Advances in zinc finger engineering Yen Choo* and Mark Isalan

411 Advances in zinc finger engineering Yen Choo and Mark Isalan Recently developments have been made in engineering sequence-specific zinc finger DNA-binding proteins. Advances in this area will soon make it routine to target proteins to specific DNA sequences associated with any given gene. The primary interest is in the regulation of gene expression using customised transcription factors. However, modular catalytic domains are also being developed in order to engineer chimaeric proteins with customised restriction enzyme, methylase and integrase activity. Addresses Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK and Gendaq Ltd., 1 3 Burtonhole Lane, London NW7 1AD, UK e-mail: choo@mrc-lmb.cam.ac.uk Current Opinion in Structural Biology 2000, 10:411 416 0959-440X/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. Abbreviation bp base pairs Introduction Designing a protein to recognise any given DNA sequence is an important goal in molecular biology. Such proteins could be used for a variety of applications, most notably in regulating gene expression. When we last reviewed this field [1,2], several groups working on the zinc finger motif had developed phage display strategies in order to isolate modules that recognised given trinucleotide sequences embedded in the DNA binding site of the protein Zif268 [3 6]. Our group had also demonstrated that such phage-selected zinc fingers could be used to assemble polymers that recognise a longer sequence (10 bp) and had thus designed a protein that could down-regulate an oncogene in mouse cells [7]. Since then, the zinc finger field has progressed substantially through basic research into the mechanism of DNA recognition by this motif [8,9] and further phage selection strategies have been developed [10,11,P1 ]. Also significant has been the engineering of finger polymers with relatively long DNA recognition sites, in order to be able to target genes more specifically [12,13]. Increasing work has also been reported in the control of gene expression using zinc finger transcription factors [6,14 18]. Most recently, a second example of gene regulation has been reported, with a zinc finger transcription factor having been designed to repress the erbb oncogenes [19 ]. Work is also underway to identify and develop promising effector domains in order to construct chimaeric enzymes, such as restriction enzymes [20], integrases [21] and DNA methylases [22]. This review focuses on these developments in zinc finger engineering. Engineering zinc fingers using phage display The zinc finger is a compact protein module that has a remarkably simple mode of interaction with DNA, making it ideal for protein engineering [23,24]. Zinc finger proteins contain tandem repeats of individual modules with a wide variety of DNA specificities, potentially allowing the targeting of any DNA sequence. Moreover, only a few positions in the recognition helix of a zinc finger need be altered to result in either variant DNA-binding specificities [3 6,9,10,11,19,25,26 ] or the ability to recognise base modifications, such as cytosine methylation [25,26 ]. The above-mentioned references are all phage display studies and it is now clear that this is a very reliable approach to zinc finger engineering. One key limitation of conventional zinc finger phage display is the restriction of library size (diversity) that arises as a consequence of the practical limitations in cloning efficiency. This has typically meant that the α-helical base-contacting residues of only one zinc finger can be fully randomised in any given library [3 6]. Protein engineering strategies have therefore been devised to assemble these zinc finger monomers (which recognise 3 4 bp) into polymeric arrays capable of recognising longer predetermined DNA sequences. The first approach to engineering polymers of zinc fingers selected by phage display was simply to preselect individual monomers and subsequently to combine them into a single polypeptide [7] (Figure 1a). We have previously pointed out the advantage of using a conformationally constrained zinc finger for this purpose (e.g. the middle finger of a three-finger array) [1]. Recently, this very strategy has been put to good purpose in selecting zinc fingers that recognise 16 different trinucleotides comprising the subset GNN (where N is any nucleotide), providing prefabricated building blocks for the construction of proteins that bind to guanine-rich sequences [11 ]. Several of these building blocks were subsequently validated by incorporation into proteins that bind the erbb oncogenes [18,19 ]. Although the general strategy of combining preselected monomers is clearly effective, the major caveat