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Zinc fingers and a green thumb: manipulating expression in plants David J Segaly, Justin T Stegez and Carlos F Barbas III§

Artificial factors can be rapidly constructed A variety of techniques have been developed to manip- from predefined -finger modules to regulate virtually any ulate gene expression in plants. Increased expression of gene. Stable, heritable up- and downregulation of endogenous is most commonly achieved through endogenous genes has been demonstrated in transgenic overexpression [1]. The introduction of tissue- plants. These advances promise new approaches for creating specific and inducible promoters has improved the utility functional knockouts and conditional overexpression, and of this approach, and efficient and robust plant transforma- for other gene discovery and manipulation applications in tion techniques have made the construction of plants. a relatively routine task. However, variable expression and co-suppression of transgenes often complicate this process. Addresses Furthermore, transgenes cannot accommodate alternative The Skaggs Institute for Chemical and the Department of splicing, which may be important for the appropriate , The Scripps Research Institute, La Jolla, function of some transgenes [2]. California 92037, USA yDepartment of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721, USA Reducing or eliminating the expression of a gene in plants zDiversa Corporation, San Diego, California 92121, USA is not as simple as overexpressing a gene. Gene disruption §The Scripps Research Institute, BCC-550, North Torrey Pines Road, by homologous recombination, tDNA insertions and che- La Jolla, California 92037, USA mical mutagenesis has been used successfully, but these e-mail: [email protected] Correspondence: Carlos F Barbas III approaches are inefficient and time-consuming technolo- gies. Several promising approaches for the repression of gene expression in animal systems have been described, Current Opinion in Plant Biology 2003, 6:163–168 operating either at the transcriptional or posttranscriptional This review comes from a themed issue on level [3–6]. Many of these techniques have also been Plant applied in plants. For example, one of the first demonstra- Edited by Wolf B Frommer and Roger Beachy tions that hammerhead could function as active endoribonucleases in vivo was performed in plant proto- 1369-5266/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. plasts [7]. Triplex-forming , which bound to homopurine/homopyrimidine tracts in a maize promo- DOI 10.1016/S1369-5266(03)00007-4 ter, have been shown to represses gene expression in vivo [8]. Chimeric RNA–DNA oligonucleotides have been Abbreviations used to cause site-specific base changes in episomal and Ap3 Apetella3 chromosomal targets in plant cells [9]. The use of antisense SID Sin3A interaction domain RNA or DNA to reduce mRNA levels was first explored in TFsZF zinc-finger-based artificial tobacco more than a decade ago [10]; today this approach remains one of the most commonly used methods to regulate genes in plants. More recently, double-stranded Introduction RNA interference (RNAi) has been employed to produce The manipulation of plant traits in basic plant biology specific and heritable genetic repression in Arabidopsis [11]. research and agricultural biotechnology would be greatly facilitated if endogenous genes-of-interest could be Back to nature: gene regulation with turned on or off in a controlled and selective manner. transcription factors Altering the expression of specific target genes could give In nature, the expression of eukaryotic nuclear genes is rise to healthier, hardier or more nutritious crop plants by tightly regulated at both the transcriptional and the increasing /stress resistance, altering metabolic translational level. Much of this control is achieved pathways or repressing the production of anti-nutritive through DNA-binding transcription factors. Transcrip- . Specific genes could be activated or repressed to tion factors are modular proteins that typically consist elucidate the complex interactions involved in many of a DNA-binding domain that localizes the to a important processes. In this review, we describe several specific DNA address and an domain that directs approaches to the manipulation of gene regulation in the type of activity to take place at the site [12,13]. plants, focusing in detail on one promising new method that involves zinc-finger-based artificial transcription fac- One conceptual approach to gene manipulation is the en- tors (TFsZF; Figure 1). gineered expression of specific endogenous transcription www.current-opinion.com Current Opinion in Plant Biology 2003, 6:163–168 164 Plant biotechnology

Figure 1 proteins in DNA recognition is perhaps best reflected by their success in natural systems. The Cys2-His2 zinc- finger domain is the most common DNA-binding motif in nature [23]. With 4500 examples identified, it is by far the most frequently encoded protein domain in the [24], and examples have also been found in various plants [24,25].

