- Email: [email protected]
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins
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Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins David J Segal and Carlos F Barbas III* Artificial transcription factors based on modified zinc-finger DNA-binding domains have been shown to activate or repress the transcription of endogenous genes in multiple organisms. Advances in both the construction of novel zinc-finger proteins and our understanding of the characteristics of a productive regulatory site have fueled these achievements.
Figure 1 F1
Addresses The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, BCC-550, North Torrey Pines Road, La Jolla, CA 92037, USA *e-mail: [email protected]
F2
Current Opinion in Biotechnology 2001, 12:632–637 0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations DBD DNA-binding domain ED effector domain
F3
Introduction The ability to wilfully manipulate gene expression holds tremendous promise for both basic and applied research. Primary methods for understanding gene function involve enhancing or abolishing the expression of a gene, followed by observation of phenotypic effects. The field of functional genomics, the effort to assign function to all of the genes identified from the sequencing of the human and other genomes, has become the driving force for the development of methodologies that are faster, simpler, and more broadly applicable than classical techniques. Knowledge of gene function coupled with improved methods to regulate gene expression is also stimulating new approaches and advances in the development of applications such as animal models for human disease and novel therapeutics. Creating a system that can both upregulate and downregulate gene expression requires control at the level of transcription. Transcriptional regulation in all living cells is mediated by protein transcription factors. These factors are typically composed of at least two parts: a sequencespecific DNA-binding domain (DBD) and an effector domain (ED) [1]. The DBD directs binding of the factor to the promoter region of a particular gene or genes, whereas the ED acts through protein–protein interactions to recruit components of activation or repression complexes. Many EDs have been shown to maintain their ability to activate or repress transcription when attached to heterologous DBDs [2,3]. Therefore, the task of creating an artificial transcription factor can be reduced to attaching an ED to a custom DNA-binding protein — one that can be targeted to any desired gene. This review describes recent
Current Opinion in Biotechnology
The structure of a three-domain zinc-finger protein (based on [43]). Fingers 1, 2 and 3 (labeled F1, F2 and F3, respectively) are shown as ribbons. Zinc atoms are depicted as spheres. The protein wraps through the major groove of the DNA (wire and shaded object).
advances in zinc-finger technology that now make custom DNA-binding proteins readily available and details how these proteins are being used to regulate the expression of transgenes and endogenous genes.
Construction methodology Among the many naturally occurring DNA-binding proteins, the Cys2-His2 zinc-finger domain has emerged as the scaffold of choice for the design of novel sequencespecific DNA-binding proteins [4]. Each 30 amino acid residue domain or finger forms a stable ββα fold (Figure 1). The N terminus of the α helix recognizes a small patch of nucleotides in the major groove of the DNA, typically three base pairs. Recognition of extended sequences is achieved by linking the domains in tandem arrays. The versatility of zinc-finger proteins in DNA recognition is perhaps best reflected by its success in natural systems. The Cys2-His2 zinc-finger domain is the most commonly found DNA-binding motif in eukaryotes, and the second most frequently encoded protein domain in the human
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Segal and Barbas
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Figure 2 Representations of (a) parallel, (b) sequential and (c) bipartite library construction methods. Individual fingers and their approximate recognition sites are color-matched. Anchor fingers are in gray. Libraries of variant fingers are circled. Fingers selected from the library are then used in the next step of construction (illustrated by arrows). Helices in (b) and (c) have magenta and cyan shading to suggest possible interactions between domains.
(a)
(b)
(c)
Current Opinion in Biotechnology
genome [5–7]. These features have attracted researchers intent on creating novel, sequence-specific DNA-binding proteins. The randomization and selection methodologies that have allowed us to endow zinc fingers with new binding specificities are also reminiscent of nature’s own evolutionary forces, albeit a few million years faster. Three successful selection methods have been described. All have been based on the display of randomized zinc-finger proteins from the surface of filamentous bacteriophage, although an alternative in vivo bacterial system has also been described [8•]. The parallel and sequential selection schemes have been compared recently [4,9]. In the parallel approach (Figure 2a), DNA-contacting residues in the middle finger of a three-finger protein are randomized to form a library of variants [10–13,14•,15••,16,17]. The two flanking fingers are unmodified and serve to anchor and appropriately orient the middle finger. Using phage display, variants are selected that recognize a new three-base pair sequence in the middle of their binding site. Following selection and optimization of fingers that can recognize each of the 64 possible three-base pair subsites, the fingers can be assembled in any order necessary to form new three- or six-finger proteins capable of binding any desired sequence. With each domain specifying three base pairs, a six-finger protein should have the capacity to bind one of almost 70 billion unique 18 base-pair sites. When presented with the 3.2 billion base pairs of DNA in the human genome, a six-finger protein has the potential to recognize a unique site.
