the interaction (31Sun L.J. Peterson B.R. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4919-4924Crossref PubMed Scopus (42) Google Scholar, 32Jain J. Loh C. Rao A. J. Immunol. 1993; 151: 837-848PubMed Google Scholar). The AP-1 binding element is a low affinity site and neither Fos/Jun heterodimers nor Jun homodimers bind easily unless recruited by NF-AT (16Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (681) Google Scholar). In contrast, Fos/Jun heterodimers and Jun homodimers readily bind a consensus TRE (12-O-tetradecanolyphorbol-13-acetate response element) in the absence of other proteins (33Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar). It has recently been shown that the overexpression of p21SNFT leads to the specific repression of both human IL-2 promoter activity and the production of IL-2 by activated Jurkat cells (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar). p21SNFT is constitutively expressed, and several lines of evidence strongly indicate that it is able to replace Fos in dimerization with Jun on AP-1 binding sites (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar). To develop an understanding of its mechanism of activity, heterodimeric complexes formed by p21SNFT with Jun were compared in detail to c-Fos/c-Jun heterodimers. Like several known bZIP transcription factors including Fos and Jun family members (35Echlin D.R. Tae H.J. Mitin N. Taparowsky E.J. Oncogene. 2000; 19: 1752-1763Crossref PubMed Scopus (85) Google Scholar, 36Aronheim A. Zandi E. Hennemann H. Elledge S.J. Karin M. Mol. Cell. Biol. 1997; 17: 3094-3102Crossref PubMed Scopus (391) Google Scholar, 37Liu J.L. Lee L.F., Ye, Y. Qian Z. Kung H.J. J. Virol. 1997; 71: 3188-3196Crossref PubMed Google Scholar), p21SNFTlocalized to the nucleus. Additionally, interactions between a p21SNFT/Jun dimer and NF-AT at the human distal NF-AT/AP-1 enhancer were shown to be highly cooperative similar to those of AP-1 proteins with NF-AT. However, a NF-AT/AP-1 complex formed more efficiently and footprinted DNA differently than a complex containing p21SNFT in place of Fos. The results suggest that a ternary complex containing p21SNFT may be conformationally distinct from one containing Fos and may explain the repression of NF-AT/AP-1 activity observed in the presence of this protein. The IL-2 luciferase (38McGuire K.L. Iacobelli M. J. Immunol. 1997; 159: 1319-1327PubMed Google Scholar), pCI/SNFT (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar), pNFATpx5 (39Jain J. Burgeon E. Badalian T.M. Hogan P.G. Rao A. J. Biol. Chem. 1995; 270: 4138-4145Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and GST-SNFT (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar) constructs have been described previously. The GFP-SNFT construct was cloned by digesting pCI/SNFT withNotI/SalI and blunting the NotI site with T4 DNA Polymerase (New England Biolabs, Beverly, MA). The resulting fragment was ligated in the appropriate reading frame into a modified pEGFP-N2 vector (CLONTECH, Inc., Palo Alto, CA) digested withSmaI/SalI. The His6-Fos and His6-Jun purification constructs pQE30-Fosdb and pQE32-Jundb contain the DNA binding domains of human c-Fos (amino acids 59–211) and human c-Jun (amino acids 223–333) cloned into the pQE His6 expression plasmids from Qiagen, Inc (Valencia, CA). Transient transfection assays in Jurkat cells were performed as described previously (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar) using 5 μg of IL-2 luciferase construct and the indicated amounts of pCI/SNFT, GFP-SNFT, or pEGFP expression constructs. Cells were stimulated with 10 ng/ml phorbol 12-myristate 13-acetate and 1.5 μm ionomycin and harvested, and luciferase assays were performed as described previously (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar). 