The rise and fall of universal recognition codes In canonical-type zinc fingers, the amino acids in posi- tions 1, 2, 3, and 6 of the a-helix make base-specific contacts [26]. Early observations of zinc-finger proteins gave rise to speculation that a particular in each position could recognize a particular base. The hope was that a simple universal one-amino-acid to one-base- pair (1aa:1bp) recognition code could be divined that would allow the construction of new DNA-binding pro- teins. Research carried out over the past several years has found, however, that simple recognition codes are insuf- ficient to predict true binding specificity [26,27,28– 31,32]. The primary weakness of simple recognition codes is that they fail to consider the influence of other amino acids within the helix and between helices. As a further complication, a given sequence can often be recognized by helices containing different amino acids.

More recent efforts have focused on the randomization of the entire recognition helix and the selection of proteins Zinc-finger-based artificial transcription factors (background) have been applied in plants such as Arabidopsis (foreground). with new binding specificities. Several successful con- struction methods have been described [32,33,34] and compared in detail elsewhere [22,35,36].Themost commonly used approach is based on the assembly of factors that have evolved to control particular genes. The pre-defined zinc-finger modules that have been pre- whole-genome of Arabidopsis [14] and more viously selected and optimized [17,30,33]. Each module recently [15], combined with informatics-based ana- originated as the middle finger in a three-finger protein, , has identified numerous putative plant transcription which was subsequently selected to recognize a new 3-bp factors [15,16]. However, the identification and charac- binding site using phage-display technology. The terization of the molecular targets of these transcription selected modules were further refined by site-directed factors is still at a very early stage. Consequently, it is not mutagenesis to provide optimal binding specificity. The yet possible to use them broadly as gene-specific tools to modules can be assembled in any order necessary to form regulate endogenous gene expression. new three- or six-finger proteins. With each domain specifying 3-bp, a six-finger protein should have the Recently, several studies have targeted endogenous capacity to bind one of almost 70 billion unique 18-base genes in cultured mammalian cells using synthetic TFsZF pair sites. The human genome contains around 3.2 billion [2,17–20,21]. Among the many naturally occurring base pairs of DNA, and so a six-finger protein has the DNA-binding proteins, the Cys2-His2 zinc-finger domain potential to recognize a unique site in the human gen- has emerged as the scaffold of choice for the design of ome. Only 64 modules are required to recognize all novel sequence-specific DNA-binding proteins [22]. possible 3-bp sites. To date, modules have been reported Within these proteins, each 30-amino-acid domain, or that can bind a majority of these sites, enabling the finger, forms a stable bba fold. The amino-terminus of construction of more than one billion different six-finger the a-helix typically recognizes a 3-bp subsite in the DNA proteins, approximately 27 000 proteins for every gene (Figure 2). The recognition of extended DNA sequences in the human genome. New proteins can now be is achieved by linking the domains into tandem arrays. constructed by any molecular biology laboratory using These target-recognizing domains can be found in arrays this modular assembly method, relying solely on the of up to 37 repeats [23], facilitating the recognition of PCR-based assembly of published zinc-finger modules extended DNA sequences. The versatility of zinc-finger (Figure 2; [37]).

Current Opinion in Plant Biology 2003, 6:163–168 www.current-opinion.com Zinc fingers and a green thumb: manipulating gene expression in plants Segal, Stege and Barbas 165

Figure 2

(a) (d) F1 F2 F3 F4 F5 F6 AP3 gene ( )

(b) (e) 5′-TAC TTC TTC AAC TCC ATC-3′ F1 F2 F3 F4 F5 F6 3′-ATG AAG AAG TTG AGG TAG-5′ SID- F1 F2 F3 F4 F5 F6 (c)