in its implementation is that the selection of individual zinc fingers (in the context of a multifinger protein) has so far yielded monomers that specifically recognise only DNA triplets of the form GNN (i.e. trinucleotides having guanine fixed in the 5 position). This, in turn, means that it is only possible to design proteins that specifically bind DNA sequences of the form GNNGNN (i.e. sequences having guanine at every third base position) [11 ]. Obviously, one would like to be able to bind a wider variety of DNA sequences, instead of being confined to a relatively small (and rarely occurring) subset. The reason for this guanine-dependent limitation has recently become clear from phage display experiments

412 Engineering and design Figure 1 (a) (b) (c) F3 Clone new library Splice Splice Clone new library Current Opinion in Structural Biology Strategies for selecting zinc finger polymers with novel DNA-binding specificities. Schematic figure showing zinc fingers binding to DNA subsites (rectangular boxes). The DNA subsites recognised by randomised phage library regions are indicated by asterisks (). Differential shading indicates the origin and final destination of the individual peptide and DNA components in each system. (a) Parallel (or pre-) selections of middle fingers from premade libraries are followed by a splicing step to make new three-finger proteins [7]. This system is currently limited to binding DNA sites of the form (GNN)n [11 ]. (b) Sequential selection of zinc fingers [10]. Following each selection step, a new C-terminal finger library is cloned onto the construct. In this way, a growing chain of new peptide walks across the gene of interest. (c) Bipartite-complementary strategy [P1 ]. Parallel selections are carried out from two premade half libraries, after which the two selected portions are spliced together to make a novel DNA-binding domain. Small arrows represent recombination sites in the proteins. This system is designed to overcome incompatibilities at the interface between adjacent fingers [9] and can be adapted for high-throughput using liquid-handling robotics. in which interactions between two adjacent fingers were studied. It emerges that the DNA subsites of adjacent zinc fingers overlap, such that the fingers frequently bind DNA synergistically. The synergistic interactions in certain zinc finger frameworks, such as that in Zif268, cause interference between adjacent fingers, unless they bind to DNA in which every third base is guanine or thymine [8]. More importantly, it emerges that different synergistic interactions between adjacent zinc fingers and DNA are the key to breaking the requirement for this periodicity [8,9]. An alternative method of engineering zinc finger proteins by phage display comprises the stepwise assembly of a DNA-binding domain by appending and selecting a series of zinc finger libraries to a growing polymer [10] (Figure 1b). This iterative approach eventually allows the assembly of a polymeric protein, essentially by walking across the target DNA sequence. As the interfinger contacts produced by this method are compatible, albeit not necessarily synergistic, no interference occurs between adjacent fingers. Accordingly, this method suffers less from the DNA sequence restriction of the previously described protocol, producing zinc finger polymers that recognise a greater diversity of nucleotide sequences. For instance, using the new protocol, zinc finger proteins were designed against DNA subsites that did not contain guanine and the specificity

Zinc finger engineering Choo and Isalan 413 Figure 2 (a) (b) C GAL4 4 C G C C C A N T G G G C G C G G G T N A C C C G...13 bp... G C T T G G G C A C C C G G C C G C C C G C G G G A T (c)... 0 2 bp... N N Linker - TGEKP - - LRQKDGERP - - LRQKDGGGSERP - F4 F5 F6 Current Opinion in Structural Biology Engineering the recognition of longer DNA sequences. Zinc finger DNA binding is represented schematically according to a binding model derived from [23]. (a) Homodimerisation of the first two fingers from Zif268 using a portion of the dimerisation domain from GAL4 [34]. (b) Homodimerisation of the first two fingers from Zif268 using phage selected peptides [35 ]. (c) Connecting three-finger domains with a variety of peptide linkers allows the recognition of approximately 18 bp sites [12,13]. Linkers that are longer than the canonical TGEKP peptide appear to enhance binding affinity and allow gaps of up to 2 bp to be spanned [12]. of these proteins was later shown to be generally good [27]. A serious disadvantage of this method, however, is that it is apparently cumbersome, owing to the requirement for multiple serial library constructions for each protein engineering project. Recently, we devised