As the selection process is not dependent on any particular DNA target sequence, all 64 required domains could be selected in parallel. An advantage of this method is that once selection and optimization are complete, new proteins can be constructed in a matter of hours using standard PCR methods. Consequently, this type of method has been adopted by our laboratory [11,15••,18••] and for commercial purposes [19••,20••]. Potential limitations of this approach lie in its underpinning assumptions: that each domain recognizes only three base pairs and that the domains can be assembled in any order. The major limitation of this strategy is seen in the phenomenon called target-site overlap that occurs in Zif268. This occurs because one of the anchor fingers of this protein actually recognizes a four base-pair subsite, overlapping the subsite of the middle finger and forcing specification of its 5′ nucleotide to be G or T [21]. Indeed, our early studies bypassed these concerns by focusing on fingers capable of recognizing members of the 5′-GNN-3′ set of DNA sequences [12,14•]. The limitations imposed by target-site overlap have, however, recently been overcome. Sequential selection (Figure 2b) was developed to address the concerns of target-site overlap [22••,23,24]. In this method, a terminal finger of a three-finger protein is randomized while the other two act as anchors. However, following selection of a finger that recognizes a new DNA subsite, one of the anchor fingers is removed and a new library is appended to the previously selected finger. A third cycle of removal, appendage addition, and selection
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Figure 3 (a) ED = VP64
DBD = Zinc finger
Activation
(b) ED = KRAB
DBD = Zinc finger
Repression
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Transcriptional activation or repression by a TFZF bound in the 5′ UTR of its target gene. (a) A TFZF composed of a custom zinc-finger DBD and a VP64 ED (a derivative of VP16 [11]) causes activation (indicated by a bold arrow). (b) A TFZF composed of a custom zinc-finger DBD fused to the KRAB ED leads to repression (indicated by a bold cross).
creates a sequence-specific three-finger protein. In this way, the new finger is always selected in the ‘context’ of the previous finger, with no assumptions about overlap or modularity. A recent crystal structure of a sequentially selected protein with its cognate DNA seems to validate these concerns. Some fingers were found to recognize not only four base pairs but five, and interdomain interactions were observed [22••]. The primary disadvantage of this system is that multiple rounds of library construction and selection are required for each new DNA target. A six-finger protein would require six sequential construction and selection steps, making the procedure extremely laborious and beyond the technical reach of most laboratories. Recently, a new method was reported that attempts to embrace the virtues of the former two approaches while overcoming their obstacles. The bipartite library approach (Figure 2c) involves randomization of one-and-a-half fingers of a three-finger protein [25•,26]. This maximizes the possibility for interdomain cooperativity. To circumvent the technical limitations of cloning and displaying such a large number of variants, randomization is limited to only a subset of the 20 amino acids. Only two pre-constructed
libraries are required to create a new three-finger protein: one randomized at the N terminus and one randomized at the C terminus. After selection, the two libraries are recombined and final sets of selections are performed to obtain the optimal recombinant protein. Although assumptions are introduced by restricting the number, type, and position of randomized amino acids, this method addresses the concerns of target-site overlap while shortening construction time, according to the authors, to 10–14 days. All three methods produce proteins of comparable affinity (when affinity data are normalized to that of the protein Zif268) [4,25•]. Early pessimism that the parallel method could produce only zinc fingers that recognize 5′-GNN-3′type sequences was recently dispelled with the report of domains that bind 5′-ANN-3′ sequences [15••]. This was accomplished by eliminating target-site overlap from the offending anchor finger. Although fingers binding 5′-GNN-3′ or 5′-ANN-3′ subsites represent only half of the domains required for comprehensive recognition, a six-finger site recognizable by these domains should occur every 32 base pairs. Using these 32 domains, over one billion different six-finger proteins can now be constructed (i.e. 27 000 proteins for every gene in the human genome). Furthermore, zinc-finger domains that bind the 5′-TNN-3′ and 5′-CNN-3′ subsites can also be prepared in this manner.
