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Synthetic promoters: genetic control through cis engineering
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Synthetic promoters: genetic control through cis engineering Mauritz Venter Institute for Plant Biotechnology, Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
Technological advances in plant genetics integrated with systems biology and bioinformatics has yielded a myriad of novel biological data and insights into plant metabolism. This unprecedented advance has provided a platform for targeted manipulation of transcriptional activity through synthetic promoter engineering, and holds great promise as a way to further our understanding of regulatory complexity. The challenge and strategy for predictive experimental gene expression is the accurate design and use of molecular ‘switches’ and modules that will regulate single or multiple plant transgenes in direct response to specific environmental, physiological and chemical cues. In particular, focusing on cis-motif rearrangement, future plant biotechnology applications and the elucidation of cis- and trans-regulatory mechanisms could greatly benefit from using plant synthetic promoters. Modifying promoters – the way forward? One of the major challenges in a plant genetic engineering program is to design a transformation-cassette that would enable the precise control of transgene activity. The choice of promoter, to confer constitutive, spatial and/or temporal transgene expression, is one of the key determinants used in plant biotechnology applications. In recent years, a wide range of different promoters from plant, viral and bacterial origin have been characterized and used extensively in regulated transgene expression systems in plant cells [1– 3]. Several plant genetic engineering strategies have incorporated the use of strong constitutive promoters in the study of gene and transcription factor (TF) function as well as for conferring transgene expression for crop improvement and bio-pharmaceutical applications. The well-described cauliflower mosaic virus (CaMV) 35S promoter [4–7] confers high-level gene activity and has been used most commonly in plant transgene expression studies. The way forward for the study and design of inducible transgene expression cassettes was laid by research investigating the modification of the 35S promoter using the core-region (essential to initiate transcription), combined with dissected cis-regulatory elements, as well as early analysis of other dicot- and monocot-promoters [8–15]. From these initial attempts it has become apparent that the use of a synthetic and regulatory module that can be tuned, that is suited for a specific application and driven by the core-transcriptional initiation region of a constitutive promoter will prove invaluable in Corresponding author: Venter, M. ([email protected]). Available online 9 February 2007. www.sciencedirect.com
future genetic engineering programs. A few exciting studies have described how changes in promoter architecture, and the targeted design of cis-motif context can improve the control of spatial and temporal gene activity, regulate multiple transgenes, and overcome drawbacks such as homology-dependent gene silencing in plant cells [16–20]. Results from these research efforts underscore the value of using synthetic promoters to assist in elucidating synergistic regulatory interactions, the role of individual cis-motifs and in biotechnological applications. The scope of several other important factors such as gene silencing and RNA interference (RNAi), introns and splicing, transgene selection, different inducible systems and transformation technologies is too large to be covered in this review although they all play major roles in plant inducible gene expression studies. There is a considerable amount of data reporting on the use of chimeric inducible systems including promoters and transcription factors. In an attempt not to summarize all the relevant literature, this review will focus specifically on key examples highlighting previous and current strategies for the accurate design of an inducible plant synthetic promoter. An integrative design strategy for analysis and putative prediction of gene expression is proposed that incorporates transcriptomics, conserved cis-motif organization and the use of intricate bioinformatic software. Re-designing the regulatory code Refined and targeted modification of promoter architecture necessary for coordinated manipulation of gene activity requires accurate deciphering of the regulatory framework. The basics of cellular processes on a nucleotide and protein level have been studied and defined, resulting in a unified theory of eukaryotic gene expression and regulation. Even with the significant understanding gained from this