a novel zinc finger engineering strategy that is both rapid and convenient, and that is also not limited to producing fingers that bind particular subsets of DNA sequences [P1 ] (Figure 1c). The key features of this method are that it makes use of parallel selections from premade libraries (resulting in speed) and that the libraries have randomised positions from two adjacent fingers (giving synergy that results in the ability to bind unrestricted DNA sequences). The protocol has been validated by selecting a number of DNA-binding domains that specifically recognise the promoter region (LTR) of HIV-1 [P1 ]. We believe that it will be possible to target any gene promoter using this method. Engineering zinc fingers by rational design A subfamily of zinc finger proteins, including Zif268 and Sp1, binds guanine/cytosine-rich DNA using a remarkably simple set of contacts that resembles a protein DNA recognition code [2]. In the past decade, site-directed mutagenesis [28,29] and phage display experiments [30] have been used to expand this code to describe binding to all four bases, allowing the rational design of zinc fingers that bind certain DNA sequences. The utility and limitations of the code have been discussed in detail elsewhere and clearly the code is not absolute [9,27,31]. Rational design is limited to fingers that recognise DNA sequences of the form GNNGNN and, additionally, in our own (unpublished) experiments, we have found it fairly cumbersome to implement (because the code is degenerate) and also quite unreliable. Now, Corbi et al. [32,33] have published two papers in which zinc finger proteins were rationally designed to bind arbitrary DNA sequences. Although binding to these sequences was fairly strong,

414 Engineering and design SELEX experiments revealed that the two proteins reliably discriminated only seven out of nine and five out of nine bases in their target recognition sites. These results are encouraging as they validate some of the rules deduced for zinc finger DNA recognition. On the other hand, this work also emphasises the current deficiencies of zinc finger rational design. Engineering the recognition of very long DNA sequences The probability of encountering a given DNA sequence in any genome is inversely proportional to the length of that sequence. A simple calculation thus predicts that a DNA sequence of 16 18 bp would occur only once in random DNA the size of an animal or plant genome. DNA-binding proteins have evolved two simple strategies to target such rare, or lengthy, sequences: they contain arrays of DNAbinding modules (e.g. zinc fingers) or they cooperate to bind adjacent DNA sites (e.g. bacterial repressor dimers). Recently, both these methods were used to extend the binding sites of zinc finger proteins (Figure 2). Firstly, structure-based design was used to create a fusion between a two-zinc-finger peptide and the dimerisation domain from Gal4 [34] (Figure 2a). Secondly, a library of zinc fingers with N-terminal randomised polypeptide extensions was created and polyvalent zinc finger phage were selected using DNA containing an inverted repeat of the zinc-fingerbinding site to search for interacting pairs [35 ] (Figure 2b). Both strategies produced zinc finger proteins that preferentially bound DNA as homodimers in solution. As the complexes were formed by a dimer of zinc finger pairs (i.e. a total of four fingers), the length of the specified DNA sequence was only 12 bp. It would be interesting to see whether functional dimers can be formed from three-finger constructs (to target 18 bp sites) and also whether heterodimer formation is possible (to target asymmetric sites). Arrays of tandemly repeated zinc fingers have been constructed by connecting pairs of three-finger domains using polypeptide linkers [12,13] (Figure 2c). A number of groups have used the canonical four amino acid zinc finger linker GEKP to add extra fingers to three-finger domains (listed in [12]). NMR [36] and crystallographic [23] studies show that this linker is flexible in solution and fully extended in protein DNA complexes. Using the canonical linker to connect three-finger domains usually results in a modest increase in affinity for a six-finger DNA binding site relative to a three-finger DNA binding site (e.g. [13,37]). Ironically, such six-finger proteins are potentially less specific than their three-finger constituents, as subsets of fingers within the six-finger array would recognise short runs of DNA without substantial loss in binding affinity. In contrast, when a much longer (eight amino acids) linker was used to connect three-finger domains, a dramatic 6000-fold increase in relative affinity was reported [12]. We note, however, that, in our own work (M Moore, A Klug, Y