Cyclophilin A and FKBP12 Interact with YY1 and Alter Its Transcriptional Activity
1995; Elsevier BV; Volume: 270; Issue: 25 Linguagem: Inglês
10.1074/jbc.270.25.15187
ISSN1083-351X
AutoresWen‐Ming Yang, Carla Inouye, Edward Seto,
Tópico(s)Virus-based gene therapy research
ResumoYY1 is a zinc finger transcription factor with unusual structural and functional features. In a yeast two-hybrid screen, two cellular proteins, cyclophilin A (CyPA) and FK506-binding protein 12 (FKBP12), interacted with YY1. These interactions are specific and also occur in mammalian cells. Cyclosporin A and FK506 efficiently disrupt the YY1-CyPA and YY1-FKBP12 interactions. Overexpression of human CyPA and FKBP12 have different effects on YY1-regulated transcription with these effects being promoter-dependent. These results suggest that immunophilins may be mediators in the functional role of YY1. YY1 is a zinc finger transcription factor with unusual structural and functional features. In a yeast two-hybrid screen, two cellular proteins, cyclophilin A (CyPA) and FK506-binding protein 12 (FKBP12), interacted with YY1. These interactions are specific and also occur in mammalian cells. Cyclosporin A and FK506 efficiently disrupt the YY1-CyPA and YY1-FKBP12 interactions. Overexpression of human CyPA and FKBP12 have different effects on YY1-regulated transcription with these effects being promoter-dependent. These results suggest that immunophilins may be mediators in the functional role of YY1. INTRODUCTIONYY1 is a zinc finger transcription factor that binds to a variety of cis DNA elements located in viral and cellular promoters. It has been implicated as a positive regulatory factor that binds to the ribosomal protein L30 and L32 gene promoters(1Hariharan N. Kelley D.E. Perry R.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9799-9803Crossref PubMed Scopus (245) Google Scholar), the B19 parvovirus P6 promoter(2Momoeda M. Kawase M. Jane S.M. Miyamura K. Young N.S. Kajigaya S. J. Virol. 1994; 68: 7159-7168Crossref PubMed Google Scholar), the transcriptional regulatory region of LINE-1(3Singer M.F. Krek V. McMillan J.P. Swergold G.D. Thayer R.E. Gene (Amst.). 1993; 135: 183-188Crossref PubMed Scopus (53) Google Scholar, 4Becker K.G. Swergold G.D. Ozato K. 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Pons G. Mader S.L. Park K. Atchison M.L. Walsh K. Mol. Cell. Biol. 1992; 12: 4209-4214Crossref PubMed Scopus (145) Google Scholar), the c-fos promoter(10Gualberto A. LePage D. Pons G. Mader S.L. Park K. Atchison M.L. Walsh K. Mol. Cell. Biol. 1992; 12: 4209-4214Crossref PubMed Scopus (145) Google Scholar, 11Natesan S. Gilman M.Z. Genes & Dev. 1993; 7: 2497-2509Crossref PubMed Scopus (210) Google Scholar), the β-casein gene promoter(12Raught B. Khursheed B. Kazansky A. Rosen J. Mol. Cell. Biol. 1994; 14: 1752-1763Crossref PubMed Google Scholar, 13Meier V.S. Groner B. Mol. Cell. Biol. 1994; 14: 128-137Crossref PubMed Google Scholar), the human cytomegalovirus major immediate early enhancer/promoter(14Liu R. Baillie J. Sissons J.G. Sinclair J.H. Nucleic Acids Res. 1994; 22: 2453-2459Crossref PubMed Scopus (115) Google Scholar), the rat serum amyloid A1 gene promoter(15Lu S.Y. Rodriguez M. Liao W.S. Mol. Cell. Biol. 1994; 14: 6253-6263Crossref PubMed Google Scholar), the human interferon-g promoter(16Ye J. Ghosh P. Cippitelli M. Subleski J. Hardy K.J. Ortaldo J.R. Young H.A. J. Biol. Chem. 1994; 269: 25728-25734Abstract Full Text PDF PubMed Google Scholar), the g and ∈ silencers(17Gumucio D.L. Heilstedt-Williamson H. Gray T.A. Tarle S.A. Shelton D.A. Tagle D.A. Slightom J.L. Goodman M. Collins F.S. Mol. Cell. Biol. 1992; 12: 4919-4929Crossref PubMed Google Scholar, 18Yost S.E. Shewchuk B. Hardison R. Mol. Cell. Biol. 1993; 13: 5439-5449Crossref PubMed Google Scholar, 19Peters B. Merezhinskaya N. Diffley J.F. Noguchi C.T. J. Biol. Chem. 1993; 268: 3430-3437Abstract Full