Cellular gene regulation The attachment of appropriate EDs to zinc-finger DBDs creates potent transcriptional activators and repressors. Activation domains such as VP16 [3] and p65 [27] and repression domains such as KRAB [2] and SID [28] are components of naturally occurring transcription factors. All have been shown to regulate a variety of promoters in a distance- and orientation-independent manner when fused to heterologous DBDs. Fusion of these domains with custom zinc-finger DBDs results in artificial transcription factors (TFZF) that can upregulate and downregulate genes (Figure 3) [11]. This is significant as, for the first time, regulation is possible using unmodified, genomic DNA sequences (i.e. without the pre-insertion of artificial binding sites). The ability to study and manipulate genes in their native chromatin environment, a previously unapproachable task, represents a major advance. Within the past year, TFZFs have been shown to regulate the endogenous chromosomal genes for ErbB-2 and ErbB-3 [15••,18••], Epo [20••], VEGF-A [19••], and AP3 [29••,30••] (Table 1). The ErbB2-specific TFZF targeted a highly conserved sequence in the gene, and was shown to function in human, mouse and monkey cells [18••]. Furthermore, by using tetracycline regulation, chemical control of an endogenous gene was imposed. The AP3-specific TFZF functions in plant cell culture and whole plants. The ErbB-2-specific TFZF was shown to upregulate (with a VP16 derivative ED) or downregulate (with a KRAB ED) erbB-2, but not erbB-3. The reverse was also true for the ErbB-3-specific TFZF. The
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Segal and Barbas
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Table 1 Endogenous genes regulated by zinc finger–effector domain fusions. Gene product
Function
Target position
Number of fingers
Species
Activation
Repression
References
ErbB-2
Oncogene
5′-UTR
6
Human, monkey, mouse
✓
✓
[15••,18••]
ErbB-3
Oncogene
5′-UTR
6
Human
✓
✓
[15••,18••]
Epo
Erythropoiesis induction
Upstream
3
Human
✓
[20••]
VEGF-A
Angiogenesis induction
5′-UTR
3
Human
✓
[19••]
Flower development
5′-UTR
6
Arabidopsis
✓
Ap3
binding sites of these two factors differed by only three out of 18 base pairs, demonstrating that regulation was extremely specific. Endogenous gene expression was reduced to background levels, suggesting that TFZF can be used to make rapid, functional gene knockouts. An issue of current importance in the field is to understand the characteristics of a productive regulatory site. Zinc fingers, even without EDs, have been shown to inhibit transcriptional initiation if targeted very close to the initiation site [31,32]. Some of the most activating TFZFs were targeted downstream of the initiation site, however, demonstrating that they do not inhibit transcriptional elongation [18••,19••]. Affinity did not seem to correlate with activity, as long as the affinity was less than 10 nM [15••,18••–20••]. Local DNA topology and cellular binding proteins, such as transcription factors and histones, present challenges to in vivo target-site selection. Using DNase I hypersensitivity as an indicator of chromatin accessibility, TFZF targeted to DNase I-protected sites failed to regulate the VEGF-A gene, whereas others targeted to hypersensitive sites were activating [19••]. However, even within accessible regions some TFZFs were more activating than others, suggesting chromatin accessibility is necessary, but not sufficient, for productive regulation. More studies will be needed to understand what works best and why.
Conclusions Our understanding of zinc-finger–DNA interactions continues to advance rapidly, augmented recently by reports that have examined the effects of non-DNA-contacting residues on affinity and specificity [33–38]. Studies such as these, in conjunction with computer modeling [14•,15••] and structural studies [22••,39•], are producing an increasingly clear picture of a recognition domain that can interact with DNA in both simple and complex ways. The frontier of zinc-finger research is now shifting beyond DNA-recognition to novel applications. Aside from gene regulation, zinc-finger-based endonucleases were recently shown to stimulate homologous recombination in eukaryotic cells [40••]. Strategies for creating ligand-dependent TFZFs have also been reported [41•,42•] (and reviewed in [9]), while improved delivery systems are being investigated.
✓
[29••,30••]
With the barriers of sequence recognition and small-molecule regulation breached, zinc-finger transcription factors are serious candidates as gene therapeutics. The robust features of this technology that provide for the rapid assembly of transcription factors from pre-defined zincfinger domains should also ensure their application in functional genomics.
Acknowledgements We thank Laurent Magnenat for his comments on this manuscript.
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.
Ptashne M: Control of gene transcription: an outline. Nat Med 1997, 3:1069-1072.
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Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ, Rauscher FJ III: Kruppel-associated boxes are potent transcriptional repression domains. Proc Natl Acad Sci USA 1994, 91:4509-4513.
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Sadowski I, Ma J, Triezenberg S, Ptashne M: GAL4-VP16 is an unusually potent transcriptional activator. Nature 1988, 335:563-564.
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Segal DJ, Barbas CF: Design of novel sequence-specific DNA-binding proteins. Curr Opin Chem Biol 2000, 4:34-39.
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Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W et al.: Initial sequencing and analysis of the human genome. Nature 2001, 409:860-921.