fundamental research, major overlaps and synergistic interactions at different levels of regulation are still poorly understood. Studies in model organisms focusing on the principles of transcriptional control, promoters, gene expression and regulatory networks [21– 28] have emphasized the importance of combining regulatory data (cis-motifs and transcription factors) and gene expression profiles to elucidate the underlying mechanisms governing genetic control. The construction of sophisticated in silico promoter models [29,30] has enabled more accurate prediction of gene expression and/or association (but not necessarily function). To predict desired gene expression patterns in response to specific cues it is necessary to understand combinatorial regulatory principles. A ground-breaking study conducted in the model eukaryotic
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organism Saccharomyces cerevisiae (yeast) revealed how conserved cis-motif-logic can be used for relatively accurate prediction (73%) of a distinct expression pattern during a specific condition [31]. Results of this study suggest that it should be possible to design a synthetic promoter model that could confer a particular expression pattern in a biotechnological application dependent on the availability of regulatory information. The strategy is simple. However, the complexity and synergistic interplay of a multitude of regulatory mechanisms (although individually well characterized) poses a significant challenge for future promoter design. Given this complexity it is evident that the integration of an improved understanding of genome-wide regulatory mechanisms of complex, multi-cellular eukaryotes and the power of systems biology, will serve as the basis for synthetic and predictive biology [32,33]. In silico predictions of gene regulatory events must ultimately be validated experimentally. Synthetic promoters play a major role in elucidating cis-motif logic (orientation, strength–weight, position) to study gene regulation in vivo [34,35] as well as advancing intricate design strategies of expression cassettes to be used for metabolic pathway engineering [16,36–39] and gene therapy applications [40–43]. With the focus on plants, the accessibility of a vast amount of genetic data that were produced as a result of sequencing the whole-genomes of model organisms such as Arabidopsis thaliana (thale cress) and Oryza sativa (rice) have enabled the rapid identification of large sets of promoter sequences. Recent years have also witnessed much progress in understanding plant promoter architecture and general TF assembly [44–47]. As a result of the preceding factors, several promoters and TFs have been selected for inducible transgene expression studies. Promoters used in these studies include unmodified wild-type, synthetic (with new combinations of cis-motifs from various sources) and truncated (reduced to cis-motifs essential for desired expression profile) modules, and are divided into the following categories: (i) constitutive, (ii) inducible, (iii) tissue- and (iv) developmental-stage specific. Most promoters have a core- or minimal-region necessary to initiate transcription. Established strategies for the isolation of promoters using approaches for the large-scale identification of candidate genes, expressed under a certain condition are effective but time-consuming. By contrast, the development of high-throughput technologies for extensive identification of TF-binding sites (such as chromatin immunoprecipitation assays – ChiPs) and advances in plant TF-database assistance has proved invaluable in characterizing native plant promoters [48]. ChiP is a strategy that enables the rapid identification of large sets of promoter regions using a specific TF as a probe [49,50]. However, until now these strategies have not been optimized for a wide variety of plant species. In addition to fundamental cis-motif elucidation, other factors considered to be stumbling blocks in applied plant genetic engineering include expression of multiple transgenes, specific inducibility of gene expression and homology-dependent gene silencing. The inability of conventional wild-type plant promoters to address these hurdles has renewed interest in the development of synthetic promoters. www.sciencedirect.com
