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BIBTEX RIS

Chemically Regulated Zinc Finger Transcription Factors

2000; Elsevier BV; Volume: 275; Issue: 42 Linguagem: Inglês

10.1074/jbc.m005108200

ISSN

1083-351X

Autores

Roger R. Beerli, Ulrich Schopfer, Birgit Dreier, Carlos F. Barbas,

Tópico(s)

RNA modifications and cancer

Resumo

Ligand-dependent transcriptional regulators were generated by fusion of designed Cys2-His2 zinc finger proteins and steroid hormone receptor ligand binding domains. To produce novel DNA binding domains, three-finger proteins binding specific 9-base pair sequences were constructed from modular building blocks. Fusion of these zinc finger proteins to a transcriptional activation domain and to modified ligand binding domains derived from either the estrogen or progesterone receptors yielded potent ligand-dependent transcriptional regulators. Together with optimized minimal promoters, these regulators provide 4-hydroxytamoxifen- or RU486-inducible expression systems with induction ratios of up to 3 orders of magnitude. These inducible expression systems are functionally independent, and each can be selectively switched on within the same cell. The potential use of zinc finger-steroid receptor fusion proteins for the regulation of natural promoters was also explored. A gene-specific six-finger protein binding an 18-base pair target sequence was converted into a ligand-dependent regulator by fusion with either two estrogen receptor ligand binding domains or one ecdysone receptor and one retinoid X receptor ligand binding domain. These single-chain receptor proteins undergo an intramolecular rearrangement, rather than intermolecular dimerization and are functional as monomers. Thus, the ability to engineer DNA binding specificities of zinc finger proteins enables the construction of ligand-dependent transcriptional regulators with potential for the regulation of virtually any desired artificial or natural promoter. It is anticipated that the novel chemically regulated gene switches described herein will find many applications in applied and basic research, where the specific modulation of gene expression can be exploited. Ligand-dependent transcriptional regulators were generated by fusion of designed Cys2-His2 zinc finger proteins and steroid hormone receptor ligand binding domains. To produce novel DNA binding domains, three-finger proteins binding specific 9-base pair sequences were constructed from modular building blocks. Fusion of these zinc finger proteins to a transcriptional activation domain and to modified ligand binding domains derived from either the estrogen or progesterone receptors yielded potent ligand-dependent transcriptional regulators. Together with optimized minimal promoters, these regulators provide 4-hydroxytamoxifen- or RU486-inducible expression systems with induction ratios of up to 3 orders of magnitude. These inducible expression systems are functionally independent, and each can be selectively switched on within the same cell. The potential use of zinc finger-steroid receptor fusion proteins for the regulation of natural promoters was also explored. A gene-specific six-finger protein binding an 18-base pair target sequence was converted into a ligand-dependent regulator by fusion with either two estrogen receptor ligand binding domains or one ecdysone receptor and one retinoid X receptor ligand binding domain. These single-chain receptor proteins undergo an intramolecular rearrangement, rather than intermolecular dimerization and are functional as monomers. Thus, the ability to engineer DNA binding specificities of zinc finger proteins enables the construction of ligand-dependent transcriptional regulators with potential for the regulation of virtually any desired artificial or natural promoter. It is anticipated that the novel chemically regulated gene switches described herein will find many applications in applied and basic research, where the specific modulation of gene expression can be exploited. ligand binding domain 4-hydroxytamoxifen DNA binding domain