Transcriptional Repression by Zinc Finger Peptides

A series of studies were performed to determine whether zinc finger peptides could efficiently repress transcription from RNA polymerase II promoters in vivo and to determine how such repression might depend on the position of the zinc finger binding site with respect to those of the TATA box or the initiator element. Promoter constructs were prepared with Zif268 binding sites inserted at various positions, and the activity of a reporter gene was measured in transfection studies. We found that the peptide containing the three zinc fingers of Zif268 could efficiently repress activated transcription when bound to a site near the TATA box (19-fold repression) or when bound to a site near the initiator element (18-fold repression). Repression was even more effective when the zinc finger peptide was bound to both of these sites (63-fold repression). Novel zinc finger peptides that had been selected via phage display also served as repressors of activated transcription, but repression with these proteins was somewhat less efficient than with the Zif268 peptide. A series of studies were performed to determine whether zinc finger peptides could efficiently repress transcription from RNA polymerase II promoters in vivo and to determine how such repression might depend on the position of the zinc finger binding site with respect to those of the TATA box or the initiator element. Promoter constructs were prepared with Zif268 binding sites inserted at various positions, and the activity of a reporter gene was measured in transfection studies. We found that the peptide containing the three zinc fingers of Zif268 could efficiently repress activated transcription when bound to a site near the TATA box (19-fold repression) or when bound to a site near the initiator element (18-fold repression). Repression was even more effective when the zinc finger peptide was bound to both of these sites (63-fold repression). Novel zinc finger peptides that had been selected via phage display also served as repressors of activated transcription, but repression with these proteins was somewhat less efficient than with the Zif268 peptide. Designer DNA-binding proteins may eventually provide novel reagents for gene therapy and for the regulation of gene expression in transgenic organisms. In the past several years, there has been remarkable progress in the design and selection of novel zinc finger proteins (1Desjarlais J.R. Berg J.M. Proteins Struct. Funct. Genet. 1992; 12: 101-104Crossref PubMed Scopus (83) Google Scholar, 2Desjarlais J.R. Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2256-2260Crossref PubMed Scopus (200) Google Scholar, 3Rebar E.J. Pabo C.O. Science. 1994; 263: 671-673Crossref PubMed Scopus (380) Google Scholar, 4Jamieson A.C. Kim S.-H. Wells J.A. Biochemistry. 1994; 33: 5689-5695Crossref PubMed Scopus (208) Google Scholar, 5Choo Y. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11163-11167Crossref PubMed Scopus (315) Google Scholar, 6Wu H. Yang W.-P. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 344-348Crossref PubMed Scopus (181) Google Scholar, 7Jamieson A.C. Wang H. Kim S.-H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12834-12839Crossref PubMed Scopus (99) Google Scholar, 8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar) and in the structure-based design of hybrid proteins that contain zinc fingers fused to other site-specific DNA-binding domains (9Pomerantz J.L. Sharp P.A. Pabo C.O. Science. 1995; 267: 93-96Crossref PubMed Scopus (121) Google Scholar, 10Kim J.-S. Kim J. Cepek K.L. Sharp P.A. Pabo C.O. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3616-3620Crossref PubMed Scopus (53) Google Scholar). A zinc finger-homeodomain fusion already is being tested for potential applications in gene therapy (11Rivera 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). Recent phage display experiments suggest that it may be possible to select zinc finger peptides that will recognize almost any desired target site on duplex DNA (8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar). Despite this rapid progress in design, there has been relatively little information about how such novel proteins might be used to regulate transcription or about the differential effects of targeting the zinc finger peptides to various promoter regions. In this study, we begin exploring these issues to develop a more systematic basis for the application of zinc finger proteins in gene therapy and in the regulation of transgenic organisms. We systematically scan the promoter region to find sites where zinc fingers