Text PDF PubMed Google Scholar), and the Epstein-Barr virus BZLF1 gene promoter (20). Similar repression was reproduced when YY1-binding sites were placed upstream of a minimal promoter or the SV40 promoter/enhancer (21).In the immunoglobulin heavy chain enhancer, YY1 can act as a repressor or an activator(22Park K. Atchison M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9804-9808Crossref PubMed Scopus (295) Google Scholar); and, in the human papillomavirus type 18 promoter, a switch region determines the positive and negative action of YY1(23Bauknecht T. Angel P. Royer H.D. zur Hausen H. EMBO J. 1992; 11: 4607-4617Crossref PubMed Scopus (122) Google Scholar). In addition, while YY1 stimulates transcription by its ability to bind to the adeno-associated virus P5 and the cytochrome c oxidase subunit Vb initiators(24Seto E. Shi Y. Shenk T. Nature. 1991; 354: 241-245Crossref PubMed Scopus (337) Google Scholar, 25Basu A. Park K. Atchison M.L. Carter R.S. Avadhani N.G. J. Biol. Chem. 1993; 268: 4188-4196Abstract Full Text PDF PubMed Google Scholar), it inhibits transcription from the human immunodeficiency virus type 1 initiator (26).YY1 was first purified from HeLa cell nuclear extracts by sequence-specific DNA affinity chromatography and analyzed by protein microsequencing(21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar). cDNA encoding YY1 was obtained using probes generated from microsequencing information. The binding property of YY1 was also used as a means to screen expression libraries for cDNA clones encoding it(1Hariharan N. Kelley D.E. Perry R.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9799-9803Crossref PubMed Scopus (245) Google Scholar, 8Flanagan J.R. Becker K.G. Ennist D.L. Gleason S.L. Driggers P.H. Levi B.Z. Appella E. Ozato K. Mol. Cell. Biol. 1992; 12: 38-44Crossref PubMed Scopus (216) Google Scholar, 22Park K. Atchison M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9804-9808Crossref PubMed Scopus (295) Google Scholar). Functional studies of YY1 included mutation of its binding sites in a number of promoters or placing YY1-binding sites upstream of a variety of natural or synthetic promoters. Studies of cloned YY1 included the general strategies of expressing YY1 transiently in tissue culture cells or adding purified YY1 to an in vitro transcription extract and assaying the transcription from reporter gene promoters containing YY1-binding sites. So far, such approaches have demonstrated that YY1 activates and represses transcription of many different promoters, but little is known about its mechanism of action.The identification and characterization of proteins in a cell with which a given protein interacts is often helpful for understanding the function of that protein. Previously, we and others have used the yeast two-hybrid screen to identify cellular proteins that interact with YY1. Among the clones analyzed were genes encoding nucleolar phosphoprotein B23 (27Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar) and the oncoprotein c-Myc(28Shrivastava A. Saleque S. Kalpana G.V. Artandi S. Goff S.P. Calame K. Science. 1993; 262: 1889-1892Crossref PubMed Scopus (231) Google Scholar). Both the nucleolar phosphoprotein B23 and c-Myc can relieve YY1-induced transcriptional repression. In addition, c-Myc inhibits the activator functions of YY1. These results suggest that a possible mechanism for transcription regulation by YY1 is by interaction with these two cellular proteins.We have now sequenced and characterized additional clones from our two-hybrid screen for encoded proteins capable of binding to YY1. Two proteins were identified, cyclophilin A (CyPA)1( 1The abbreviations used are: CyPAcyclophilin AFKBP12FK506-binding protein 12CsAcyclosporin ADBDDNA-binding domainADactivation domainCATchloramphenicol acetyltransferaseCMVcytomegalovirus.) and FK506-binding protein 12 (FKBP12). CyPs and