ecdysone receptor estrogen receptor maltose-binding protein ponasterone A progesterone receptor retinoid X receptor base pair(s) amino acid(s) bovine serum albumin enzyme-linked immunosorbent assay Designed transcription factors with defined target specificity and regulatory function could provide invaluable tools for basic and applied research and for gene therapy. Accordingly, the design of sequence-specific DNA binding domains has been the subject of intense interest for the last 2 decades. Of the many classes of DNA-binding proteins studied, the modular Cys2-His2 zinc finger DNA binding motif has shown the most promise for the production of proteins with tailored DNA binding specificity (1Desjarlais J.R. Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7345-7349Crossref PubMed Scopus (193) Google Scholar, 2Choo Y. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11163-11167Crossref PubMed Scopus (314) Google Scholar, 3Rebar E.J. Pabo C.O. Scienc. 1994; 263: 671-673Crossref PubMed Scopus (379) Google Scholar, 4Wu H. Yang W.-P. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 344-348Crossref PubMed Scopus (181) Google Scholar, 5Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (346) Google Scholar, 6Segal D.J. Dreier B. Beerli R.R. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2758-2763Crossref PubMed Scopus (366) Google Scholar, 7Segal D.J. Barbas III, C.F. Curr. Opin. Chem. Biol. 2000; 4: 34-39Crossref PubMed Scopus (60) Google Scholar). The novel architecture of this class of proteins provides for the rapid construction of gene-specific targeting devices. Polydactyl zinc finger proteins are most readily prepared by assembly of modular zinc finger domains recognizing predefined three-nucleotide sequences (6Segal D.J. Dreier B. Beerli R.R. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2758-2763Crossref PubMed Scopus (366) Google Scholar, 8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar, 9Beerli R.R. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1495-1500Crossref PubMed Scopus (303) Google Scholar). Polydactyl proteins may be assembled using variable numbers of zinc finger domains of varied specificity providing DNA-binding proteins that not only recognize novel sequences but also sequences of varied length. By combining six zinc finger domains, proteins have been produced that recognize 18 contiguous base pairs of DNA sequence, a DNA address sufficiently complex to specify any locus in the 4 billion-base pair human genome (or any other genome). Fusion of polydactyl zinc finger proteins of this type to activation or repression domains provides transcription factors that efficiently and specifically modulate the expression of both transgenes and endogenous genes (8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar, 9Beerli R.R. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1495-1500Crossref PubMed Scopus (303) Google Scholar). While the availability of designed transcription factors with tailored DNA binding specificities provides novel opportunities in transcriptional regulation, additional applications would be available to ligand-dependent transcription factors. Designer zinc finger proteins dependent on small molecule inducers would have a number of applications, both for the regulation of endogenous genes and for the development of inducible expression systems for the regulation of transgenes. Natural transcription factors are regulated by a number of different mechanisms, including posttranslational modification such as phosphorylation (10Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar, 11Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3330) Google Scholar), or by ligand binding. The prototype ligand-activated transcription factors are members of the nuclear hormone receptor family, including the receptors for sex steroids or adrenocorticoids (12Carson-Jurica M.A. Schrader W.T. O'Malley W. Endocr. Rev. 1990; 11: 201-220Crossref PubMed Scopus (738) Google Scholar, 13Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6276) Google Scholar). These receptors are