are most effective as repressors. We also show that novel zinc finger proteins selected via phage display can function effectively as repressors in vivo.DISCUSSIONStudies with novel zinc finger proteins may help elucidate transcriptional regulatory mechanisms (earlier studies using zinc fingers to block RNA polymerase III transcription helped delineate key regulatory sites) (24McBryant S.J. Kassavetis G.A. Gottesfeld J.M. J. Mol. Biol. 1995; 250: 315-326Crossref PubMed Scopus (13) Google Scholar), and these studies also may provide a basis for using zinc finger proteins in gene therapy. (As mentioned in the Introduction, a designer zinc finger/homeodomain fusion (9Pomerantz J.L. Sharp P.A. Pabo C.O. Science. 1995; 267: 93-96Crossref PubMed Scopus (121) Google Scholar) already is being tested for applications in gene therapy (11Rivera 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).) In the studies reported here, we have shown that zinc finger peptides can repress both basal and VP16-activated transcription when bound close to the TATA box or the initiator element. Computer modeling studies suggest that the Zif268 zinc fingers, when bound to DNA sites between the TATA box and the initiator element, may interfere with proper binding of TBP and TFIIB to the promoter. The Zif268 peptide also functioned effectively as a repressor when bound to DNA at position +18 (but not at position +49). It seems plausible that binding of the Zif268 peptide at position +18 may interfere with the binding of TBP-associated factors. (Drosophila TAF150 binds around the initiator and the footprint extends to position +33 (25Verrijzer C.P. Yokomori K. Chen J.-L. Tjian R. Science. 1994; 264: 933-941Crossref PubMed Scopus (178) Google Scholar).) We find, with several different promoter constructs, that positions −22 and +18 give essentially equivalent levels of repression (Fig. 3). When both a TATA box and an initiator element were present, the Zif peptide gave 19-fold (position −22) and 18-fold (position +18) repression. When only the initiator element was present, each site gave about 4-fold repression. When only the TATA box was present, these sites gave 9-fold (position −22) and 10-fold (position +18) repression. Agreement of these repression levels may be fortuitous, but since 1) other proteins are tightly associated with TBPin vivo to form the multiprotein TFIID complex, and since 2) the TFIID footprint covers both the −22 and +18 sites, it seems plausible that binding of the Zif peptide at these positions may actually block the same step (i.e. binding of TFIID to the promoter), explaining why similar levels of repression are observed for these two positions.Phage display systems (3Rebar E.J. Pabo C.O. Science. 1994; 263: 671-673Crossref PubMed Scopus (380) Google Scholar, 4Jamieson A.C. Kim S.-H. Wells J.A. Biochemistry. 1994; 33: 5689-5695Crossref PubMed Scopus (208) Google Scholar, 5Choo Y. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11163-11167Crossref PubMed Scopus (315) Google Scholar, 6Wu H. Yang W.-P. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 344-348Crossref PubMed Scopus (181) Google Scholar, 7Jamieson A.C. Wang H. Kim S.-H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12834-12839Crossref PubMed Scopus (99) Google Scholar, 8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar) and design strategies (1Desjarlais J.R. Berg J.M. Proteins Struct. Funct. Genet. 1992; 12: 101-104Crossref PubMed Scopus (83) Google Scholar, 2Desjarlais J.R. Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2256-2260Crossref PubMed Scopus (200) Google Scholar) have been successfully used to change the DNA-binding specificities of zinc finger proteins. Recent results suggest that it may be possible to select zinc fingers that will specifically recognize almost any desired site (8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar). This should allow the design of transcription factors that target critical sites in the promoters of viral genes and of oncogenes. Such novel zinc finger peptides may be used alone (as tested here), or possibly as fusions with repression domains Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar) to target genes in a of the DNA binding proteins may be critical in gene since it will be to repression of regulation of other genes might to effects in a In this we that it may be possible to the and of transcriptional repression targeting sites in a promoter We found that the Zif268 when bound to two critical sites, gave repression when bound to a when activated transcription from a promoter containing a TATA box and an initiator element, binding