FKBPs comprise two families of ubiquitous and often abundant proteins conserved from prokaryotes to eukaryotes (reviewed in Refs. 29-40). CyPA was first identified and purified from bovine spleen on the basis of its high affinity for cyclosporin A (CsA)(41Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1442) Google Scholar). It is an abundant, cytosolic protein of 18 kDa found in all tissues in eukaryotic cells. It possesses rotamase activity, which enables it to catalyze the cis-trans isomerization of peptide bonds involving a prolyl residue, and might facilitate protein folding(42Takahashi N. Hayano T. Suzuki M. Nature. 1989; 337: 473-475Crossref PubMed Scopus (936) Google Scholar). It has been shown that CyPA binds the human immunodeficiency virus type 1 (HIV-1) Gag protein and is specifically incorporated into HIV-1 virion particles(43Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Cell. 1993; 73: 1067-1078Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 44Franke E.K. Yuan H.E.H. Luban J. Nature. 1994; 372: 359-362Crossref PubMed Scopus (645) Google Scholar, 45Thali M. Bukovsky A. Kondo E. Rosenwirth B. Walsh C.T. Sodroski J. Gottlinger H.G. Nature. 1994; 372: 363-365Crossref PubMed Scopus (559) Google Scholar). Although it has no sequence homology with CyPs, FKBPs are also abundant cytosolic proteins that possess cis-trans peptidyl-prolyl isomerase activities that are inhibited by FK506 but not by CsA (reviewed in Refs. 34 and 46). FKBP12 has been shown to interact with the type I receptors of the transforming growth factor-β family(47Wang T. Donahoe P.K. Zervos A.S. Science. 1994; 265: 674-676Crossref PubMed Scopus (310) Google Scholar), and two FKBP-related proteins are associated with progesterone receptor complexes(48Smith D.F. Baggenstoss B.A. Marion T.N. Rimerman R.A. J. Biol. Chem. 1993; 268: 18365-18371Abstract Full Text PDF PubMed Google Scholar). Also, FKBP25 is associated with nucleolin(49Jin Y.J. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7769-7773Crossref PubMed Scopus (112) Google Scholar), a major nucleolar phosphoprotein in exponentially growing cells. Both CyPA-CsA and FKBP12-FK506 complexes prevent T-cell response to antigen, bind and modulate activity of the protein phosphatase calcineurin(50Liu J. Farmer J.J. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3566) Google Scholar), and prevent nuclear import of a subunit of NFAT(51Flanagan W.M. Corthesy B. Bram R.J. Crabtree G.R. Nature. 1991; 352: 803-807Crossref PubMed Scopus (946) Google Scholar), a T-cell activation transcription factor.In this report, we present evidence that YY1 interacts specifically with CyPA and FKBP12 in both yeast and mammalian cells, suggesting that YY1 may be a common natural ligand for both immunophilins. Consistent with the ability of these immunophilins to interact with YY1, overexpression of CyPA or FKBP12 alters the transcription activity of YY1.MATERIALS AND METHODSPlasmidsThe following plasmids have previously been described: pAS-YY1(27Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar), which encodes a Gal4 DNA-binding domain (DBD)-human YY1 fusion protein; pAS-Rb2(52Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar), which encodes a Gal4 DBD-retinoblastoma fusion protein; pAS-SNF1(52Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar), which encodes a Gal4 DBD-yeast SNF1 fusion protein; Y14(27Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar), which encodes a Gal4 activation domain (AD)-CyPA fusion protein; Y20(27Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar), which encodes a Gal4 AD-FKBP fusion protein; pG5E1BCAT(53Lillie J.W. Green M.R. Nature. 