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Even better than the real wild-type thing A synthetic promoter designed to initiate transcription of protein-encoding genes should adhere to the universal requirements necessary for eukaryotic gene transcription. Such a promoter is a stretch of DNA comprising a corepromoter region and multiple repeats or combinations of heterologous upstream regulatory elements (cis-motifs or TF-binding sites). The core-promoter region (also known as the minimal-region) usually contains a TATA-box necessary for recruiting RNA polymerase II and the orchestrated assembly of general transcription factors to form the preinitiation complex (PIC) [51]. The CaMV 35S core-promoter is ideal for transcription initiation and has been used in several plant promoter engineering strategies. The two most frequently used strategies are (i) combinatorial engineering of cis-motifs upstream of the core-promoter and/or (ii) combined with bidirectionalization of a unidirectional promoter (Figure 1). Synthetically organized motifs include enhancers, activators and/or repressors that bind to TFs expressed in response to specific conditions. Promoters that respond to inducers can be regulated by (i) environmental cues (i.e. light, cold and heat stress); (ii) biotic and abiotic stress (pathogens, wounding, insects, drought and salinity); (iii) hormones (i.e. ethylene, auxin, abscisic and salicylic acid); and (iv) chemicals (i.e. tetracycline, copper, estradiol and dexamethasone). The use of synthetic promoters that allow for targeted inducibility is of considerable interest for plant engineering strategies. Non-plant transactivated- and chemical-inducible systems (using transformation cassettes incorporating a core-promoter and multimers of the upstream activation sequences) have gained much attention, although not emphasized in this discussion. Some of these systems have proven highly flexible and could be used to either repress or activate plant transgene expression. Comparative and indepth analyses of these systems have recently been reviewed [52,53]. cis-Motif engineering An in-depth study using synthetic promoters illustrated the usefulness of combining TF knowledge to ‘cut and paste’ pathogen-inducible cis-motifs [17,54]. Results from this study showed that promoter inducibility and strength varied depending on motif copy number and, more specifically, spacing of motifs (with the same core-sequence) relative to the TATA-box. Moreover, one of the main observations in this detailed study revealed that promoter activity was not necessarily enhanced with an increase in motif copy number [17] and, in several instances, it was shown that a single copy of a specific cis-motif was sufficient for a pathogen-induced response. The functionality of defense-related plant TF binding sites appears to be conserved among different plant species [17]. This shared regulatory function will assist in the future design and application of pathogen-inducible promoters across the borders of plant species [54]. There are still several combinatorial mechanisms of regulatory context and signaling that are largely unknown, which prevents the optimal design of a synthetic pathogen-inducible promoter. We are still in the early stages of understanding plant– pathogen interactions and it is evident that the use of
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Figure 1. Simplified representation of a plant synthetic promoter. Core-region containing TATA-box (green block arrow) of a wild-type constitutive promoter, such as CaMV 35S, is used to drive transcription. A uni- or bidirectional synthetic promoter comprises the stretch of DNA containing multiple copies of a specific cis-motif (blue ellipses) upstream of 35S core-promoter to bind transcription factors (red block arc) expressed in response to specific stimuli.
synthetic promoters will not only be valuable for genetic engineering of disease resistant plants, but also contribute to elucidating pathogen-induced motif function out of native context [17,54]. Homology-dependent gene silencing (HDGS) is of great concern in plant genetic engineering strategies and is thought to be caused by multiple copies of homologous transgene and promoter sequences. Transgene silencing can occur on a transcriptional and posttranscriptional level [55,56]. Investigations in Nicotiana tabacum (tobacco) have shown that the use of synthetic promoters with minimal sequence similarity could serve as a valuable tool to overcome HDGS in plant transgenic strategies [18]. Comparison was made between the ‘reshuffling’ (or swapping) of previously described functional domains of 35S [7] and the incorporation of cis-motifs in a synthetic stretch of DNA, both driven by 35S core-promoter sequence [18]. Both strategies were evaluated in comparison to high-level expression of the wild-type CaMV 35S promoter. Results from this study showed that domain swapping led to a less efficient promoter activity compared with insertion of cis-elements in a synthetic DNA-sequence [18]. However, it is suggested that repetitive use of ciselements with identical core-sequences and homologous intervening regions (within a functional domain) might cause depletion of TFs, consequently reducing endogenous gene expression [18]. Therefore, design strategies using multimers of cis-motifs need to be optimized to achieve the desirable inducibility of the transgene without compromising endogenous ‘house-keeping’ regulation in the plant cell. An important study investigated individual and combinatorial functions of light responding elements (LREs) in a synthetic stretch of DNA driven by the core-promoters of www.sciencedirect.com