to position −22 gave 19-fold binding to position +18 gave 18-fold binding to both positions gave repression (Fig. results suggest that it may be to select several zinc finger critical but in a and to these peptides for repression. This has with the mechanisms of that are in gene strategies and results reported in this should provide a for zinc finger peptides as repressors for in gene therapy and in the regulation of gene expression in transgenic organisms. We find that zinc finger peptides can effectively repress transcription when to key of the and phage display should allow selection of zinc finger peptides that recognize the of studies (24McBryant S.J. Kassavetis G.A. Gottesfeld J.M. J. Mol. Biol. 1995; 250: 315-326Crossref PubMed Scopus (13) Google Scholar) had shown that zinc fingers could efficiently block RNA polymerase III transcription in to with of the binding We show that levels can be in vivo at RNA polymerase II results about two other studies that have tested designer zinc finger proteins as repressors. A Y. Klug A. 1994; PubMed Scopus Google Scholar) a zinc finger that recognize a site the region of the reported that this designer gave effective repression in in as for of the it is to that had a relatively for the DNA site is about than and that the binding site for this zinc finger peptide was of the transcription to block transcriptional than The results of to be in to the Zif268 peptide binds more tightly than peptide and Zif268 at a on transcription when binding at position The basis for these is not but results about the of targeting zinc fingers to binding sites the region of a also is to results with those reported Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar) proteins containing zinc fingers that these might be specifically to sites in a also a peptide the repression J.F. J.R. H. III, Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). repression for when it binds to a promoter that of the binding site for this have for repression of the but about the and of these proteins and about the most effective of using zinc finger proteins as repressors. repression of activated transcription, seems at as we can or levels of repression with a and a binding of zinc finger proteins promoter should allow and the potential for application of Designer DNA-binding proteins may eventually provide novel reagents for gene therapy and for the regulation of gene expression in transgenic organisms. In the past several years, there has been remarkable progress in the design and selection of novel zinc finger proteins (1Desjarlais J.R. Berg J.M. Proteins Struct. Funct. Genet. 1992; 12: 101-104Crossref PubMed Scopus (83) Google Scholar, 2Desjarlais J.R. Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2256-2260Crossref PubMed Scopus (200) Google Scholar, 3Rebar E.J. Pabo C.O. Science. 1994; 263: 671-673Crossref PubMed Scopus (380) Google Scholar, 4Jamieson A.C. Kim S.-H. Wells J.A. Biochemistry. 1994; 33: 5689-5695Crossref PubMed Scopus (208) Google Scholar, 5Choo Y. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11163-11167Crossref PubMed Scopus (315) Google Scholar, 6Wu H. Yang W.-P. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 344-348Crossref PubMed Scopus (181) Google Scholar, 7Jamieson A.C. Wang H. Kim S.-H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12834-12839Crossref PubMed Scopus (99) Google Scholar, 8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar) and in the structure-based design of hybrid proteins that contain zinc fingers fused to other site-specific DNA-binding domains (9Pomerantz J.L. Sharp P.A. Pabo C.O. Science. 1995; 267: 93-96Crossref PubMed Scopus (121) Google Scholar, 10Kim J.-S. Kim J. Cepek K.L. Sharp P.A. Pabo C.O. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3616-3620Crossref PubMed Scopus (53) Google Scholar). A zinc finger-homeodomain fusion already is being tested for potential applications in gene therapy (11Rivera 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). Recent phage display experiments suggest that it may be possible to select zinc finger peptides that will recognize almost any desired target site on duplex DNA (8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar). Despite this rapid progress in design, there has been relatively little information about how such novel proteins might be used to regulate transcription or about the differential effects of targeting the zinc finger peptides to various promoter regions. In this study, we begin exploring these issues to develop a more systematic basis for the application of zinc finger proteins in gene therapy and in