1989; 338: 39-44Crossref PubMed Scopus (471) Google Scholar), which contains five Gal4-binding sites upstream of the adenovirus E1B TATA box and the chloramphenicol acetyltransferase (CAT) reporter gene; pGal4-RB(52Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar), which contains the DBD (amino acids 1-147) of the Gal4 protein fused to the retinoblastoma protein under the control of the simian virus 40 (SV40) early promoter/enhancer; pYY1/VP16(27Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar), which expresses the full-length YY1 protein fused to the acidic activating domain of the herpes simplex virus-1 VP16 (amino acids 413-490); pHCMV-LAP348(54Baim S.B. Labow M.A. Levine A.J. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5072-5076Crossref PubMed Scopus (91) Google Scholar), which contains a modified lac repressor gene fused to the VP16 acidic activation domain under the control of the human cytomegalovirus (CMV) immediate early promoter; pP5-60SVECAT and pP5-60(mt2)SVECAT(21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar), which carry wild-type or mutant YY1-binding sites 5′ to the SV40 enhancer and CAT gene; pSG424(55Sadowski I. Ptashne M. Nucleic Acids Res. 1989; 17: 7539Crossref PubMed Scopus (470) Google Scholar), which contains the Gal4 DBD under the control of the SV40 promoter/enhancer; pGal4-YY1(21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar), which contains the Gal4 DBD fused to YY1 cDNA; and pGal4TKCAT(21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar), which contains five Gal4-binding sites upstream of the thymidine kinase TATA box in plasmid pBLCAT2(56Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar).pGH289 (obtained from Gary Hayward, Johns Hopkins University) contains the full-length VP16 driven by the CMV promoter; pGal4DBD-CyPA was constructed by taking the human CyPA cDNA, an EcoRI fragment from pGCyPA(43Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Cell. 1993; 73: 1067-1078Abstract Full Text PDF PubMed Scopus (700) Google Scholar), and ligating it to the EcoRI site of pSG424. pGal4DBD-FKBP was constructed by first isolating an EcoRI fragment from pJDB-GAP-FKBP-A (57Maki N. Sekiguchi F. Nishimaki J. Miwa K. Hayano T. Takahashi N. Suzuki M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5440-5443Crossref PubMed Scopus (111) Google Scholar) and subcloning it into pGEM3Z (Promega) and then using EcoRI and XbaI to isolate the FKBP12 cDNA and subcloning the final fragment into pSG424. pGal4AD-YY1 was constructed by first subcloning the Gal4 AD from pGAD424 (52Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar) into pcDNAI/Amp (Invitrogen) and then fusing the YY1 cDNA in-frame with the activation domain downstream from the CMV promoter. pGal4AD-YY1Δ is identical to pGal4AD-YY1, with the exception that nucleotides 1236-1513 were deleted from the YY1 cDNA, resulting in expression of a Gal4 fusion with YY1 amino acids 1-331. pGal4DBD-LR was constructed by placing the human laminin receptor (amino acids 27-295) (58Yow H.K. Wong J.M. Chen H.S. Lee C.G. Davis S. Steele G.J. Chen L.B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6394-6398Crossref PubMed Scopus (177) Google Scholar) downstream of and in-frame with the Gal4 DBD. pCMV-CyPA was constructed by ligation of an EcoRI fragment from pGCyPA into pcDNAI/Amp. pCMV-FKBP was similarly constructed by taking an EcoRI fragment from pJDB-GAP-FKBP-A and subcloning it into pcDNAI/Amp. pSG5-YY1 was constructed by taking pYY1/VP16 and digesting it with BglII and then religating the plasmid to remove the VP16 sequence. All recombinants were verified by dideoxy sequencing.Yeast Two-hybrid Interaction ExperimentsTo assay systematically the pairwise interactions, library-derived Y14 or Y20 plasmids were transformed into Y153 alone or Y153 harboring pAS-YY1, pAS-Rb2, or pAS-SNF1 as described(27Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar, 52Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar). Transformants were assayed for the presence of β-galactosidase activity using the colony filter lift method(59Breeden L. Nasmyth K. Cold Spring