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Zinc Fingers and a Green Thumb: Manipulating Gene Expression in Plants Segal, Stege and Barbas 165

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Zinc Fingers and a Green Thumb: Manipulating Gene Expression in Plants Segal, Stege and Barbas 165 163 Zinc fingers and a green thumb: manipulating gene expression in plants David J Segaly, Justin T Stegez and Carlos F Barbas IIIç Artificial transcription factors can be rapidly constructed A variety of techniques have been developed to manip- from predefined zinc-finger modules to regulate virtually any ulate gene expression in plants. Increased expression of gene. Stable, heritable up- and downregulation of endogenous genes is most commonly achieved through endogenous genes has been demonstrated in transgenic transgene 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 transgenes plants. a relatively routine task. However, variable expression and co-suppression of transgenes often complicate this process. Addresses Furthermore, transgenes cannot accommodate alternative ÃThe Skaggs Institute for Chemical Biology and the Department of splicing, which may be important for the appropriate Molecular Biology, 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 biotechnology applied in plants. For example, one of the first demonstra- Edited by Wolf B Frommer and Roger Beachy tions that hammerhead ribozymes 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 oligonucleotides, 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 transcription factor 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 disease/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- proteins. 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 protein to a important processes. In this review, we describe several specific DNA address and an effector 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 human genome [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 amino acid 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 sequencing of Arabidopsis [14] and more viously selected and optimized [17,30,33]. Each module recently rice [15], combined with informatics-based ana- originated as the middle finger in a three-finger protein, lysis, 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.
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