held inactive in the absence of hormone, by association with a number of inactivating factors including hsp90 (14Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1514) Google Scholar). Upon ligand binding, nuclear hormone receptors dissociate from the inactivating complex, dimerize, and become able to bind DNA and activate transcription (12Carson-Jurica M.A. Schrader W.T. O'Malley W. Endocr. Rev. 1990; 11: 201-220Crossref PubMed Scopus (738) Google Scholar, 13Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6276) Google Scholar, 14Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1514) Google Scholar). Significantly, not only hormone binding but also inactivation and dimerization functions reside within the ligand binding domain (LBD)1 of these proteins (15Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2838) Google Scholar). This fact has been exploited experimentally and steroid hormone receptor LBDs have found wide use as tools to render heterologous proteins hormone-dependent. In particular, the estrogen receptor (ER) LBD has been used to render the functions of c-Myc (16Eilers M. Picard D. Yamamoto K.R. Bishop J.M. Nature. 1989; 340: 66-68Crossref PubMed Scopus (388) Google Scholar), c-Fos (17Superti-Furga G. Bergers G. Picard D. Busslinger M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5114-5118Crossref PubMed Scopus (78) Google Scholar), and even the cytoplasmic kinase c-Raf (18Samuels M.L. Weber M.J. Bishop J.M. McMahon M. Mol. Cell. Biol. 1993; 13: 6241-6252Crossref PubMed Scopus (322) Google Scholar) hormone-dependent. To develop an inducible expression system for use in basic research and gene therapy, the availability of ligand-dependent transcriptional regulators is a prerequisite. Preferentially, these regulators would be activated by a small molecule inducer with no other biological activity, bind specific sequences present only in the target promoter, and have low immunogenicity. A number of ligand-regulated artificial transcription factors have been generated by various means, using functional domains derived from either prokaryotes (19Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4214) Google Scholar, 20Gossen M. Freundlieb S. Bender G. Müller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2011) Google Scholar, 21Labow M.A. Baim S.B. Shenk T. Levine A.J. Mol. Cell. Biol. 1990; 10: 3343-3356Crossref PubMed Scopus (79) Google Scholar, 22Baim 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) or eukaryotes (23Christopherson K.S. Mark M.R. Bajaj V. Godowski P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6314-6318Crossref PubMed Scopus (140) Google Scholar, 24No D. Yao T.-P. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3346-3351Crossref PubMed Scopus (752) Google Scholar, 25Wang Y. O'Malley Jr., B.W. Tsai S. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8180-8184Crossref PubMed Scopus (391) Google Scholar, 26Wang Y. Xu J. Pierson T. O'Malley B.W. Tsai S.Y. Gene Ther. 1997; 4: 432-441Crossref PubMed Scopus (75) Google Scholar, 27Braselmann S. Graninger P. Busslinger M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1657-1661Crossref PubMed Scopus (141) Google Scholar, 28Louvion J.F. Havaux-Copf B. Picard D. Gene (Amst. ). 1993; 131: 129-134Crossref PubMed Scopus (138) Google Scholar, 29Rivera V.M. Clackson T. Natesan S. Pollock R. Amara J.F. Keenan T. Magari S.R. Phillips T. Courage N.L. Cerasoli Jr., F. Holt D.A. Gilman M. Nat. Med. 1996; 2: 1028-1032Crossref PubMed Scopus (453) Google Scholar). Of the functional domains derived from eukaryotic proteins, nuclear hormone receptor LBDs have been the most widely used. In particular, regulators based on the Gal4 DNA binding domain (DBD) fused to a human ER (27Braselmann S. Graninger P. Busslinger M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1657-1661Crossref PubMed Scopus (141) Google Scholar, 28Louvion J.F. Havaux-Copf B. Picard D. Gene (Amst. ). 1993; 131: 129-134Crossref PubMed Scopus (138) Google Scholar) or progesterone receptor (PR) LBD (25Wang Y. O'Malley Jr., B.W. Tsai S. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8180-8184Crossref PubMed Scopus (391) Google Scholar,26Wang Y. Xu J. Pierson T. O'Malley B.W. Tsai S.Y. Gene Ther. 1997; 4: 432-441Crossref PubMed Scopus (75) Google Scholar), as well as the ecdysone-inducible system based on theDrosophila ecdysone receptor (EcR) and the mammalian retinoid X receptor (RXR) (23Christopherson K.S. Mark M.R. Bajaj V. Godowski P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6314-6318Crossref PubMed Scopus (140) Google Scholar, 24No D. Yao T.-P. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3346-3351Crossref PubMed Scopus (752) Google Scholar) have been described. Compared with the heterodimeric EcR/RXR system, regulators based on the ER and PR LBDs have the important advantage that they function as homodimers and require the delivery of only one cDNA. However, while ecdysone has no known biological effect on mammalian cells, estrogen and progesterone elicit a biological response in cells or tissues that express the endogenous steroid receptors. With the availability of a modified ER and PR LBDs that have lost responsiveness to their natural ligands but not to synthetic antagonists such as 4-hydroxytamoxifen (4-OHT) (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (695) Google Scholar) or RU486 (31Vegeto E. Allan G.F. Schrader W.T. Tsai M.-J. McDonnell D.P. O'Malley B.W. Cell. 1992; 69: 703-713Abstract Full Text PDF PubMed Scopus (334) Google Scholar), respectively, this is no longer of great concern. Thus, steroid hormone receptor LBD-based inducible expression systems can be developed that function independently of the endogenous steroid receptors. To date, this has been shown for the PR LBD through the development of an RU486-inducible expression system based on the Gal4 DBD (25Wang Y. O'Malley Jr., B.W. Tsai S. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8180-8184Crossref PubMed Scopus (391) Google Scholar, 26Wang Y. Xu J. Pierson T. O'Malley B.W. Tsai S.Y. Gene Ther. 1997; 4: 432-441Crossref PubMed Scopus (75) Google Scholar). An inducible expression system based on a point-mutated (G525R) ER LBD (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (695) Google Scholar) that has lost the responsiveness to estrogen but not the antagonist 4-OHT has not been described to date. Designed zinc finger proteins have a number of advantages as compared with other DBDs, including the one derived from Gal4, since the ability to engineer DNA binding specificities allows ligand-dependent regulators to be directed to any desired artificial or natural promoter. Here we explore the utility of fusion proteins between designed zinc finger proteins and nuclear hormone receptor LBDs for the inducible control of gene expression. For the construction of the B3 and N1 zinc finger proteins, DNA recognition helices from the Zif268 finger 2 variants pmGAA, pmGAC, pmGGA, pmGGG, and pGTA were utilized (6Segal D.J. Dreier B. Beerli R.R. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2758-2763Crossref PubMed Scopus (366) Google Scholar). 2Dreier, B., Segal, D, J., and Barbas, C. F., III (2000) J. Mol. Biol., in press. Three-finger proteins binding the respective 9-bp target sites were constructed by grafting the appropriate DNA recognition helices into the framework of the three-finger protein Sp1C (33Desjarlais J.R. Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2256-2260Crossref PubMed Scopus (200) Google Scholar); DNA fragments encoding the two three-finger proteins were assembled from six overlapping oligonucleotides as described (8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar). The three-finger protein coding regions were then cloned into the bacterial expression vector pMal-CSS, a derivative of the bacterial expression plasmid pMal-C2 (New England Biolabs) using SfiI restriction sites. Maltose-binding protein (MBP) fusion proteins were purified to >90% homogeneity using the Protein Fusion and Purification System (New England Biolabs), except that zinc buffer A (10 mm Tris, pH 7.5, 90 mm KCl, 1 mm MgCl2, 90 μmZnCl2), 1% BSA, 5 mm dithiothreitol