GAA QSSNLVR GCA QSGDLRR GGA QRAHLER GTA QSSSLVR (f) GAC DPGNLVR GCC DCRDLAR GGC DPGHLVR GTC DPGALVR GAG RSDNLVR GCG RSDDLVR GGG RSDKLVR GTG RSDELVR SID- AP3 gene GAT TSGNLVR GCT TSGELVR GGT TSGHLVR GTT TSGSLVR ( )

Current Opinion in Plant Biology

Modular construction of artificial transcription factors. (a) The DNA sequence of the gene to be regulated (in this case AP3) is searched for (b) an 18-bp binding site that can be recognized using a combination of existing zinc-finger domains. Sequence-specific recognition domains (F1–F6) are selected for each 3-bp subsite in the target sequence using (c) a table of optimized zinc-finger modules. (Note: 3-bp subsites in (c) are written 50 to 30, those in (b) appear in the opposite orientation.) A more complete table is provided in [37]. (d) The for the new DNA-binding protein is assembled from overlapping primers using PCR. (e) Appending an activation or repression domain (a SID repression domain is shown) creates an artificial transcription factor that is capable of (f) endogenous gene regulation.

Into the plant become a reality: the function and stability of TFsZF The attachment of an appropriate effector domain to a in transgenic multicellular organs and the ability of these zinc-finger protein creates potent transcriptional activa- genes to be stably inherited by subsequent generations. tors and . Activation domains such as VP16 [38] Several studies on the regulation of plant genes by and p65 [39] and repression domains such as KRAB TFsZF have been recently published. An in vitro assay (Kru¨ pple-associated box) [40] and SID (Sin3A interaction has been used to evaluate the activity of several different domain) [41] are components of naturally occurring tran- TFsZF constructs on a variety of target reporter config- scription factors. All of these domains are able to regulate urations in plant cells. The effective repression and a variety of mammalian promoters in a distance- and activation of reporter genes was dependent on the pro- orientation-independent manner when fused to zinc-fin- moter strength and the location of the site that binds the ger proteins [2,17–20,21]. These domains exert their zinc-finger protein [42].Ab-glucuronidase (GUS) effect by recruiting global activation and repression com- stably integrated in tobacco plants was plexes to the site. Because this recruitment activated to high levels in transgenic plants using an  often involves specific protein–protein interactions, effec- artificial TFsZF [43 ]. This activation was tor domains that are found in one type may not stable over multiple generations, indicating that TFsZF function in a different cell type or species. For example, functions are stably inherited and non-toxic in plants. the KRAB domain is a potent transcriptional repression These results were supported by evidence from a study  domain in mammalian systems. This domain is not found by Guan et al. [44 ] in which TFsZF were designed to in melanogaster, , Sacchar- target the Apetella3 (Ap3)promoterinArabidopsis for omyces cerevisiae,orArabidopsis thaliana [24], however, and activation or repression. When expressed in a tissue- would therefore be a poor candidate for gene regulation in specific manner, these reporters were able to activate or plants. By contrast, the SID domain interacts directly repress an Ap3::GUS reporter in a stable, inheritable with the highly conserved Sin3A protein and its homo- manner. Furthermore, homeotic transformations of the logues [41], and is therefore a more appropriate candidate floral organs were evident in these transgenic plants, to regulate endogenous plant genes. consistent with the well-established model for Arabidop- sis floral-organ identity [45]. These results indicate that Plant-specificTFsZF have a diverse range of potential the Ap3-specificTFsZF is able to manipulate the expres- applications for agriculture. Until recently, however, two sion of the endogenous AP3 gene (Figure 3). Again, the crucial issues remained to be addressed before the TFsZF used in these studies were constructed from application of artificial plant-specificTFsZF could predefined and engineered domains. www.current-opinion.com Current Opinion in Plant Biology 2003, 6:163–168 166 Plant biotechnology

Figure 3

(b) AP1::VP64::AP3 p Ap3 activator VP64- (a) f

Activation of Ap3 in sepals causes partial sepal → petal transformation

se pe ca st (c)

Wildtype flower SID-

AP1::SID::AP3 Repression of Ap3 in petals causes Ap3 partial petal → sepal transformation