the nopaline synthase (NOS) gene and CaMV 35S [15]. The NOS core-promoter confers no basal transcriptional activity, enabling an ‘ON or OFF’ phenotype to be used and proved to be ideal for engineering inducible synthetic systems [15]. Owing to limitations in our understanding and shortfalls in current design strategies only a limited number of studies have shed light on multimeric cis-motif design. Inducible transgene expression in both directions In many plant biotechnological applications (e.g. metabolic engineering or pathogen resistance) the advantages of expressing multiple transgenes are not only advantageous, but necessary. Gene-stacking, or pyramiding, remains a challenge for crop improvement and several different systems and strategies have been reviewed [57,58]. There are unique reports describing naturally occurring bidirectional promoters in different organisms, expressing two genes simultaneously [59–62]. A few key studies have focused on the usefulness of modifying a unidirectional promoter to a bidirectional promoter for specific biotechnological applications in plants (e.g. simultaneously expressing sense and antisense transcripts to mediate gene silencing) [16,63]. Comparative analysis of bi-directional promoters in Vitis vinifera (grape) and Nicotiana tabacum (tobacco), demonstrated higher reporter-gene expression efficiency compared with a unidirectional expression system incorporating similar enhancer and core-promoter complexes [64]. Furthermore, this study supported previous views on how the formation of transcription machinery is more efficient in a bidirectional mode compared with that in a unidirectional regulatory structure. In addition to the
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above, recent investigations shed light on how the synthetic cis-motif context (of a synthetically designed activation module) and combinatorial interactions regulate gene expression and influence the stability of transcriptional assembly on the TATA- box of the corepromoter [19]. It appears that there is a fine balance between complete occupancy of synthetic cis-motifs and success-rate of complete transcriptional activation and stability of the initiation complex assembled on the TATA-box. This suggests that depletion of endogenous TFs because of an excess of synthetic TF-binding sites can cause transcriptional inactivation [18,19]. This is one of the major challenges that could greatly limit future promoter engineering strategies. Nevertheless, advances in bioinformatics (incorporating plant TF-databases) [65] combined with plant TF-function and accurate in silico promoter models could assist in a more refined strategy for designing synthetic promoters. A recent investigation successfully integrated computational assistance and bidirectionalization to construct a synthetic transcriptional unit for high-level reporter-gene expression in response to specific elicitors, which yielded exciting results [20]. The complete synthetic stretch of DNA, known as the transcription activation module (TAM), was constructed from regulatory sequences compiled from a database of highly expressed plant genes [66]. Stepwise construction of this TAM consisted of: (i) screening a computational database for genes with high expression levels in plants and (ii) identification of conserved TATA-box proximal regions and motifs 500 base pairs upstream from the transcriptional start site. Initial analysis showed that this TAM could express the b-glucuronidase (GUS) reporter gene in stably transformed tobacco (Nicotiana tabacum) at higher levels compared with those expressed by the wild-type CaMV 35S promoter [66]. As already discussed in this review, it has been shown that the TAM could facilitate stable enhancer complex (enhanceosome) assembly for accurate transcriptional initiation [19]. The TAM was modified in a more recent study to confer bidirectional expression of reporter genes, b-glucuronidase (GUS) and green fluorescence protein (GFP), and evaluated in transient and stably transformed tobacco (Nicotiana tabacum) leaf discs [20]. Expression of both reporter genes was up-regulated in a bidirectional mode in response to specific elicitors, suggesting that enhanceosome assembly to form a stable PIC on both TATA-boxes (modulated by a DNA-bending sequence within the TAM) could initiate transcription in both directions [20]. Specific design strategies for the development of synthetic promoters are in their infancy. The investigations described here, which were performed by several different research groups, are some of the major examples of the strategies being used in plant promoter engineering, and could serve as important technical contributions for future biotechnological applications as well as in the elucidation of synergistic motif-TF interplay. In silico analysis to assist in the grand design The combination of high-throughput gene expression profiles with promoter architecture and bioinformatics is a powerful approach to compile data for the possible prediction of coordinated gene expression during a specific www.sciencedirect.com