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Tupler R, Perini G: Green MR: expressing the human genome. Nature 2001, 409:832-833.
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Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA et al.: The sequence of the human genome. Science 2001, 291:1304-1351.
8. •
Joung JK, Ramm EI, Pabo CO: A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions. Proc Natl Acad Sci USA 2000, 97:7382-7387. A novel survival-based selection strategy similar to the yeast two-hybrid system was developed for the selection of zinc-finger proteins. This strategy is a unique alternative to phage display for the selection of novel zinc-finger domains. 9.
Beerli RR, Barbas CF III: The age of polydactyl zinc finger transcription factors is upon us. Nat Biotechnol 2001, in press.
10. Barbas CF III, Rader C, Segal DJ, List B, Turner JM: From catalytic asymmetric synthesis to the transcriptional regulation of genes: in vivo and in vitro evolution of proteins. Adv Protein Chem 2000, 55:317-366. 11. Beerli RR, Segal DJ, Dreier B, Barbas CF III: 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.
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12. Segal DJ, Dreier B, Beerli RR, Barbas CF III: Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′′-GNN-3′′ DNA target sequences. Proc Natl Acad Sci USA 1999, 96:2758-2763. 13. 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. 14. Dreier B, Segal DJ, Barbas CF III: Insights into the molecular • recognition of the 5′′-GNN-3′′ family of DNA sequences by zinc finger domains. J Mol Biol 2000, 303:489-502. This paper describes insights gained from the characterization of 84 domains en route to the selection of the 16 optimal domains for specific recognition of the 5′-GNN-3′ family of DNA sequences described in [12]. Using this information, and methods described in [11], any laboratory can rapidly construct a protein to bind a unique site in virtually any gene. 15. Dreier B, Beerli RR, Segal DJ, Flippin JD, Barbas CF III: •• Development of zinc finger domains for recognition of the 5′′-ANN-3′′ family of DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem 2001, 276:29466-29478. This paper describes domains optimized by parallel selection and mutagenesis for specific recognition of the 5′-ANN-3′ family of DNA sequences. An effective strategy for avoiding the limitations of target-site overlap is presented. The efficacy of this approach is demonstrated with transgene and endogenous gene regulation studies using these novel domains. Together with domains described in [12,14•], over one billion unique 18 base pair DNA-binding proteins can be rapidly constructed. 16. Choo Y, Klug A: Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage. Proc Natl Acad Sci USA 1994, 91:11163-11167. 17.
Choo Y, Klug A: Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. Proc Natl Acad Sci USA 1994, 91:11168-11172.
18. Beerli RR, Dreier B, Barbas CF III: Positive and negative regulation •• of endogenous genes by designed transcription factors. Proc Natl Acad Sci USA 2000, 97:1495-1500. This paper provides the first demonstration that endogenous genes can be both activated and repressed using zinc-finger-based transcription factors. The efficacy of the approach is demonstrated by targeting and regulating the erbB-2 oncogene in multiple species. In addition, the study demonstrates for the first time that even endogenous genes can be placed under chemical control so that they may be activated or repressed by the addition of a small molecule. Regulation of another erb family member, erbB-3, is also reported. Highly specific regulation of the two erb genes was observed despite a 15 (out of a total of 18) base-pair match between the sites targeted in these two genes. 19. Liu PQ, Rebar EJ, Zhang L, Liu Q, Jamieson AC, Liang Y, Qi H, Li PX, •• Chen B, Mendel MC et al.: Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J Biol Chem 2001, 276:11323-11334. See comments for [20••]. 20. Zhang L, Spratt SK, Liu Q, Johnstone B, Qi H, Raschke EE, •• Jamieson AC, Rebar EJ, Wolffe AP, Case CC: Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene. J Biol Chem 2000, 275:33850-33860. This paper and [19••] describe the third and fourth endogenous genes to be regulated by artificial transcription factors, proving that the methods are generally applicable. Specific gene regulation is achieved with three-finger proteins that bind nine base pairs of DNA. Excellent studies of chromatin accessibility are presented and discussed in relation to the application of the zinc-finger approach to gene regulation. 21. 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. 22. Wolfe SA, Grant RA, Elrod-Erickson M, Pabo CO: Beyond the •• ‘recognition code’: structures of two Cys2His2 zinc finger/TATA box complexes. Structure 2001, 9:717-723. Crystal structures of sequentially selected proteins are described. The results show that even fingers in the standard framework of Zif268 can support unexpected DNA contacts and show extensive target-site overlap. Current recognition codes for understanding zinc-finger–DNA interactions are therefore context-dependent and not generally applicable. 23. Wolfe SA, Greisman HA, Ramm EI, Pabo CO: Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code. J Mol Biol 1999, 285:1917-1934.