Current Opinion in Plant Biology

(a) In wildtype flowers, AP1 is expressed in the sepals (se) and petals (pe) whereas AP3 is expressed in the petals and stamen (st). When AP1 alone is expressed in a floral organ, a sepal is formed. When AP1 and AP3 are expressed together, a petal is formed. (b) Expression of an Ap3-specific activator (VP64::AP3) with the Ap1 promoter (in sepals and petals) causes a partial homeotic transformation of the sepals into petals. (c) Expression of an Ap3-specific repressor (SID::AP3) with the Ap1 promoter (again in sepals and petals) causes a partial homeotic transformation of the petals into sepals. Photographs reproduced from [44]. ca, carpel.

Conclusions genome sequencing to dissect the complex genetic inter- Recent reports demonstrate the potential of artificial actions involved in many important processes such as transcription factor technology to target specific plant disease/stress resistance, and metabolite genes for up- or downregulation. Further research will biosynthesis. Taken a step further, complex physiological focus on the identification of potent, robust, plant-specific changes could be achieved through the tissue-specificor activation and repression domains, and on the optimiza- inducible expression of a TFsZF that is designed to control tion of TFsZF design to provide effective, specific reg- a pathway or family of genes. Biosynthetic pathways ulation of target gene(s). Once this approach has been might be engineered at the level of transcription, with demonstrated to be a robust technology that can be used competing pathways silenced in an orchestrated fashion. to manipulate the expression of any given plant gene, it will have many applications in agricultural biotechnology. Future studies will also take advantage of libraries of combinatorial transcription factors with predefined spe- Repressing the expression of anti-nutritive or allergenic cificities. In this approach, libraries of TFsZF are intro- proteins would increase the value and quality of many duced into cells potentially to turn every gene in the important crop plants. The expression of -specific genome either off or on/up within a population of cells in TFsZF could provide effective resistance to plant which each cell is modulated by a unique transcription that replicate through double-stranded DNA intermedi- factor. This forward-genetic approach then allows cells ates. TFsZF could also be used to activate the expression of that display a desired to be selected. The key genes that are involved in disease and stress resistance TFsZF responsible for the desired phenotypic modifica- to make more hardy, productive crop plants. As the genes tion can then be recovered. This method has further involved in metabolite biosynthesis are characterized, potential as a powerful tool for gene discovery. As the TFsZF could be used to make tissue-specific changes to 18-bp binding site for six-finger TFsZF can be deduced improve the flavor or nutritional value of many plants. from their primary sequence, their unique genomic bind- ing sites and associated gene of action can be identified. TFsZF could be designed to alter the expression of key The use of libraries of three-finger TFsZF that recognize regulatory gene(s), and used alongside information from 9-bp binding sites would make the elucidation of the