Choo, unpublished data), we have only observed increases of 100 500-fold using even the longer (eight amino acids) linkers to construct zinc finger polymers. Nevertheless, these studies demonstrate that zinc finger proteins can be constructed to bind very tightly and specifically to long DNA sequences, for targeting unique sites in complex genomes. Gene regulation using zinc finger transcription factors There are three general methods that have been used to affect the expression of genes using zinc finger gene switches. Firstly, it is possible to cause blockage of transcription by overexpressing a zinc finger DNA-binding domain that occupies the coding sequence of a gene [7]. Secondly, it is possible to inhibit transcription by binding to a gene promoter, presumably by occupying the DNA that is normally bound by other transcription factors [16]. Thirdly, synthetic transcription factors can be constructed by fusing transcription-regulating domains to zinc finger DNA-binding domains [14,17,19,38]. Our experience is that each of these methods can be effective; however, the magnitude of the effect depends on the particular system that is studied. In principle, the most advantageous type of gene switch is that which is most frequently used by nature, namely transcription factors containing effector domains. Such zinc finger proteins are generally very reliable and effective regulators of gene expression, and can be used either as inducers or as inhibitors, depending on the effector domain. Moreover, the zinc fingers need not target a particular site in a promoter or gene, but instead may be engineered to any proximal DNA sequence. The gene regulation experiments carried out so far have mainly involved transient co-transfections of reporters containing synthetic promoters with different numbers of binding sites for zinc fingers and various dosages of plasmids expressing zinc finger transcription factors [7,12 18,19,20]. In future, we expect that there will be an increasing number of experiments performed on systems that more closely approximate the major applications of designer transcription factors, for example, functional genomics or gene therapy applications. This will involve using integrated reporters or endogenous genes, and more practical methods of zinc finger delivery. An impressive step in this direction was the recent use of an engineered zinc finger transcription factor fused to a potent repression domain to silence the erbb2/3 oncogenes in a tumourigenic cell line [19 ]. Amongst the experiments presented were the viral delivery of the zinc finger transcription factor and the use of the standard tetracycline-inducible system to control the dosage of transcription factor (see also Update). Emerging applications In addition to controlling gene expression, DNA-binding proteins are important in DNA restriction and modification,

Zinc finger engineering Choo and Isalan 415 DNA recombination, DNA integration and a number of other functions. As some of these proteins also comprise modular DNA-binding and effector domains, there is every hope that zinc finger chimaeras will provide the basis for further manipulations of DNA. A particularly good example of this is the fusion of zinc fingers to various endonuclease domains [20,39,40] that have been used to cleave DNA both in vitro and in vivo [41 ]. Zinc finger fusions have also been made with an integrase domain from HIV [21] and with methylase M SssI [22]. Interestingly, zinc fingers have also been recently engineered to bind selectively to specifically methylated DNA the methylation can be introduced either by naturally occurring methylases [26 ] or presumably by using chimaeric zinc finger methylases. Therefore, the prospect exists of creating circuits of interdependent DNA-binding proteins containing zinc fingers, in order to manipulate gene expression or DNA itself. Update A thorough study into the repression of a target reporter gene in cell culture has recently been published [42]. By using an ecdysone-dependent zinc finger expression system, control of the target gene is both inducible and reversible. Kim s group also showed that designer zinc finger proteins can access binding sites that are integrated into the genome, demonstrating that these transcription factors can access DNA promoters that are stably packaged into chromatin. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Choo Y, Klug A: Designing DNA-binding proteins on the surface of filamentous phage. Curr Opin Biotechnol 1995, 6:431-436. 2. Choo Y, Klug A: Physical basis of a protein-dna recognition code. Curr Opin Struct Biol 1997, 7:117-125. 3. Rebar EJ, Pabo CO: Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 1994, 263:671-673. 