24. Greisman HA, Pabo CO: A general strategy for selecting highaffinity zinc finger proteins for diverse DNA target sites. Science 1997, 275:657-661. 25. Isalan M, Klug A, Choo Y: A rapid, generally applicable method to • engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat Biotechnol 2001, 19:656-660. This work demonstrates the utility of the bipartite library selection method for zinc-finger design. The authors use the approach to derive three-finger proteins that bind an HIV-1 promoter sequence. 26. Isalan M, Patel SD, Balasubramanian S, Choo Y: Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation. Biochemistry 2001, 40:830-836. 27.
Fujita T, Nolan GP, Ghosh S, Baltimore D: Independent modes of κB. transcriptional activation by the p50 and p65 subunits of NF-κ Genes Dev 1992, 6:775-787.
28. Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M, Laherty CD, Torchia J, Yang WM, Brard G, Ngo SD, Davie JR et al.: A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 1997, 387:43-48. 29. Stege J, Guan X, Briggs S, Barbas CF III: Software and hardware •• for genomes: polydactyl zinc finger proteins and the regulation of endogenous genes. Keystone Symposia on a Systems Approach to Plant Biology: 2001 Jan 26–31; Big Sky, Montana. This study demonstrates the efficacy of zinc-finger technology to plant systems with the activation or repression of the Arabidopsis AP3 gene in plant cell culture and in transgenic plants. 30. Eckardt NA: The new biology. Genomics fosters a ‘systems •• approach’ and collaborations between academic, government, and industry scientists. Plant Cell 2001, 13:725-732. This meeting report discusses the potential of zinc-finger transcription factors in agricultural applications and functional genomics. 31. Kim JS, Pabo CO: Transcriptional repression by zinc finger peptides. Exploring the potential for applications in gene therapy. J Biol Chem 1997, 272:29795-29800. 32. Kang JS, Kim JS: Zinc finger proteins as designer transcription factors. J Biol Chem 2000, 275:8742-8748. 33. Nagaoka M, Nomura W, Shiraishi Y, Sugiura Y: Significant effect of linker sequence on DNA recognition by multi-zinc finger protein. Biochem Biophys Res Commun 2001, 282:1001-1007. 34. Uno Y, Matsushita K, Nagaoka M, Sugiura Y: Finger-positional change in three zinc finger protein Sp1: influence of terminal finger in DNA recognition. Biochemistry 2001, 40:1787-1795. 35. Nagaoka M, Kaji T, Imanishi M, Hori Y, Nomura W, Sugiura Y: Multiconnection of identical zinc finger: implication for DNA binding affinity and unit modulation of the three zinc finger domain. Biochemistry 2001, 40:2932-2941. 36. Imanishi M, Hori Y, Nagaoka M, Sugiura Y: DNA-bending finger: artificial design of 6-zinc finger peptides with polyglycine linker and induction of DNA bending. Biochemistry 2000, 39:4383-4390. 37.
Moore M, Choo Y, Klug A: Design of polyzinc finger peptides with structured linkers. Proc Natl Acad Sci USA 2001, 98:1432-1436.
38. Moore M, Klug A, Choo Y: Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units. Proc Natl Acad Sci USA 2001, 98:1437-1441. 39. Wang BS, Grant RA, Pabo CO: Selected peptide extension • contacts hydrophobic patch on neighboring zinc finger and mediates dimerization on DNA. Nat Struct Biol 2001, 8:589-593. This study provides a fascinating structural glimpse at a peptide selected and evolved from a random phage-display library to facilitate the dimerization of zinc-finger domains on a DNA template. 40. Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, •• Chandrasegaran S: Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 2001, 21:289-297. This study demonstrates for the first time that a zinc finger–Fok I endonuclease fusion can cleave chromatinized DNA and stimulate recombination in eukaryotic cells. It also brilliantly explores the requirements for both the DNA target and the protein to produce optimal cleavage.