was used as the column buffer. Protein purity and concentration were determined from Coomassie Blue-stained 15% SDS-PAGE gels by comparison with BSA standards. In 96-well ELISA plates, 0.2 μg of streptavidin (Pierce) was applied to each well for 1 h at 37 °C and then washed twice with water. Biotinylated target oligonucleotide (0.025 μg) was applied in the same manner. Zinc buffer A plus 3% BSA was applied for blocking, but the wells were not washed after incubation. All subsequent incubations were performed at room temperature. Starting with 2 μg of purified MBP fusion protein in the top wells, 2-fold serial dilutions were applied in 1× binding buffer (zinc buffer A, 1% BSA, 5 mm dithiothreitol, 0.12 μg/μl sheared herring sperm DNA). The samples were incubated 1 h, followed by 10 washes with water. Mouse anti-maltose binding protein monoclonal antibody (Sigma) in zinc buffer A plus 1% BSA was applied to the wells for 30 min, followed by 10 washes with water. Goat anti-mouse IgG monoclonal antibody conjugated to alkaline phosphatase (Sigma) was applied to the wells for 30 min, followed by 10 washes with water. Alkaline phosphatase substrate (Sigma) was applied, and theA 405 was quantitated with SOFTmax 2.35 (Molecular Devices). Target oligonucleotides were labeled at their 3′-ends with 32P and gel-purified. Eleven 3-fold serial dilutions of protein were incubated in 20 μl of binding reactions (1× binding buffer, 10% glycerol, 1 pM target oligonucleotide) for 3 h at room temperature and then resolved on a 5% polyacrylamide gel in 0.5× TBE buffer. Quantitation of dried gels was performed using a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA) The K D was determined by Scatchard analysis. The VP16 coding region was PCR-amplified from pcDNA3/C7-VP16 using the primers VPNhe-F (5′-GAG GAG GAG GAG GCT AGC GCC ACC ATG GGG CGC GCC GGC GCT CCC CCG ACC GAT GTC AGC CTG-3′) and VPHind-B (5′-GAG GAG GAG GAG AAG CTT GTT AAT TAA ACC GTA CTC GTC AAT TCC AAG GGC ATC G-3′). The C7 coding region was amplified from the same plasmid, using the primers C7Hind-F (5′-GAG GAG GAG GAG AAG CTT GGG GCC ACG GCG GCC CTC GAG CCC TAT GC-3′) and C7Bam-B (5′-GAG GAG GGA TCC CCC TGG CCG GCC TGG CCA CTA GTT CTA GAG TC-3′). The truncated human PR LBD (aa 645–914) was amplified from PAPCMVGL914VPc′-SV (26Wang Y. Xu J. Pierson T. O'Malley B.W. Tsai S.Y. Gene Ther. 1997; 4: 432-441Crossref PubMed Scopus (75) Google Scholar) using the primers PRBam-F (5′-GAG GAG GAG GAG GGA TCC AGT CAG AGT TGT GAG AGC ACT GGA TGC TG-3′) and PREco-B (5′-GAG GAG GAA TTC TCA AGC AAT AAC TTC AGA CAT CAT TTC TGG AAA TTC-3′). The VP16-C7-PR coding region was then assembled in pcDNA3.1(+)Zeo (Invitrogen) using the NheI,HindIII, BamHI, and EcoRI restriction sites incorporated in the PCR primers. In the resulting constructs, the C7 coding region was flanked by two SfiI sites, and the VP16 coding region was flanked by AscI and PacI sites. These restriction sites were introduced to facilitate the exchange of DBDs and effector domains, respectively. To generate VP16-C7-ER, the point-mutated murine ER LBD coding region (aa 282–599, G525R) was excised from pBSKS+ER (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (695) Google Scholar) and used to replace the PR LBD coding region via BamHI–EcoRI restriction digestion. Fusion constructs containing a VP64 effector domain were produced by replacing VP16 with the VP64 coding region viaAscI–PacI digestion. To generate fusion constructs with B3 or N1 DBDs, C7 was replaced by the B3 or N1 coding regions via SfiI digestion. The truncated human PR LBD was amplified from PAPCMVGL914VPc′-SV (26Wang Y. Xu J. Pierson T. O'Malley B.W. Tsai S.Y. Gene Ther. 1997; 4: 432-441Crossref PubMed Scopus (75) Google Scholar) using the primers PRFse-F (5′-GAG GAG GAG GAG GAG GGC CGG CCG CGT CGA CCA GGT CAG AGT TGT GAG AGC ACT GGA TGC-3′) and PRAsc-B (5′-GAG GAG GAG GAG GAG GGC GCG CCC CGT CGA CCC AGC AAT AAC TTC AGA CAT CAT TTC TGG-3′). The point-mutated mouse ER LBD was amplified from pBSKS+ER (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (695) Google