the regulation of transgenic organisms. We systematically scan the promoter region to find sites where zinc fingers are most effective as repressors. We also show that novel zinc finger proteins selected via phage display can function effectively as repressors in with novel zinc finger proteins may help elucidate transcriptional regulatory mechanisms (earlier studies using zinc fingers to block RNA polymerase III transcription helped delineate key regulatory sites) (24McBryant S.J. Kassavetis G.A. Gottesfeld J.M. J. Mol. Biol. 1995; 250: 315-326Crossref PubMed Scopus (13) Google Scholar), and these studies also may provide a basis for using zinc finger proteins in gene therapy. (As mentioned in the Introduction, a designer zinc finger/homeodomain fusion (9Pomerantz J.L. Sharp P.A. Pabo C.O. Science. 1995; 267: 93-96Crossref PubMed Scopus (121) Google Scholar) already is being tested for applications in gene therapy (11Rivera 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).) In the studies reported here, we have shown that zinc finger peptides can repress both basal and VP16-activated transcription when bound close to the TATA box or the initiator element. Computer modeling studies suggest that the Zif268 zinc fingers, when bound to DNA sites between the TATA box and the initiator element, may interfere with proper binding of TBP and TFIIB to the promoter. The Zif268 peptide also functioned effectively as a repressor when bound to DNA at position +18 (but not at position +49). It seems plausible that binding of the Zif268 peptide at position +18 may interfere with the binding of TBP-associated factors. (Drosophila TAF150 binds around the initiator and the footprint extends to position +33 (25Verrijzer C.P. Yokomori K. Chen J.-L. Tjian R. Science. 1994; 264: 933-941Crossref PubMed Scopus (178) Google Scholar).) We find, with several different promoter constructs, that positions −22 and +18 give essentially equivalent levels of repression (Fig. 3). When both a TATA box and an initiator element were present, the Zif peptide gave 19-fold (position −22) and 18-fold (position +18) repression. When only the initiator element was present, each site gave about 4-fold repression. When only the TATA box was present, these sites gave 9-fold (position −22) and 10-fold (position +18) repression. Agreement of these repression levels may be fortuitous, but since 1) other proteins are tightly associated with TBPin vivo to form the multiprotein TFIID complex, and since 2) the TFIID footprint covers both the −22 and +18 sites, it seems plausible that binding of the Zif peptide at these positions may actually block the same step (i.e. binding of TFIID to the promoter), explaining why similar levels of repression are observed for these two positions.Phage display systems (3Rebar E.J. Pabo C.O. Science. 1994; 263: 671-673Crossref PubMed Scopus (380) Google Scholar, 4Jamieson A.C. Kim S.-H. Wells J.A. Biochemistry. 1994; 33: 5689-5695Crossref PubMed Scopus (208) Google Scholar, 5Choo Y. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11163-11167Crossref PubMed Scopus (315) Google Scholar, 6Wu H. Yang W.-P. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 344-348Crossref PubMed Scopus (181) Google Scholar, 7Jamieson A.C. Wang H. Kim S.-H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12834-12839Crossref PubMed Scopus (99) Google Scholar, 8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar) and design strategies (1Desjarlais J.R. Berg J.M. Proteins Struct. Funct. Genet. 1992; 12: 101-104Crossref PubMed Scopus (83) Google Scholar, 2Desjarlais J.R. Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2256-2260Crossref PubMed Scopus (200) Google Scholar) have been successfully used to change the DNA-binding specificities of zinc finger proteins. Recent results suggest that it may be possible to select zinc fingers that will specifically recognize almost any desired site (8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar). This should allow the design of transcription factors that target critical sites in the promoters of viral genes and of oncogenes. Such novel zinc finger peptides may be used alone (as tested here), or possibly as fusions with repression domains Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar) to target genes in a of the DNA binding proteins may be critical in gene since it will be to repression of regulation of other genes might to effects in a In this we that it may be possible to the and of transcriptional repression targeting sites in a promoter We found that the Zif268 when bound to two critical sites, gave repression when bound to a when