Harbor Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (467) Google Scholar).DNA Transfections and CAT AssaysHeLa cells were grown on 60-mm tissue culture dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Transfections were performed using the calcium phosphate method(60Wigler M. Pellicer A. Silverstein S. Axel R. Cell. 1978; 14: 725-731Abstract Full Text PDF PubMed Scopus (775) Google Scholar). Forty-eight hours after transfection, cells were lysed by repeated freeze/thaw cycles and extracts assayed for CAT activity by thin-layer chromatography (61Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5288) Google Scholar) and quantified with the PhosphorImager (Molecular Dynamics). To determine the effects of CsA and FK506 on YY1-immunophilin interaction, different amounts of CsA or FK506 (1 mg/ml stock in 50% phosphate-buffered saline and ethanol) were added to the culture media 4-8 h after transfection.RESULTSY14 and Y20 Are Identical to CyPA and FKBP12Previously, we have used the yeast two-hybrid screen to detect mouse cDNAs coding for products that interact with YY1(27Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar). Of approximately 106 transformants, 14 clones showed specificity in binding to YY1 but not Rb or the yeast SNF1 protein. Preliminary DNA sequence analysis revealed eight novel sequences and six that were highly homologous to known sequences. Of the six known sequences, we have now completely sequenced two additional cDNA inserts designated Y14 and Y20. The Y14 clone is identical to the previously published mouse cyclophilin cDNA sequence (nucleotides 289-679; Ref. 62 (GenBank accession number X52803)). The insert in this clone begins at the 84th codon of CyPA and continues through the open reading frame past the 3′-noncoding sequences of the gene. The Y20 clone is identical to the previously published mouse FK506-binding protein (nucleotides 50-849; Ref. 63 (GenBank accession number X60203)). The insert in Y20 begins in the 5′-untranslated portion of the FKBP RNA 47 nucleotides before the initiator methionine and continues through the entire open reading frame past the noncoding sequences at the 3′-end of the gene.Confirmation That Y14 and Y20 Are Dependent on YY1 Hybrid ExpressionTo confirm that the phenotype observed in the original screen was reproducible and dependent on the YY1 hybrid, Y14 and Y20 plasmids were recovered and transformed into Y153 either alone or with test fusions in pAS1. As shown in Fig. 1A, transformants were assayed and β-galactosidase activities were positive only in the presence of pAS-YY1.CyPA or FKBP12 Fused to the Gal4 DNA-binding Domain Can Support Transcriptional Activation Mediated by a YY1-Gal4 Activation Domain Fusion ProteinIf a true interaction exists between YY1 and CyPA or FKBP12, this interaction should occur independent of the Gal4 DBD and the Gal4 AD. Specifically, activation of a reporter gene should still occur if the two Gal4 domains are switched. In addition, since YY1 is a mammalian transcription factor, it is important to confirm that this interaction occurs in mammalian cells. To address this issue, we fused human CyPA or FKBP12 to the Gal4 DBD, Gal4(1-147), and tested, in HeLa cell cotransfection assays, the ability of each Gal4 fusion protein to support Gal4 AD-YY1-mediated transcriptional activation using reporter constructs that contained Gal4-binding sites (Fig. 1B).Fig. 1C shows that neither the Gal4 DBD-CyPA fusion or the Gal4 AD-YY1 fusion significantly activated the reporter gene (lanes2 and 4), but the two proteins together activated CAT activity 10-fold (lane3). A deletion mutation of amino acids 332-414 in YY1 did not have any effect on the interaction (lane5). Similarly, a Gal4 DBD-FKBP12 fusion and the Gal4 AD-YY1 fusion together activated the CAT reporter gene (lane8). However, unlike the YY1-CyPA interaction, deletion of amino acids 332-414 in