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Segal and Barbas
41. Beerli RR, Schopfer U, Dreier B, Barbas CF III: Chemically regulated • zinc finger transcription factors. J Biol Chem 2000, 275:32617-32627. Fusion of 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 provided 4-hydroxytamoxifen- or RU486-inducible expression systems with induction ratios of up to three orders of magnitude. These inducible expression systems were functionally independent and could be selectively switched on within the same cell. The potential of single-chain steroid hormone receptors is reported for the first time, as shown by the fusion of either two estrogen receptor ligand-binding domains or one ecdysone receptor and one retinoid X receptor ligand-binding domain. These single-chain receptor proteins
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undergo intramolecular, rather than intermolecular, dimerization and are functional as monomers. 42. Xu L, Zerby D, Huang Y, Ji H, Nyanguile OF, de los Angeles JE, • Kadan MJ: A versatile framework for the design of liganddependent, transgene-specific transcription factors. Mol Ther 2001, 3:262-273. This report demonstrates the efficacy of the type of steroid hormone systems described in [41•] in the regulation of an adenovirus-delivered endostatin transgene in mice. 43. 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–DNA interactions. Structure 1996, 4:1171-1180.
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins David J Segal and Carlos F Barbas III* Artificial transcription factors based on modified zinc-finger DNA-binding domains have been shown to activate or repress the transcription of endogenous genes in multiple organisms. Advances in both the construction of novel zinc-finger proteins and our understanding of the characteristics of a productive regulatory site have fueled these achievements.
Figure 1 F1
Addresses The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, BCC-550, North Torrey Pines Road, La Jolla, CA 92037, USA *e-mail: [email protected]
F2
Current Opinion in Biotechnology 2001, 12:632–637 0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations DBD DNA-binding domain ED effector domain
F3
Introduction The ability to wilfully manipulate gene expression holds tremendous promise for both basic and applied research. Primary methods for understanding gene function involve enhancing or abolishing the expression of a gene, followed by observation of phenotypic effects. The field of functional genomics, the effort to assign function to all of the genes identified from the sequencing of the human and other genomes, has become the driving force for the development of methodologies that are faster, simpler, and more broadly applicable than classical techniques. Knowledge of gene function coupled with improved methods to regulate gene expression is also stimulating new approaches and advances in the development of applications such as animal models for human disease and novel therapeutics. Creating a system that can both upregulate and downregulate gene expression requires control at the level of transcription. Transcriptional regulation in all living cells is mediated by protein transcription factors. These factors are typically composed of at least two parts: a sequencespecific DNA-binding domain (DBD) and an effector domain (ED) [1]. The DBD directs binding of the factor to the promoter region of a particular gene or genes, whereas the ED acts through protein–protein interactions to recruit components of activation or repression complexes. Many EDs have been shown to maintain their ability to activate or repress transcription when attached to heterologous DBDs [2,3]. Therefore, the task of creating an artificial transcription factor can be reduced to attaching an ED to a custom DNA-binding protein — one that can be targeted to any desired gene. This review describes recent
Current Opinion in Biotechnology
The structure of a three-domain zinc-finger protein (based on [43]). Fingers 1, 2 and 3 (labeled F1, F2 and F3, respectively) are shown as ribbons. Zinc atoms are depicted as spheres. The protein wraps through the major groove of the DNA (wire and shaded object).
advances in zinc-finger technology that now make custom DNA-binding proteins readily available and details how these proteins are being used to regulate the expression of transgenes and endogenous genes.
Construction methodology Among the many naturally occurring DNA-binding proteins, the Cys2-His2 zinc-finger domain has emerged as the scaffold of choice for the design of novel sequencespecific DNA-binding proteins [4]. Each 30 amino acid residue domain or finger forms a stable ββα fold (Figure 1). The N terminus of the α helix recognizes a small patch of nucleotides in the major groove of the DNA, typically three base pairs. Recognition of extended sequences is achieved by linking the domains in tandem arrays. The versatility of zinc-finger proteins in DNA recognition is perhaps best reflected by its success in natural systems. The Cys2-His2 zinc-finger domain is the most commonly found DNA-binding motif in eukaryotes, and the second most frequently encoded protein domain in the human
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Segal and Barbas
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Figure 2 Representations of (a) parallel, (b) sequential and (c) bipartite library construction methods. Individual fingers and their approximate recognition sites are color-matched. Anchor fingers are in gray. Libraries of variant fingers are circled. Fingers selected from the library are then used in the next step of construction (illustrated by arrows). Helices in (b) and (c) have magenta and cyan shading to suggest possible interactions between domains.