4. Jamieson AC, Kim S-H, Wells JA: In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry 1994, 33:5689-5695. 5. Choo Y, Klug A: Toward a code for the interactions of zinc fingers with DNA: selection of randomised zinc fingers displayed on phage. Proc Natl Acad Sci USA 1994, 91:11163-11167. 6. Wu H, Yang W-P, Barbas CF III: Building zinc fingers by selection: toward a therapeutic application. Proc Natl Acad Sci USA 1995, 92:344-348. 7. Choo Y, Sanchez-Garcia I, Klug A: In vivo repression by a sitespecific DNA-binding protein designed against an oncogenic sequence. Nature 1994, 372:642-645. 8. Isalan M, Choo Y, Klug A: Synergy between adjacent zinc fingers in sequence-specific DNA recognition. Proc Natl Acad Sci USA 1997, 94:5617-5621. 9. Isalan M, Klug A, Choo Y: Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers. Biochemistry 1998, 37:12026-12033. 10. Greisman HA, Pabo CO: A general strategy for selecting highaffinity zinc finger proteins for diverse DNA target sites. Science 1997, 275:657-661. 11. Segal DJ, Dreier B, Beerli RR, Barbas CF: Toward controlling gene expression at will: selection and design of zinc finger domains recognising each of the 5 -GNN-3 DNA target sequences. Proc Natl Acad Sci USA 1999, 96:2758-2763. A library of the middle zinc finger of Zif268 was used to select variants that recognise the 16 different trinucleotides of the form GNN. These can be spliced to construct zinc finger polymers that bind DNA sequences in which guanine is repeated every three nucleotides. See also [18]. 12. Kim J-S, Pabo CO: Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. Proc Natl Acad Sci USA 1998, 95:2812-2817. 13. Liu Q, Segal DJ, Ghiara JB, Barbas CF III: Design of polydactyl zincfinger proteins for unique addressing within complex genomes. Proc Natl Acad Sci USA 1997, 94:5525-5530. 14. Pomerantz JL, Sharp PL, Pabo CO: Structure based design of transcription factors. Science 1995, 267:93-96. 15. Kim J-S, Kim J, Cepek KL, Sharp PA, Pabo CO: Design of TATA boxbinding protein/zinc finger fusions for targeted regulation of gene expression. Proc Natl Acad Sci USA 1997, 94:3616-3620. 16. Kim J-S, Pabo CO: Transcriptional repression by zinc finger peptides. J Biol Chem 1997, 272:29795-29800. 17. Choo Y, Castellanos A, Garcia-Hernandez B, Sanchez-Garcia I, Klug A: Promoter-specific activation of gene expression directed by bacteriophage-selected zinc fingers. J Mol Biol 1997, 273:525-532. 18. Beerli RR, Segal DJ, Dreier B, Barbas CF: Toward controlling gene expression at will: specific regulation of the erbb-2/her-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci USA 1998, 95:14628-14633. 19. Beerli RR, Dreier B, Barbas CF: Positive and negative regulation of endogenous genes by designed transcription factors. Proc Natl Acad Sci USA 2000, 97:1495-1500. Following on from the results described in [18], transcription factors designed against the erbb-2 and erbb-3 oncogene promoters were used to regulate these genes in transient transfection of the breast cancer cell line SKBR3. 20. Chandrasegaran S, Smith J: Chimaeric restriction enzymes: what is next? Biol Chem 1999, 380:841-848. 21. Bushman FD, Miller MD: Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites. J Virol 1997, 71:458-464. 22. Xu G-L, Bestor TH: Cytosine methylation targeted to predetermined sequences. Nat Genet 1997, 17:376-378. 23. Pavletich NP, Pabo CO: Zinc finger-dna recognition: crystal structure of a Zif268-DNA complex at 2.1 Å. Science 1991, 252:809-817. 24. Elrod-Erickson M, Rould MA, Nekludova L, Pabo CO: Zif268 protein- DNA complex refined at 1.6 Å: a model system for understanding zinc finger interactions. Structure 1996, 4:1171-1180. 25. Choo Y: Recognition of DNA methylation by zinc fingers. Nat Struct Biol 1998, 5:264-265. 26. Isalan M, Choo Y: Engineered zinc finger proteins that recognise DNA modification by HaeIII and HhaI methyltransferase enzymes. J Mol Biol 2000, 295:471-477. Following on from the results in [25], it is shown that bacterial methyltransferases can be used to induce zinc finger DNA binding through the modification of the DNA. 27. Wolfe SA, Greisman HA, Ramm EI, Pabo CO: Analysis of zinc fingers optimised via phage display: evaluating the utility of a recognition code. J Mol Biol 1999, 285:1917-1934. 28. Desjarlais JR, Berg JM: Toward rules relating zinc finger protein sequences and DNA binding site preferences. Proc Natl Acad Sci USA 1992, 89:7345-7349. 29. Desjarlais JR, Berg JM: Use of a zinc finger consensus sequence framework and specificity rules to design specific DNA binding proteins. Proc Natl Acad Sci USA 1993, 90:2256-2260. 30. Choo Y, Klug A: Selection of DNA binding sites for zinc fingers using rationally randomised DNA reveals coded interactions. Proc Natl Acad Sci USA 1994, 91:11168-11172.