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condition [31,67,68]. In this section, a simplified representation will be used to illustrate how this strategy could facilitate a practical and refined design of a plant synthetic promoter able to confer differential gene expression during defined conditions (Figure 2). Promoter regions of gene-clusters co-expressed during a specific condition contain conserved cis-motifs. The biggest challenge is to dissect which conserved motif is associated directly with the specific condition under investigation and which are associated with co-expression [21]. The emergence of plant TF-binding site database-assistance [65] has greatly facilitated large-scale plant promoter analysis. The three major plant TF-binding site databases, PLACE [69], PlantCARE [70] and TRANSFAC [71], are updated constantly and provide a plethora of possible cis-motif combinations. To analyze and/or construct accurate in silico promoter models, it is essential to distinguish between overrepresented motifs and background ‘noise’. Several computational methods have been developed for this purpose [72–74], and assessments of the most prominent cis-motif detection and/or classification methods and statistical tools have been reviewed [75]. Identification of cis-regulatory codes remain complex, particularly given that within a promoter sequence of 500 to 5000 base pairs the core sequence of a cis-regulatory motif can range between 4 to 10 base pairs. Gibbs sampling [76] and expectation maximization (MEME) [77] are powerful methods for predicting over-represented motifs. There are numerous other methods based on different operating principles [75] and some are modified and/or improved versions combined with statistical modeling techniques such as hidden Markov models (HMMs) [78]. Initial promoter sequence output, using plant TF-database assistance, can be refined by these probabilistic algorithms to select over-represented cis-motifs in a multiple set of sequences upstream of coexpressed genes. Furthermore, it should be possible to identify universal or common cis-motifs that are synergistically associated with cis-motifs of promoters that are induced during different conditions [21]. Probabilistic fine-tuning of promoter architecture is followed by construction of a motif synergy map using design ‘rules’ AND-NOT-OR cis-motif logic. This simplified approach accentuates a combination of previous studies conducted in S. cerevisiae [21,31] and Arabidopsis [79] that could assist in the design of one or several plant synthetic promoters to facilitate a more accurate prediction of gene expression in response to each specific condition (Figure 2). Single copies of a cis-motif in a synthetic stretch of DNA might be sufficient for a desired gene expression profile [17]; however, the operation principles of the current computational methods are more useful for identifying a cluster of over-represented cis motifs in a contiguous segment of DNA [75]. By contrast, conventional views regarding the nature of combinatorial control appears to be inconsistent with recent and exciting experimental evidence showing that quantitative transcriptional control of the Drosophila melanogaster (fruitfly) even skipped gene (eve) is operative outside a compact arrangement of cis-motifs [80]. It is evident that precise genetic control is governed by TF binding specificity [80] and a complex network of regulatory mechanisms on different levels (chromatin, histone and
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Figure 2. Combinatorial cis-motif engineering for the accurate design of synthetic promoters. (a) DNA microarrays enable large-scale identification of induced gene expression (indicated by blue lightning bolt) in response to a specific condition (e.g. stress, chemical induction or plant development or growth). Upstream promoter regions (black line and white block arrow) of gene clusters contain a multitude of conserved and non-conserved cis-motifs (black circles, triangles, rectangles and squares) [67]. (b) Probabilistic motif detection algorithms (MEME and Gibbs sampling) combined with plant TF-database assistance enable the identification of conserved motifs (indicated