2 μg of pEGFP or GFP-SNFT DNA were used in transient transfection assays in HeLa cells using the LipofectAMINE Plus Kit (Invitrogen) according to manufacturer's protocol. Mock transfections included no DNA. 20 h posttransfection, cells were analyzed by fluorescence microscopy, and images were saved to disk using C-View for Windows (DVC Company, Austin, TX). Transfections were then stimulated with 50 ng/ml phorbol 12-myristate 13-acetate or left unstimulated, and images were taken 24 h poststimulation. Cell lysates were harvested for Western blots 24 h poststimulation. Lysate preparation and Western blots for p21SNFT were done as described previously (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar). Western blots for green fluorescent protein (GFP) were identical with the exceptions that the blocking buffer used was 5% milk/TBST (20 mm Tris, pH 7.6, 140 mm NaCl, 0.1% Tween 20), the primary antibody was a rabbit anti-full-length GFP antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) diluted 1:2000 in 5% milk/TBST, and incubation with this antibody was done overnight at 4 °C. GST-SNFT was purified as described previously (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar). DH5α Escherichia coli containing the pNFATpx5 plasmid expressing the DNA binding domain of NF-ATp were cultured in Luria Broth in the presence of 100 μg/ml ampicillin at 37 °C, 255 rpm to an A 600 of 0.6. Protein expression was induced with 0.5 mmisopropyl-β-d-thiogalactopyranoside for 3 h, and cells were harvested by centrifugation for 15 min at 5000 rpm at 4 °C. His6-NF-AT was purified to ∼90% according to the QiaExpressionist protocol for purification from bacterial cells under denaturing conditions (Qiagen) using 5-ml columns from the QiaExpress IV kit. pQE30-Fosdb and pQE32-Jundb were transformed into M15 E. coli cells, selected using 100 μg/ml ampicillin and 25 μg/ml kanamycin, and grown to an A 600 of 0.6. Proteins were purified to ∼80 and 90%, respectively, using the QiaExpressionist protocol for purification from mammalian cells under native conditions and 5-ml columns from the QiaExpress IV kit. The protocol was modified to include 40 mm imidazole in the wash buffer. To renature proteins, purified proteins were dialyzed against 20 mm HEPES, pH 7.4, 1 mm dithiothreitol, 100 mm NaCl, 2 mm EDTA, 20% glycerol, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, and 0.1 mg/ml phenylmethylsulfonyl fluoride. Protein purities were assessed using SDS-PAGE followed by Coomassie Blue staining. Activities were verified by EMSA analysis. Consensus TRE and NF-AT/AP-1 double-stranded oligonucleotide probes used for EMSA analysis were described previously (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar). Purified proteins were incubated for 15 min at room temperature in 25 μl of total volume of 25 mm Tris, pH 8.0, 50 mm KCl, 6.25 mm MgCl2, 0.5 mm EDTA, 10% glycerol, 1.3 mm dithiothreitol, and 10 μg/ml poly(dI·dC). 10,000–25,000-cpm probe (doubly end-labeled with [32P]dATP) was added to each, and reactions were incubated for an additional 15 min prior to gel loading. Samples were loaded onto a 6.6% native acrylamide gel and run at 170 V for 2.5–3.5 h in 0.5× Tris borate EDTA (45 mm Tris borate, 1 mm EDTA) running buffer. Gels were dried and exposed to Kodak X-Omat AR film (Eastman Kodak Co.) or PhosphorImager cassette (Amersham Biosciences). To determine the ratios of specific unbound DNA (Ds) to specific bound DNA (PDs), EMSA analyses were performed as described above using purified proteins. A protocol by Emerson et al. (40Emerson B.M. Lewis C.D. Felsenfeld G. Cell. 1985; 41: 21-30Abstract Full Text PDF PubMed Scopus (136) Google Scholar) was modified to calculate specificversus nonspecific DNA affinities for the conditions used. Labeled TRE or NF-AT/AP-1 site (specific DNA) was used at 216 pm, and proteins were