Scholar) using the primers ERFse-F (5′-GAG GAG GAG GAG GAG GGC CGG CCG CCG AAA TGA AAT GGG TGC TTC AGG AGA C-3′) and ERAsc-B (5′- GAG GAG GAG GAG GAG GGC GCG CCC GAT CGT GTT GGG GAA GCC CTC TGC TTC-3′). The resulting PCR products were then inserted into pcDNA3/E2C-VP16 (8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar), in between the E2C and VP16 coding regions, via digestion with the restriction endonucleases FseI andAscI. To generate fusion constructs with B3 or N1 DBDs, E2C was replaced by the B3 or N1 coding regions via SfiI digestion. Fusion constructs containing a VP64 effector domain were produced by replacing VP16 by the VP64 coding region via AscI–PacI digestion. For construction of the E2C-ER fusion, the point-mutated mouse ER LBD was amplified from pBSKS+ER (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (695) Google Scholar) using the primers ERFse-F and ERPac-B (5′-GAG GAG GAG GAG GAG TTA ATT AAG ATC GTG TTG GGG AAG CCC TCT GCT TC-3′). The PCR product was then inserted into the construct pcDNA3/E2C-VP64, replacing the VP64 coding region, via FseI–PacI digestion. To generate the ER-VP64 fusion, the ER LBD was amplified using the primers ERATGBam-F (5′-GAG GAG GAG GAG GGA TCC GCC ACC ATG CGA AAT GAA ATG GGT GCT TCA GGA GAC-3′) and ERAsc-B. The PCR product was then inserted into pcDNA3/E2C-VP64 (8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar), replacing the E2C coding region, viaBamHI–AscI digestion. For construction of single-chain fusions with two ER LBDs, the point-mutated mouse ER LBD was amplified from pBSKS+ER (30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (695) Google Scholar) either using the primers ERFse-F and ERSpeI-B (5′-GAG GAG GAG GAG GAG GAG ACT AGT GGA ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA GAT CGT GTT GGG GAA GCC CTC TGC-3′) or using the primers ERNheI-F1 (for the 18-aa linker construct; 5′-GAG GAG GAG GAG GAG GAG GCT AGC GGC GGT GGC GGT GGC TCC TCT GGT GGC GGT GGC GGT TCT TCC AAT GAA ATG GGT GCT TCA GGA GAC-3′) or ERNheI-F2 (for the 30-aa linker construct; 5′- GAG GAG GAG GAG GAG GAG GCT AGC TCT TCC AAT GAA ATG GGT GCT TCA GGA GAC-3′) and ERAsc-B. The PCR products were then digested with, respectively,FseI and SpeI, or NheI andAscI, and inserted intoFseI–AscI-linearized pcDNA3/E2C-VP64 (8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar). For construction of RXR-EcR single-chain fusions, the ligand binding domain of the human retinoid X receptor (hRXRα; aa 373–654) was PCR-amplified from pVgRXR (Invitrogen) using the primers RXRFse-F (5′-GAG GAG GAG GGC CGG CCG GGA AGC CGT GCA GGA GGA GCG GC-3′) and RXRSpe-B (5′-GAG GAG GAG GAG GAG ACT AGT GGA ACC ACC CCC ACC ACC GCC CGA GCC ACC GCC ACC AGA GGA AGT CAT TTG GTG CGG CGC CTC CAG C-3′). The ligand binding domain of the ecdysone receptor (EcR, aa 202–462;Drosophila melanogaster) was PCR-amplified from pVgRXR using the primers EcRNhe-F1 (for the 18-aa linker construct; 5′-GAG GAG GAG GAG GCT AGC TCT TCC GGT GGC GGC CAA GAC TTT GTT AAG AAG G-3′) or EcRNhe-F2 (for the 30-aa linker construct; 5′-GAG GAG GAG GAG GCT AGC GGC GGT GGC GGT GGC TCC TCT GGT GGC GGT GGC GGT TCT TCC GGT GGC GGC CAA GAC TTT GTT AAG AAG G-3′) and EcRAsc-B (5′-GAG GAG GAG GGC GCG CCC GGC ATG AAC GTC CCA GAT CTC CTC GAG-3′). The PCR products were then digested with, respectively, FseI and SpeI, orNheI and AscI, and inserted intoFseI–AscI-linearized pcDNA3/E2C-VP64 (8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar). DNA binding domains were exchanged via SfiI digestion, and effector domains were exchanged viaAscI–PacI digestion. To generate the 36-aa linker E2C-RLLE-VP64 fusion construct, the RXR LBD was PCR-amplified from pcDNA3/E2C-RE-VP64 using the primers RXRFse-F and RXRSpeLL-B (5′-GAG GAG GAG GAG GAG ACT AGT AGA GCC ACC GCC CCC TTC AGA ACC GCC CGA GCC ACC GCC ACC AGA GG-3′). The EcR LBD was amplified from the same plasmid, using the primers EcRNheLL-F (5′-GAG GAG GAG GAG GCT AGC GGG GGT TCG GAG GGT GGC GGG TCT GAG GGT GGG GGT GGT TCC ACT AGC TCT TCC-3′) and EcRAsc-B. The PCR products were inserted into pcDNA3/E2C-VP64 as described