YY1 reduced the interaction of YY1-FKBP12 significantly (compare lanes8 and 9). These data confirm that YY1 binds CyPA and FKBP12 and further suggest that the YY1 carboxyl-terminal domain is required for its interaction with FKBP12 but not with CyPA.As with all experiments presented in this paper, in place of using an internal control (which often gets activated or repressed by YY1, CyPA, or FKBP12), each experiment shown is representative of at least three (and sometimes as many as 20) similar experiments, and all results were reproduced with more than one transfection reagent and DNA preparation.Additional Evidence for Interaction between YY1 and CyPA or FKBP12If the ability of Gal4 DBD-CyPA or Gal4 DBD-FKBP12 to support Gal4 AD-YY1 is specific, then other non-related Gal4 DBD fusion proteins should not support a YY1-AD fusion response. Furthermore, a Gal4 DBD-CyPA or Gal4 DBD-FKBP12 should not activate transcription in the presence of other non-related AD fusion proteins. In particular, we found that a Gal4 DBD-CyPA or Gal4 DBD-FKBP12 can synergize with a YY1 protein fused to the AD of VP16 (Fig. 2, B, lane4 and C, lane4), but neither the Gal4 DBD-RB nor Gal4 DBD-LR can activate transcription with YY1-VP16AD (Fig. 2, B, lanes6 and 8 and C, lanes6 and 8). Also, a Lac I-VP16AD fusion or an entire VP16 protein cannot stimulate transcription with Gal4 DBD-CyPA or with Gal4 DBD-FKBP12 (Fig. 2, B, lanes10 and 12 and C, lane10). Taken together, these results argue that the interaction between YY1 and CyPA or FKBP12 is highly specific.Figure 2:CyPA- or FKBP12-mediated transcriptional activation by activation domain-YY1 fusion is highly specific. A, schematic drawing of plasmids used in transfection assays. B and C, HeLa cells were cotransfected with 5 μg each of the indicated plasmids. All transfections were normalized to equal amounts of DNA with parental expression vectors. Transfection and CAT assays were done as described under “Materials and Methods” and for Fig. 1, except that smaller amount of extracts and a lower reaction time were used to keep the assay linear. The results are the mean ± S.D. from at least three separate transfections.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CsA and FK506 Inhibit CyPA-YY1 and FKBP12-YY1 Interactions, RespectivelyThe immunosuppressive drug CsA, a cyclic undecapeptide of fungal origin, has been shown to bind CyPA with high affinity (reviewed in Refs. 29-40). This binding results in an inhibition of peptidyl-prolyl cis-trans isomerase activity. FK506 is a macrolide (cyclic ester) that is structurally distinct from CsA but also blocks T-cell activation in the same way as CsA (reviewed in Refs. 34 and 46). FK506 does not bind to CyPA, but it does bind to FKBP12. Both CsA-CyPA and FK506-FKBP12 bind to and inhibit protein phosphatase 2B, the cellular target of calcineurin. To determine whether CsA has any effect on YY1-CyPA interaction, we transfected pGal4DBD-CyPA, pYY1/VP16, and pG5E1BCAT into HeLa cells and added different amounts of CsA into the media. As shown in Fig. 3A, CsA inhibited the YY1-CyPA interaction in a dose-dependent manner. Similarly, FK506 inhibited the interaction between YY1 and FKBP12 (Fig. 3B). In both of these experiments, the concentrations of CsA and FK506 are below the cellular toxicity levels(64Berger R. Majdic O. Meingassner J.G. Knapp W. Immunopharmacology. 1982; 5: 123-127Crossref PubMed Scopus (16) Google Scholar, 65Reem G.H. J. Autoimmun. 