used at the concentrations found to maximally shift this concentration of probe as described under “Results” dimerization studies. These parameters were held constant, and specific DNA (unlabeled) was titrated over several reactions to 108 nm. In addition, 0.01 μg/μl unlabeled double-stranded poly(dI·dC) (Amersham Biosciences) was included in each reaction. The concentration of nonspecific sites (Dn) is 0.01 μg/μl/660 μg/μmol (15.1 μm) in each reaction (∼140-fold greater than PDs + Ds). In EMSA analyses, the total concentration of specific sites (PDs + Ds) is equal to the input concentrations of labeled plus unlabeled specific DNA. EMSAs were exposed to PhosphorImager cassette. Free (unshifted) and bound (shifted) specific DNA were quantitated using ImageQuant software (Amersham Biosciences), and percent free versus percent bound was calculated. PDs and Ds were determined in Equations 1 and2. Ds=(concentration of specific sites)(%free)Equation 1 PDs=(concentration of specific sites)(%bound)Equation 2 The ratio of specific to nonspecific affinity (K r) is determined by Equation 3 (40Emerson B.M. Lewis C.D. Felsenfeld G. Cell. 1985; 41: 21-30Abstract Full Text PDF PubMed Scopus (136) Google Scholar), (PDs)(Dn)(Ds)=(Kr)(P0−PDs)Equation 3 where K r is identified as the negative slope of the line produced when these values are plotted. Footprinting probes were generated by cloning the matrix metalloproteinase 1 (MMP-1) or IL-2 promoter regions into the pCR2.1 TA cloning vector (Invitrogen). Singly end-labeled probes were prepared by digesting to produce a 5′ overhang filling in with [32P]dATP or dCTP and Klenow (New England Biolabs), and 3′ blunt-digesting to yield the appropriate fragments, which were subsequently gel purified. Binding reactions were similar to those in EMSAs using 25,000-cpm probe/sample. Following the reactions, 4 μl of RQ DNase I (Promega, Madison, WI) diluted 1:5 in 10 mmTris, pH 8.0, was added to the sample, and samples were incubated for 2 min at room temperature. Reactions were stopped in 25 μl of 200 mm NaCl, 20 mm EDTA, pH 8.0, 1% SDS, 50 μg/ml tRNA, and 150 μl of 0.3 m NaOAc, pH 5.5, 1% SDS, 50 μg/ml tRNA were added. MgCl2 was added to 10 mm final concentration, and reactions were ethanol-precipitated either immediately or following phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform extractions. The DNA was pelleted, counted, and resuspended to equal cpm/μl (∼5000) in 95% formamide, 20 mm EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol. Samples were boiled, and 5 μl each (∼25,000 cpm) were loaded to 1× Tris borate EDTA, 8% acrylamide, 42% urea-sequencing gels. Gels were run in 1× Tris borate EDTA at 1600 V for 2.5–4.5 h, dried, and exposed to PhosphorImager cassette. Sequencing reactions using T7 Sequenase version 2.0 DNA ddNTP sequencing kit (Amersham Life Sciences, Cleveland, OH) were run alongside to identify the sequences of footprinted regions. To match the sequence with the 5′ singly end-labeled probe, each sequencing primer was the reverse complement of the labeled strand such that the 5′ end of the primer matched the 3′ blunt-digested end of the probe. Consequently, each sequence read was the reverse complement of the digested footprinting probe. Singly end-labeled DNA probes were prepared, and binding reactions were performed as described above. Following the reactions, 7 μl of MPE reagent (100 μm MPE, 200 μmFe2+(NH4)2(SO4)2) and 2.5 μl of 0.1 m dithiothreitol were added, and the reactions were incubated for 7 min at room temperature. 150 μl of stop solution was added (70 mm EDTA, pH 8.0, 0.82m NH4OAc, 1% SDS, 50 μg/ml tRNA), and reactions were phenol:chloroform:isoamyl alcohol-extracted twice and then chloroform-extracted. 