activated transcription from a promoter containing a TATA box and an initiator element, binding to position −22 gave 19-fold binding to position +18 gave 18-fold binding to both positions gave repression (Fig. results suggest that it may be to select several zinc finger critical but in a and to these peptides for repression. This has with the mechanisms of that are in gene strategies and results reported in this should provide a for zinc finger peptides as repressors for in gene therapy and in the regulation of gene expression in transgenic organisms. We find that zinc finger peptides can effectively repress transcription when to key of the and phage display should allow selection of zinc finger peptides that recognize the of studies (24McBryant S.J. Kassavetis G.A. Gottesfeld J.M. J. Mol. Biol. 1995; 250: 315-326Crossref PubMed Scopus (13) Google Scholar) had shown that zinc fingers could efficiently block RNA polymerase III transcription in to with of the binding We show that levels can be in vivo at RNA polymerase II results about two other studies that have tested designer zinc finger proteins as repressors. A Y. Klug A. 1994; PubMed Scopus Google Scholar) a zinc finger that recognize a site the region of the reported that this designer gave effective repression in in as for of the it is to that had a relatively for the DNA site is about than and that the binding site for this zinc finger peptide was of the transcription to block transcriptional than The results of to be in to the Zif268 peptide binds more tightly than peptide and Zif268 at a on transcription when binding at position The basis for these is not but results about the of targeting zinc fingers to binding sites the region of a also is to results with those reported Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar) proteins containing zinc fingers that these might be specifically to sites in a also a peptide the repression J.F. J.R. H. III, Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). repression for when it binds to a promoter that of the binding site for this have for repression of the but about the and of these proteins and about the most effective of using zinc finger proteins as repressors. repression of activated transcription, seems at as we can or levels of repression with a and a binding of zinc finger proteins promoter should allow and the potential for application of with novel zinc finger proteins may help elucidate transcriptional regulatory mechanisms (earlier studies using zinc fingers to block RNA polymerase III transcription helped delineate key regulatory sites) (24McBryant S.J. Kassavetis G.A. Gottesfeld J.M. J. Mol. Biol. 1995; 250: 315-326Crossref PubMed Scopus (13) Google Scholar), and these studies also may provide a basis for using zinc finger proteins in gene therapy. (As mentioned in the Introduction, a designer zinc finger/homeodomain fusion (9Pomerantz J.L. Sharp P.A. Pabo C.O. Science. 1995; 267: 93-96Crossref PubMed Scopus (121) Google Scholar) already is being tested for applications in gene therapy (11Rivera 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).) In the studies reported here, we have shown that zinc finger peptides can repress both basal and VP16-activated transcription when bound close to the TATA box or the initiator element. Computer modeling studies suggest that the Zif268 zinc fingers, when bound to DNA sites between the TATA box and the initiator element, may interfere with proper binding of TBP and TFIIB to the promoter. The Zif268 peptide also functioned effectively as a repressor when bound to DNA at position +18 (but not at position +49). It seems plausible that binding of the Zif268 peptide at position +18 may interfere with the binding of TBP-associated factors. (Drosophila TAF150 binds around the initiator and the footprint extends to position +33 (25Verrijzer C.P. Yokomori K. Chen J.-L. Tjian R. Science. 1994; 264: 933-941Crossref PubMed Scopus (178) Google Scholar).) We find, with several different promoter constructs, that positions −22 and +18 give essentially equivalent levels of repression (Fig. 3). When both a TATA box and an initiator element were present, the Zif peptide gave 19-fold (position −22) and 18-fold (position +18) repression. When only the initiator element was present, each site gave about 4-fold repression. When only the TATA box was present, these sites gave 9-fold (position −22) and 10-fold (position +18) repression. Agreement of these repression levels may be fortuitous, but since 1) other proteins are tightly associated with TBPin vivo to form the multiprotein TFIID complex, and since 2) the TFIID footprint covers both the −22 and +18 sites, it seems plausible that binding of the Zif peptide at these positions may actually block the same step (i.e. binding of TFIID to the promoter), explaining why similar levels of repression are observed for these two display systems (3Rebar E.J. Pabo C.O. Science. 