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target gene more challenging. However, the reduced 13. Ptashne M, Gann A: Transcriptional activation by recruitment. Nature 1997, 386:569-577. specificity of three-finger TFsZF in comparison with 14. The Arabidopsis Genome Initiative: Analysis of the genome six-finger TFsZF potentially allows several genes or gene sequence of the flowering plant Arabidopsis thaliana. families to be regulated potentially by a single TFZF. Nature 2000, 408:796-815. The use of three-finger TFs may then induce more ZF 15. Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook complex . The power of this combinatorial J, Sessions A, Oeller P, Varma H et al.: A draft sequence of the rice approach is that potent transcriptional regulators can be genome (Oryza sativa L. ssp. japonica). Science 2002, obtained even when the targeted gene(s) is unknown. 296:92-100. The potential of this approach has been demonstrated in 16. Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR et al.: Arabidopsis cell lines in which endogenous regulators for a variety of transcription factors: genome-wide comparative analysis human genes have been selected. In plants, the combi- among . Science 2000, 290:2105-2110. natorial approach may be applied at the organismal level 17. Dreier B, Beerli RR, Segal DJ, Flippin JD, Barbas CF III: [46]. Libraries of TFs could readily be delivered using Development of zinc finger domains for recognition of the ZF 50-ANN-30 family of DNA sequences and their use in the Agrobacterium transformation to create libraries of construction of artificial transcription factors. J Biol Chem 2001, whole plants that could be screened or selected for novel 276:29466-29478. plant phenotypes. 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. References and recommended reading Papers of particular interest, published within the annual period of 19. Zhang L, Spratt SK, Liu Q, Johnstone B, Qi H, Raschke EE, review, have been highlighted as: Jamieson AC, Rebar EJ, Wolffe AP, Case CC: Synthetic zinc finger transcription factor action at an endogenous  of special interest chromosomal site. Activation of the human  of outstanding interest gene. J Biol Chem 2000, 275:33850-33860. 1. Zuo J, Chua NH: Chemical-inducible systems for regulated 20. Xu D, Ye D, Fisher M, Juliano RL: Selective inhibition of expression of plant genes. Curr Opin Biotechnol 2000, P-glycoprotein expression in multidrug-resistant tumor cells 11:146-151. by a designed transcriptional regulator. J Pharmacol Exp Ther 2002, 302:963-971. 2. 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 21. Ren D, Collingwood TN, Rebar EJ, Wolffe AP, Camp HS: PPARc locus using a panel of designed zinc finger proteins targeted  knockdown by engineered transcription factors: exogenous to accessible regions. Activation of vascular PPARc2 but not PPARc1 reactivates adipogenesis. Genes Dev endothelial growth factor A. JBiolChem2001, 2002, 16:27-32. 276:11323-11334. A six-finger TFZF repressor, similar to that described in [18], was used to This study demonstrates the regulation of the endogenous vascular target the gene for PPARg, a nuclear receptor involved in endothelial growth factor A (VEGF-A) gene in mammalian cells by a adipogenesis. By selectively repressing one splice variant but not the TFs . It also shows that all of the multiple splice variants of this gene other, the authors were able to differentiate unique roles for the two ZF isoforms and showed that only the PPARg2 isoform was responsible for can be upregulated by TFsZF in contrast to cDNA-based overexpression methods. ligand-independent adipogenesis. 3. Gottesfeld JM, Neely L, Trauger JW, Baird EE, Dervan PB: 22. Segal DJ, Barbas CF III: Design of novel sequence-specific Regulation of gene expression by small molecules. Nature 1997, DNA-binding proteins. Curr Opin Chem Biol 2000, 4:34-39. 387:202-205. 23. Rhodes D, Klug A: Zinc fingers. Sci Am 1993, 268:56-59,62-65. 4. Cooney M, Czernuszewicz G, Postel EH, Flint SJ, Hogan ME: Site- specific oligonucleotide binding represses transcription of the 24. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, human c-myc gene in vitro. Science 1988, 241:456-459. Smith HO, Yandell M, Evans CA, Holt RA et al.: The sequence of the human genome. 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Chuang CF, Meyerowitz EM: Specific and heritable genetic finger domains. J Mol Biol 2000, 303:489-502. interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 2000, 97:4985-4990. 30. Segal DJ, Dreier B, Beerli RR, Barbas CF III: Toward controlling gene expression at will: selection and design of zinc finger 12. Cowell IG: Repression versus activation in the control of gene domains recognizing each of the 50-GNN-30 DNA target transcription. Trends Biochem Sci 1994, 19:38-42. sequences. Proc Natl Acad Sci USA 1999, 96:2758-2763. www.current-opinion.com Current Opinion in Plant Biology 2003, 6:163–168 168 Plant biotechnology