1992; 5: 159-165Crossref PubMed Scopus (11) Google Scholar).Figure 3:CsA and FK506 block binding of YY1 to CyPA and FKBP12, respectively. Transfections of pGal4DBD-CyPA (A and C), pGal4DBD-FKBP (B and C), pGal4DBD-VP16 (C), pYY1/VP16 (A-C), and pG5E1BCAT (A-C) into HeLa cells and CAT assays were performed as described for Fig. 2. CsA or FK506 was added to the culture medium 4-8 h after transfection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To be certain that the inhibition of interaction observed is not due to a general inhibitory effect of the two drugs, we repeated the transfection experiments but switched the two drugs. Specifically, we transfected HeLa cells with pG5E1BCAT, pGal4DBD-CyPA, and pYY1/VP16, followed by treatment with 1000 ng/ml FK506 but not CsA. As expected, FK506 did not affect CyPA-YY1 interaction (Fig. 3C, lane9). Similarly, CsA did not affect FKBP-YY1 interaction because cells treated with 500 ng/ml CsA and transfected with pG5E1BCAT, pGal4DBD-FKBP, and pYY1/VP16 did not result in a reduction of CAT activity (Fig. 3C, lane5). Furthermore, neither 500 ng/ml CsA nor 1000 ng/ml FK506 inhibited activation of pG5E1BCAT by Gal4 DBD-VP16 (Fig. 3C, lanes1-3). These two sets of experiments, then, strongly argue that inhibition of CyPA-YY1 interaction by CsA and inhibition of FKBP-YY1 interaction by FK506 is not due to a generally nonspecific effect of the two drugs.CyPA and FKBP12 Enhance Transcriptional Repression Mediated by YY1-binding SitesTo determine the effects of CyPA and FKBP12 on YY1-related transcription, plasmids pP5-60(mt2)SVECAT and pP5-60SVECAT were transfected into HeLa cells separately in the presence or absence of another plasmid expressing either CyPA or FKBP12. In agreement with our earlier study(21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar), the presence of YY1-binding sites 5′ to the transcriptional control region of the SV40 promoter/enhancer repressed transcription (Fig. 4, compare lanes1 and 4). Interestingly, overexpression of CyPA but not FKBP12 caused an increase in CAT activity in the absence of YY1-binding sites (compare lane1 to lanes2 and 3). However, in the presence of YY1-binding sites, both CyPA and FKBP12 repressed transcription (compare lane4 to lanes5 and 6). Subtracting the effects of CyPA and FKBP12 on the SV40 promoter/enhancer, there is a 4- and 20-fold reduction in CAT activity, respectively. In summary, overexpression of CyPA and FKBP12 represses transcription from the SV40 promoter/enhancer containing YY1-binding sites.Figure 4:Overexpression of CyPA or FKBP12 can repress transcription from YY1-binding sites. HeLa cells were cotransfected with 5 μg each of pP5-60(mt2)SVECAT (lanes 1-3) or pP5-60SVECAT (lanes 4-6) and pCMV-CyPA or pCMV-FKBP, as indicated. All transfections and CAT assays were done as described for Fig. 1 and under “Materials and Methods.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)FKBP12 Can Reverse Transcriptional Repression by YY1Previously, we have shown that a Gal4 DBD-YY1 fusion protein represses transcription when directed to a promoter containing Gal4-binding sites and that the adenovirus E1A or nucleolar phosphoprotein B23 can relieve the repression(21Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Abstract Full Text PDF PubMed Scopus (809) Google Scholar, 27Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar). To determine the effects of FKBP12 on YY1-induced transcriptional repression, we targeted YY1 to a promoter containing Gal4-binding sites and cotransfected a plasmid expressing FKBP12. As shown in Fig. 5B (lane3), the addition of a plasmid that expressed FKBP12 relieved the repression by YY1 and further activated CAT expression. This is somewhat surprising given the fact that in the previous experiment overexpression of FKBP12 can further repress a YY1-binding site (Fig. 4).Figure 5:FKBP12 can relieve YY1-induced transcriptional repression. A, schematic drawing of plasmids used in transfection assays. B, HeLa cells were cotransfected with 5 μg each of plasmids pSG424 expressing Gal4 DBD, pGal4-YY1 expressing Gal4 DBD-YY1, pCMV-FKBP expressing FKBP12, and pGal4TKCAT as indicated. All transfections and CAT assays were done as described for Fig. 1 and under “Materials and Methods.”View Large
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