1994; 263: 671-673Crossref PubMed Scopus (380) Google Scholar, 4Jamieson A.C. Kim S.-H. Wells J.A. Biochemistry. 1994; 33: 5689-5695Crossref PubMed Scopus (208) Google Scholar, 5Choo Y. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11163-11167Crossref PubMed Scopus (315) Google Scholar, 6Wu H. Yang W.-P. Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 344-348Crossref PubMed Scopus (181) Google Scholar, 7Jamieson A.C. Wang H. Kim S.-H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12834-12839Crossref PubMed Scopus (99) Google Scholar, 8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar) and design strategies (1Desjarlais J.R. Berg J.M. Proteins Struct. Funct. Genet. 1992; 12: 101-104Crossref PubMed Scopus (83) Google Scholar, 2Desjarlais J.R. Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2256-2260Crossref PubMed Scopus (200) Google Scholar) have been successfully used to change the DNA-binding specificities of zinc finger proteins. Recent results suggest that it may be possible to select zinc fingers that will specifically recognize almost any desired site (8Greisman H.A. Pabo C.O. Science. 1997; 275: 657-661Crossref PubMed Scopus (347) Google Scholar). This should allow the design of transcription factors that target critical sites in the promoters of viral genes and of oncogenes. Such novel zinc finger peptides may be used alone (as tested here), or possibly as fusions with repression domains Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar) to target genes in a of the DNA binding proteins may be critical in gene since it will be to repression of regulation of other genes might to effects in a In this we that it may be possible to the and of transcriptional repression targeting sites in a promoter We found that the Zif268 when bound to two critical sites, gave repression when bound to a when activated transcription from a promoter containing a TATA box and an initiator element, binding to position −22 gave 19-fold binding to position +18 gave 18-fold binding to both positions gave repression (Fig. results suggest that it may be to select several zinc finger critical but in a and to these peptides for repression. This has with the mechanisms of that are in gene The strategies and results reported in this should provide a for zinc finger peptides as repressors for in gene therapy and in the regulation of gene expression in transgenic organisms. We find that zinc finger peptides can effectively repress transcription when to key of the and phage display should allow selection of zinc finger peptides that recognize the of studies (24McBryant S.J. Kassavetis G.A. Gottesfeld J.M. J. Mol. Biol. 1995; 250: 315-326Crossref PubMed Scopus (13) Google Scholar) had shown that zinc fingers could efficiently block RNA polymerase III transcription in to with of the binding We show that levels can be in vivo at RNA polymerase II results about two other studies that have tested designer zinc finger proteins as repressors. A Y. Klug A. 1994; PubMed Scopus Google Scholar) a zinc finger that recognize a site the region of the reported that this designer gave effective repression in in as for of the it is to that had a relatively for the DNA site is about than and that the binding site for this zinc finger peptide was of the transcription to block transcriptional than The results of to be in to the Zif268 peptide binds more tightly than peptide and Zif268 at a on transcription when binding at position The basis for these is not but results about the of targeting zinc fingers to binding sites the region of a It also is to results with those reported Barbas III, C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar) proteins containing zinc fingers that these might be specifically to sites in a also a peptide the repression J.F. J.R. H. III, Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). repression for when it binds to a promoter that of the binding site for this have for repression of the but about the and of these proteins and about the most effective of using zinc finger proteins as repressors. repression of activated transcription, seems at as we can or levels of repression with a and a binding of zinc finger proteins promoter should allow and the potential for application of We H. A. and S. T. for used in these and A. Sharp for to in the for at