(a)
(b)
(c)
Current Opinion in Biotechnology
genome [5–7]. These features have attracted researchers intent on creating novel, sequence-specific DNA-binding proteins. The randomization and selection methodologies that have allowed us to endow zinc fingers with new binding specificities are also reminiscent of nature’s own evolutionary forces, albeit a few million years faster. Three successful selection methods have been described. All have been based on the display of randomized zinc-finger proteins from the surface of filamentous bacteriophage, although an alternative in vivo bacterial system has also been described [8•]. The parallel and sequential selection schemes have been compared recently [4,9]. In the parallel approach (Figure 2a), DNA-contacting residues in the middle finger of a three-finger protein are randomized to form a library of variants [10–13,14•,15••,16,17]. The two flanking fingers are unmodified and serve to anchor and appropriately orient the middle finger. Using phage display, variants are selected that recognize a new three-base pair sequence in the middle of their binding site. Following selection and optimization of fingers that can recognize each of the 64 possible three-base pair subsites, the fingers can be assembled in any order necessary to form new three- or six-finger proteins capable of binding any desired sequence. With each domain specifying three base pairs, a six-finger protein should have the capacity to bind one of almost 70 billion unique 18 base-pair sites. When presented with the 3.2 billion base pairs of DNA in the human genome, a six-finger protein has the potential to recognize a unique site.
As the selection process is not dependent on any particular DNA target sequence, all 64 required domains could be selected in parallel. An advantage of this method is that once selection and optimization are complete, new proteins can be constructed in a matter of hours using standard PCR methods. Consequently, this type of method has been adopted by our laboratory [11,15••,18••] and for commercial purposes [19••,20••]. Potential limitations of this approach lie in its underpinning assumptions: that each domain recognizes only three base pairs and that the domains can be assembled in any order. The major limitation of this strategy is seen in the phenomenon called target-site overlap that occurs in Zif268. This occurs because one of the anchor fingers of this protein actually recognizes a four base-pair subsite, overlapping the subsite of the middle finger and forcing specification of its 5′ nucleotide to be G or T [21]. Indeed, our early studies bypassed these concerns by focusing on fingers capable of recognizing members of the 5′-GNN-3′ set of DNA sequences [12,14•]. The limitations imposed by target-site overlap have, however, recently been overcome. Sequential selection (Figure 2b) was developed to address the concerns of target-site overlap [22••,23,24]. In this method, a terminal finger of a three-finger protein is randomized while the other two act as anchors. However, following selection of a finger that recognizes a new DNA subsite, one of the anchor fingers is removed and a new library is appended to the previously selected finger. A third cycle of removal, appendage addition, and selection
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Figure 3 (a) ED = VP64
DBD = Zinc finger
Activation
(b) ED = KRAB
DBD = Zinc finger
Repression
Current Opinion in Biotechnology
Transcriptional activation or repression by a TFZF bound in the 5′ UTR of its target gene. (a) A TFZF composed of a custom zinc-finger DBD and a VP64 ED (a derivative of VP16 [11]) causes activation (indicated by a bold arrow). (b) A TFZF composed of a custom zinc-finger DBD fused to the KRAB ED leads to repression (indicated by a bold cross).
creates a sequence-specific three-finger protein. In this way, the new finger is always selected in the ‘context’ of the previous finger, with no assumptions about overlap or modularity. A recent crystal structure of a sequentially selected protein with its cognate DNA seems to validate these concerns. Some fingers were found to recognize not only four base pairs but five, and interdomain interactions were observed [22••]. The primary disadvantage of this system is that multiple rounds of library construction and selection are required for each new DNA target. A six-finger protein would require six sequential construction and selection steps, making the procedure extremely laborious and beyond the technical reach of most laboratories. Recently, a new method was reported that attempts to embrace the virtues of the former two approaches while overcoming their obstacles. The bipartite library approach (Figure 2c) involves randomization of one-and-a-half fingers of a three-finger protein [25•,26]. This maximizes the possibility for interdomain cooperativity. To circumvent the technical limitations of cloning and displaying such a large number of variants, randomization is limited to only a subset of the 20 amino acids. Only two pre-constructed
libraries are required to create a new three-finger protein: one randomized at the N terminus and one randomized at the C terminus. After selection, the two libraries are recombined and final sets of selections are performed to obtain the optimal recombinant protein. Although assumptions are introduced by restricting the number, type, and position of randomized amino acids, this method addresses the concerns of target-site overlap while shortening construction time, according to the authors, to 10–14 days. All three methods produce proteins of comparable affinity (when affinity data are normalized to that of the protein Zif268) [4,25•]. Early pessimism that the parallel method could produce only zinc fingers that recognize 5′-GNN-3′type sequences was recently dispelled with the report of domains that bind 5′-ANN-3′ sequences [15••]. This was accomplished by eliminating target-site overlap from the offending anchor finger. Although fingers binding 5′-GNN-3′ or 5′-ANN-3′ subsites represent only half of the domains required for comprehensive recognition, a six-finger site recognizable by these domains should occur every 32 base pairs. Using these 32 domains, over one billion different six-finger proteins can now be constructed (i.e. 27 000 proteins for every gene in the human genome). Furthermore, zinc-finger domains that bind the 5′-TNN-3′ and 5′-CNN-3′ subsites can also be prepared in this manner.