300 μl of 2.92 mNH4OAc was added followed by 500 μl of isopropyl alcohol. Following isopropyl alcohol precipitations, samples were counted and resuspended, and gel was run as described above. To determine the subcellular localization of p21SNFT, a p21SNFTexpression construct containing a N-terminal GFP tag, was generated and used in transient transfection assays in HeLa cells. Because HeLa cells are adherent and contain a relatively small nucleus, the nuclear and cytoplasmic areas can be easily distinguished. Cells transfected with either GFP (27 kDa) or GFP-SNFT (48 kDa) were examined under both phorbol 12-myristate 13-acetate-stimulated and unstimulated conditions, and fluorescence microscopy was used to locate GFP. Transfections were harvested, and the presence of GFP and GFP-SNFT was verified by Western blotting (Fig. 1 A). In Western blots probed for GFP (Fig. 1 A, top), both GFP (lanes 3 and 6) and GFP-SNFT (lanes 4and 7) are constitutively expressed in the appropriate transfectants but not in untransfected cells (lanes 2 and5). An identical blot probed for p21SNFT (Fig.1 A, bottom) confirms the presence of GFP-SNFT in cells transfected with this construct (lanes 4 and7). Fig. 1 B demonstrates that GFP-SNFT localizes to the nucleus in transfected cells (right panels), whereas GFP remains cytoplasmic (left panels). These data demonstrate that the presence of the p21SNFT protein causes translocation of GFP from the cytoplasm to the nucleus. The results suggest that like other bZIP transcription factors, endogenous p21SNFT is probably a nuclear protein. To determine the response of IL-2 promoter activity to GFP-tagged p21SNFT relative to untagged p21SNFT, transient transfection assays were performed in the Jurkat transformed T cell line. Cells were transfected with a luciferase construct driven by the proximal 300-bp region of the IL-2 promoter in the presence or absence of p21SNFT expression constructs. As shown in Fig.1 C, IL-2 promoter activity is 70–85% reduced in response to p21SNFT expression; the GFP-SNFT expression construct represses the IL-2 promoter comparably to the pCI/SNFT construct. In contrast, the GFP protein alone produces no significant repression. The data demonstrate that the repression observed is specifically attributed to the presence of p21SNFT and suggest that the 27-kDa N-terminal GFP tag does not affect the localization or activity of the p21SNFT protein. EMSA analyses were performed to demonstrate the in vitro interactions of p21SNFT with DNA and with other proteins. Bacterially expressed c-Fos and c-Jun bZIP domains and full-length p21SNFT were purified and examined for binding to a consensus TRE (Fig. 2 B).Lane 1 shows the migration of TRE probe in the absence of proteins, and lanes 2–4 demonstrate that all three proteins were used at concentrations insufficient to form homodimers under the given conditions. This is particularly important for Jun, which homodimerizes on a consensus TRE at higher concentrations. When Fos and Jun are present together, an AP-1 heterodimer forms as expected (lane 5). The titration of increasing amounts of p21SNFT into this complex results in the gradual appearance of a higher molecular weight complex, coinciding with a gradual decrease in the AP-1 dimer (lanes 6–8). Lane 9demonstrates that the higher molecular weight complex is formed by a p21SNFT/Jun dimer. The data are consistent with previous observations (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar) that p21SNFT can bind a TRE with Jun and strongly suggest that it can physically replace Fos in the AP-1 complex. In vitro interactions of p21SNFT were also assessed on the distal NF-AT/AP-1 enhancer element from the IL-2 promoter (Fig. 2 C). EMSA analysis was performed using purified bacterially expressed proteins as indicated. Fos, Jun, and p21SNFT were used at concentrations that do not form homodimers or heterodimers in the absence of NF-AT (lanes 2–7). In