416 Engineering and design 31. Elrod-Erickson M, Benson TE, Pabo CO: High-resolution structures of variant Zif268-DNA complexes: implications for understanding zinc finger-dna recognition. Structure 1998, 6:451-464. 32. Corbi N, Perez M, Maione R, Passananti C: Synthesis of a new zinc finger peptide; comparison of its code deduced and CASTing derived binding sites. FEBS Lett 1997, 417:71-74. 33. Corbi N, Libri V, Fanciulli M, Passananti C: Binding properties of the artificial zinc fingers coding gene Sint1. Biochem Biophys Res Commun 1998, 253:686-692. 34. Pomerantz JL, Wolfe SA, Pabo CO: Structure-based design of a dimeric zinc finger protein. Biochemistry 1998, 37:965-970. 35. Wang BS, Pabo CO: Dimerisation of zinc fingers mediated by peptides evolved in vitro from random sequences. Proc Natl Acad Sci USA 1999, 96:9568-9573. This paper reports the imaginative use of polyvalent phage display to select peptide sequences that are capable of mediating zinc finger homodimerisation on DNA. The DNA-binding domains recognise perfectly adjacent inverted repeats (c.f. [34]). 36. Nakaseko Y, Neuhaus D, Klug A, Rhodes D: Adjacent zinc-finger motifs in multiple zinc-finger peptides from SWI5 form structurally independent, flexibly linked domains. J Mol Biol 1992, 228:619-636. 37. Kamiuchi T, Abe E, Imanishi M, Kaji T, Nagaoka M, Sugiura Y: Artificial nine zinc-finger peptide with 30 base pair binding sites. Biochemistry 1998, 37:13827-13834. 38. Thiesen HJ: From repression domains to designer finger proteins: a novel strategy of intracellular immunisation against HIV. Gene Expr 1996, 5:229-243. 39. Nahon E, Raveh D: Targeting a truncated HO-endonuclease of yeast to novel DNA sites with foreign zinc fingers. Nucleic Acids Res 1998, 26:1233-1239. 40. Smith J, Berg JM, Chandrasegaran S: A detailed study of the substrate specificity of a chimaeric restriction enzyme. Nucleic Acids Res 1999, 27:674-681. 41. Carroll D, Segal DJ, Trautman JK, Smith J, Kim Y-G, Chandrasegaran S: Stimulation of homologous recombination through targeted cleavage by a chimaeric nuclease. Proc Natl Acad Sci USA 2000, in press. A FokI zinc finger chimaeric nuclease was used to cut microinjected DNA targets in frog oocytes. Site-specific cleavage directs homologous recombination to specific sites within the genome. 42. Kang JS, Kim J-S: Zinc finger proteins as designer transcription factors. J Biol Chem 2000, 275:8742-8748. Patent P1. Isalan MD, Klug A, Choo Y: International Patent Application Number WO98/53057. A universal method to engineer customised DNA-binding domains. Premade libraries allow the rapid screening of target sites, while harnessing the synergy between adjacent zinc fingers (see Figure 1c). The process is illustrated by targeting multiple sites in the HIV-1 promoter.