above. C7 dimer-TATA fragments were generated by PCR amplification with C7 dimer-TATA primers (5′-GAG GGT ACC GCG TGG GCG A0–5GCG TGG GCGAGT CGA CTC TAG AGG GTA TAT AAT GG-3′ for direct repeats; 5′-GAG GGT ACC GCG TGG GCG A0–5CGC CCA CGCAGT CGA CTC TAG AGG GTA TAT AAT GG-3′ for inverted repeats; 5′-GAG GGT ACC CGC CCA CGC A0–5GCG TGG GCGAGT CGA CTC TAG AGG GTA TAT AAT GG-3′ for everted repeats) and GLprimer2 (5′-CTT TAT GTT TTT GGC GTC TTC C-3′; Promega), using p17x4TATA-luc (gift from S. Y. Tsai) as a template. PCR products were cloned into pGL3-Basic (Promega) via digestion with the restriction endonucleases KpnI and NcoI. 10xB3-TATA and 10xN1-TATA fragments were assembled from two pairs of complementary oligonucleotides each and cloned intoSacI–XbaI-linearized pGL3-Basic (Promega), upstream of the firefly luciferase coding region, creating the plasmids 10xB3-TATA-luc and 10xN1-TATA-luc. To generate the 10xN1-TATA-lacZ reporter construct, the lacZ coding region was excised from pβgal-Basic (CLONTECH) and used to replace the luciferase coding region of 10xN1-TATA-luc viaHindIII–BamHI digestion. For all transfections, HeLa cells were plated in 24-well dishes and used at a confluency of 40–60%. For luciferase reporter assays, 175 ng of reporter plasmid (promotor constructs in pGL3 or, as negative control, pGL3-Basic) and 25 ng of effector plasmid (zinc finger-steroid receptor fusions in pcDNA3 or, as negative control, empty pcDNA3) were transfected using the LipofectAMINE reagent (Life Technologies, Inc.). After approximately 24 h, expression was induced by the addition of 10 nm RU486 (Biomol), 100 nm 4-OHT (Sigma), or 5 μm ponasterone A (Invitrogen). Cell extracts were prepared approximately 48 h after transfection and assayed for luciferase activity using the Promega luciferase assay reagent in a MicroLumat LB96P luminometer (EG&G Berthold, Gaithersburg, MD). For dual reporter assays, 85 ng of luciferase reporter plasmid, 85 ng of β-galactosidase reporter plasmid, and 15 ng of each of the two effector plasmids were transfected. β-Galactosidase activity was measured using the luminescent β-galactosidase detection kit II (CLONTECH). Previous studies have demonstrated the potential of engineered C2-H2 zinc finger proteins for the regulation of target gene expression (8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar, 9Beerli R.R. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1495-1500Crossref PubMed Scopus (303) Google Scholar, 34Liu Q. Segal D.J. Ghiara J.B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5525-5530Crossref PubMed Scopus (236) Google Scholar, 35Kim J.S. Pabo C.O. J. Biol. Chem. 1997; 272: 29795-29800Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, to fully realize the potential of engineered zinc finger proteins, it is desirable that their otherwise constitutive DNA binding activity be rendered ligand-dependent. The LBDs of the human PR and the murine ER have previously been used for the regulation of heterologous proteins, after having been modified to remove their responsiveness to their natural hormone inducers while retaining activity with synthetic antagonists (26Wang Y. Xu J. Pierson T. O'Malley B.W. Tsai S.Y. Gene Ther. 1997; 4: 432-441Crossref PubMed Scopus (75) Google Scholar, 30Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (695) Google Scholar). In our initial study, the Zif268 variant C7 (4Wu H. Yang W.-P. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 344-348Crossref PubMed Scopus (181) Google Scholar) was fused to a transcriptional activation domain plus the LBD of either of the two nuclear hormone receptors. The VP64-C7-PR fusion protein contains an N-terminal VP64 activation domain (8Beerli R.R. Segal D.J. Dreier B. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14628-14633Crossref PubMed Scopus (400) Google Scholar) and a C-terminal human PR LBD (aa 645–914) lacking amino acids 915–933. This LBD is responsive to the progesterone antagonist RU486/mifepristone but not to progesterone (26Wang Y. Xu J. Pie

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