31. Corbi N, Libri V, Fanciulli M, Passananti C: Binding properties of transcriptional repression domains. Proc Natl Acad Sci USA the artificial zinc fingers coding gene Sint1. Biochem Biophys 1994, 91:4509-4513. Res Commun 1998, 253:686-692. 41. Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M, Laherty CD, 32. Isalan M, Choo Y: Rapid, high-throughput engineering of TorchiaJ,YangWM,BrardG,NgoSD,DavieJRet al.:  sequence-specific zinc finger DNA-binding proteins. A complex containing N-CoR, mSin3 and Methods Enzymol 2001, 340:593-609. deacetylase mediates transcriptional repression. The authors of this article describe a method for creating novel TFsZF that Nature 1997, 387:43-48. contrasts with the modular assembly described in the present review. They claim that the method can generate a new binding protein for 42. Stege JT, Guan X, Ho T, Beachy RN, Barbas CF III: Controlling virtually any DNA sequence within two weeks.  gene expression in plants using synthetic zinc finger transcription factors. Plant J 2002, 32:1077-1086. 33. Beerli RR, Segal DJ, Dreier B, Barbas CF III: Toward controlling The authors describe in vitro assays to evaluate TFsZF in plant cells. They gene expression at will: specific regulation of the erbB-2/ demonstrate the effective repression and activation of reporter genes in HER-2 promoter by using polydactyl zinc finger proteins plant cells and identify the human SID domain as a potent repression constructed from modular building blocks. Proc Natl Acad Sci domain in plant cells. The authors also explore the effects of binding-site USA 1998, 95:14628-14633. position and promoter strength on the regulation of gene expression by TFsZF. 34. Greisman HA, Pabo CO: A general strategy for selecting high- affinity zinc finger proteins for diverse DNA target sites. 43. Ordiz MI, Barbas CF III, Beachy RN: Regulation of transgene Science 1997, 275:657-661.  expression in plants with polydactyl zinc finger transcription factors. Proc Natl Acad Sci USA 2002, 99:13290-13295. 35. Segal D, Barbas CF III: Custom DNA-binding proteins come of This article describes regulation of reporter constructs by TFsZF in age: polydactyl zinc-finger proteins. Curr Opin Biotech 2001, transgenic tobacco. Target genes were reproducibly regulated in plants 12:632-637. expressing TFsZF. These plants were phenotypically normal through two generations, suggesting that the TFs were expressed in a stable, 36. Beerli RR, Barbas CF III: Engineering polydactyl zinc-finger ZF heritable manner and exerted no adverse effects. Together with [44], transcription factors. Nat Biotechnol 2002, 20:135-141.  this report demonstrates the power of expressing TFs using tissue- This is an excellent review of TFs construction and their use to regulate ZF ZF specific promoters. endogenous genes in mammalian cells. 44. Guan X, Stege J, Kim M, Dahmani Z, Fan N, Heifetz P, Barbas CF III, 37. Segal DJ: The use of zinc finger to study the role of Briggs SP: Heritable endogenous gene regulation in plants with specific factor binding sites in the chromatin environment.   designed polydactyl zinc finger transcription factors. Methods 2002, 26:76-83. Proc Natl Acad Sci USA 2002, 99:13296-13301. This article provides step-by-step instructions on building new modular This article describes the first regulation of an endogenous plant gene by zinc-finger proteins, along with critical parameters and considerations as TFs . The AP3 gene was up- and downregulated in fertile Arabidopsis determined by the Barbas laboratory. No phage display is required. ZF plants using six-finger TFs . This is also the first demonstration of Table 1 of this paper provides the most current list of recognition domains ZF endogenous gene regulation through multiple generations in a multi- and the 3-bp sites that they recognize. cellular . 38. Sadowski I, Ma J, Triezenberg S, Ptashne M: GAL4–VP16 is an 45. Irish VF: Patterning the flower. Dev Biol 1999, 209:211-220. unusually potent transcriptional activator. Nature 1988, 335:563-564. 46. Blancafort P, Magnenat L, Barbas CF III: Scanning the human genome with combinatorial transcription factor libraries. 39. Fujita T, Nolan GP, Ghosh S, Baltimore D: Independent modes of  Nat Biotechnol 2003, in press. transcriptional activation by the p50 and p65 subunits of NF-kappa B. Genes Dev 1992, 6:775-787. This article describes a potentially powerful combinatorial approach to gene regulation that uses libraries of TFsZF in human cells. This approach 40. Margolin JF, Friedman JR, Meyer WK, Vissing H, Thiesen HJ, allows libraries of TFsZF to be applied for forward in cells and Rauscher FJ III: Kruppel-associated boxes are potent whole .

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