Cellular gene regulation The attachment of appropriate EDs to zinc-finger DBDs creates potent transcriptional activators and repressors. Activation domains such as VP16 [3] and p65 [27] and repression domains such as KRAB [2] and SID [28] are components of naturally occurring transcription factors. All have been shown to regulate a variety of promoters in a distance- and orientation-independent manner when fused to heterologous DBDs. Fusion of these domains with custom zinc-finger DBDs results in artificial transcription factors (TFZF) that can upregulate and downregulate genes (Figure 3) [11]. This is significant as, for the first time, regulation is possible using unmodified, genomic DNA sequences (i.e. without the pre-insertion of artificial binding sites). The ability to study and manipulate genes in their native chromatin environment, a previously unapproachable task, represents a major advance. Within the past year, TFZFs have been shown to regulate the endogenous chromosomal genes for ErbB-2 and ErbB-3 [15••,18••], Epo [20••], VEGF-A [19••], and AP3 [29••,30••] (Table 1). The ErbB2-specific TFZF targeted a highly conserved sequence in the gene, and was shown to function in human, mouse and monkey cells [18••]. Furthermore, by using tetracycline regulation, chemical control of an endogenous gene was imposed. The AP3-specific TFZF functions in plant cell culture and whole plants. The ErbB-2-specific TFZF was shown to upregulate (with a VP16 derivative ED) or downregulate (with a KRAB ED) erbB-2, but not erbB-3. The reverse was also true for the ErbB-3-specific TFZF. The
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Segal and Barbas
635
Table 1 Endogenous genes regulated by zinc finger–effector domain fusions. Gene product
Function
Target position
Number of fingers
Species
Activation
Repression
References
ErbB-2
Oncogene
5′-UTR
6
Human, monkey, mouse
✓
✓
[15••,18••]
ErbB-3
Oncogene
5′-UTR
6
Human
✓
✓
[15••,18••]
Epo
Erythropoiesis induction
Upstream
3
Human
✓
[20••]
VEGF-A
Angiogenesis induction
5′-UTR
3
Human
✓
[19••]
Flower development
5′-UTR
6
Arabidopsis
✓
Ap3
binding sites of these two factors differed by only three out of 18 base pairs, demonstrating that regulation was extremely specific. Endogenous gene expression was reduced to background levels, suggesting that TFZF can be used to make rapid, functional gene knockouts. An issue of current importance in the field is to understand the characteristics of a productive regulatory site. Zinc fingers, even without EDs, have been shown to inhibit transcriptional initiation if targeted very close to the initiation site [31,32]. Some of the most activating TFZFs were targeted downstream of the initiation site, however, demonstrating that they do not inhibit transcriptional elongation [18••,19••]. Affinity did not seem to correlate with activity, as long as the affinity was less than 10 nM [15••,18••–20••]. Local DNA topology and cellular binding proteins, such as transcription factors and histones, present challenges to in vivo target-site selection. Using DNase I hypersensitivity as an indicator of chromatin accessibility, TFZF targeted to DNase I-protected sites failed to regulate the VEGF-A gene, whereas others targeted to hypersensitive sites were activating [19••]. However, even within accessible regions some TFZFs were more activating than others, suggesting chromatin accessibility is necessary, but not sufficient, for productive regulation. More studies will be needed to understand what works best and why.
Conclusions Our understanding of zinc-finger–DNA interactions continues to advance rapidly, augmented recently by reports that have examined the effects of non-DNA-contacting residues on affinity and specificity [33–38]. Studies such as these, in conjunction with computer modeling [14•,15••] and structural studies [22••,39•], are producing an increasingly clear picture of a recognition domain that can interact with DNA in both simple and complex ways. The frontier of zinc-finger research is now shifting beyond DNA-recognition to novel applications. Aside from gene regulation, zinc-finger-based endonucleases were recently shown to stimulate homologous recombination in eukaryotic cells [40••]. Strategies for creating ligand-dependent TFZFs have also been reported [41•,42•] (and reviewed in [9]), while improved delivery systems are being investigated.
✓
[29••,30••]
With the barriers of sequence recognition and small-molecule regulation breached, zinc-finger transcription factors are serious candidates as gene therapeutics. The robust features of this technology that provide for the rapid assembly of transcription factors from pre-defined zincfinger domains should also ensure their application in functional genomics.
Acknowledgements We thank Laurent Magnenat for his comments on this manuscript.
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Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Segal and Barbas
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