contrast, NF-AT binds alone (lane 8).Lanes 9–12 demonstrate that at the concentrations used, NF-AT does not interact with Fos, Jun, or p21SNFThomodimers or with Fos/p21SNFT heterodimers. This is particularly relevant for Jun (lane 10), because at higher Jun concentrations, a NF-AT/Jun/Jun complex forms at this site (Figs.4, 6, and 7) (16Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (681) Google Scholar). On the other hand, the same concentrations of NF-AT, Fos, and Jun incubated together produce a NF-AT/AP-1 complex (lane 13), whereas NF-AT, p21SNFT, and Jun produce a higher molecular weight complex (lane 14). If all four proteins are incubated together, the NF-AT/Fos/Jun and NF-AT/p21SNFT/Jun complexes are both apparent, but no new complex forms (lane 15), indicating that only one complex or the other can form on a single piece of DNA. These data support previous results suggesting that p21SNFT competes with Fos on the NF-AT/AP-1 distal enhancer for dimerization with Jun (34Iacobelli M. Wachsman W. McGuire K. J. Immunol. 2000; 165: 860-868Crossref PubMed Scopus (52) Google Scholar).Figure 6An NF-AT/p21SNFT/Jun complex footprints differently than a NF-AT/AP-1 complex. Purified proteins were used in DNase I footprinting analyses on the antisense strand (A) and the sense strand (B) of the human IL-2 promoter region (−314 to −219). The NF-AT and AP-1 binding elements of the distal NF-AT/AP-1 enhancer region located at −275 (sequences 5′-GGAAAA-3′ and 5′-TCATACA-3′, respectively) are marked on both strands. Jun (J) was titrated from 320 to 1280 nm in lanes 3–5 and held constant at 320 nm in lanes 6–14. p21SNFT(S) and Fos (F) were titrated from 61 to 976 nm and 55 to 440 nm, respectively, where indicated. NF-AT was used at 1.7 μm in lane 2and held constant at 256 nm in lanes 3–13.View Large Image Figure ViewerDownload (PPT)Figure 7The difference in footprint observed between NF-AT/AP-1 and NF-AT/p21SNFT/Jun complexes is confirmed using MPE:Fe. Purified proteins were used in MPE:Fe footprinting analyses on the antisense strand (A) and the sense strand (B) of the IL-2 promoter in binding reactions identical to those in Fig. 6. C, EMSA analysis of identical binding reactions was used to verify the formation of a NF-AT/p21SNFT/Jun complex under the conditions footprinted. NF-AT/probe (N), NF-AT/p21SNFT/Jun (N/S/J), and NF-AT/AP-1 (N/A) complexes are indicated.View Large Image Figure ViewerDownload (PPT) It is particularly significant that a p21SNFT/Jun dimer does not bind DNA in the absence of NF-AT at this site (lane 7) despite the fact p21SNFT and Jun will readily dimerize on a consensus TRE using proteins from the same preparations at these concentrations (Fig. 2 B). This finding demonstrates that interactions between the p21SNFT/Jun dimer and NF-AT are highly cooperative as has been shown for NF-AT and AP-1 (16Jain J. McCaffrey P.G. Miner Z. Kerppola T.K. Lambert J.N. Verdine G.L. Curran T. Rao A. Nature. 1993; 365: 352-355Crossref PubMed Scopus (681) Google Scholar). To compare the ability of Jun to dimerize with p21SNFTrelative to Fos or with itself, purified proteins were used in quantitative EMSA analyses. The absolute amounts of each protein used in these studies are dependent on the percent of each individual preparation that is active. The studies are based on the assumption that if more Jun from a single protein preparation is required to optimally dimerize with p21SNFT than with Fos, the overall ability of Jun to interact with p21SNFT is lower than its ability to interact with Fos. The first study compared the formation of p2" @default.
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• W1530082693 title "Correlation of Transcriptional Repression by p21SNFTwith Changes in DNA·NF-AT Complex Interactions" @default.
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