by blue circles, yellow rectangles, red triangles and green squares) within multiple promoter sequences associated with a particular condition [65,76,77]. (c) A cis-motif synergy map is constructed to gain a holistic view of: (i) motif occurrence in relation to other motifs associated with a specific condition [21,23] and (ii) motif spacing relative to the TATA-box of the corepromoter. Computational promoter modeling [29,30] based on the combined regulatory organization associated with different conditions and gene expression patterns enable the use of a more refined synthetic design strategy. (d) Plant synthetic promoters are engineered by dissection of cis-regulatory context (crossed out elements are removed) specific for induction of transgene activity in response to different conditions. Transcription is initiated by the 35S core-promoter sequence (indicated by green block arrow). www.sciencedirect.com
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nuclear organization) [24] and over long genomic distances [81,82]. Given that a single TF can bind to cis-motifs of different genes, permitting combinatorial control [45,46], the possibilities for future inducible expression systems are exciting and challenging. Concluding remarks The goal of this review was to prioritize specific past and more recent research investigations that have focused on design strategies and applications of plant synthetic promoters. It is evident that the complexity in dissecting cisregulatory architecture alone (where spacing, relative to the TATA-box and other motifs, orientation, copy number and function of the motif) poses a major challenge for synthetic promoter design. Advances in bioinformatics and a more holistic deciphering of regulatory processes including plant TF networks and cis- and/or trans-synergistic interactions, could greatly accelerate design strategies for the construction of a synthetic promoter with enhanced inducibility and selectivity. The well established examples described have been discussed in the context of relatively new strategies that serve as small ‘stepping stones’ in the burgeoning and exciting field of plant synthetic biology. Acknowledgements I thank Sue Bosch for constructive comments and critical review of this manuscript.
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64 Li, Z.T. et al. (2004) Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Transgenic Res. 13, 143–154 65 Hehl, R. and Wingender, E. (2001) Database-assisted promoter analysis. Trends Plant Sci. 6, 251–255 66 Sawant, S. et al. (2001) Designing of an artificial expression cassette for the high-level expression of transgenes in plants. Theor. Appl. Genet. 102, 635–644 67 Venter, M. and Botha, F.C. (2004) Promoter analysis and transcription profiling: integration of genetic data enhances understanding of gene expression. Physiol. Plant. 120, 74–83 68 Wang, D. et al. (2005) Induction of protein secretory pathway is required for systemic acquired resistance. Science 308, 1036–1040 69 Higo, K. et al. (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 27, 297–300 70 Lescot, M. et al. (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30, 325–327 71 Matys, V. et al. (2003) TRANSFAC: transcriptional regulation, from patterns to profiles. Nucleic Acids Res. 31, 374–378 72 Van Helden, J. (2003) Regulatory sequence analysis tools. Nucleic Acids Res. 31, 3593–3596 73 Steffens, N.O. et al. (2004) AthaMap: an online resource for in silico transcription factor binding sites in the Arabidopsis thaliana genome. Nucleic Acids Res. 32, D368–D372 74 Holt, K.E. et al. (2006) ModuleFinder and CoReg: alternative tools for linking gene expression modules with promoter sequences motifs to uncover gene regulation mechanisms in plants. Plant Methods 2, 8 75 Tompa, M. et al. (2005) Assessing computational tools for the discovery of transcription factor binding sites. Nat. Biotechnol. 23, 137–144 76 Lawrence, C.E. et al. (1993) Detecting subtle sequence signals: a Gibbs sampling strategy for multiple alignment. Science 262, 208–214 77 Bailey, T.L. and Elkan, C. (1995) Unsupervised learning of multiple motifs in biopolymers using Expectation Maximization. Mach. Learn. 21, 51–80 78 Eddy, S.R. (2004) What is a hidden Markov model? Nat. Biotechnol. 22, 1315–1316 79 Geisler, M. et al. (2006) A universal algorithm for genome-wide in silico identification of biologically significant gene promoter putative cisregulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis. Plant J. 45, 384–398 80 Janssens, H. et al. (2005) Quantitative and predictive model of transcriptional control of the Drosophila melanogaster even skipped gene. Nat. Genet. 38, 1159–1165 81 West, A.G. and Fraser, P. (2005) Remote control of gene transcription. Hum. Mol. Genet. 14, R101–R111 82 Fraser, P. (2006) Transcriptional control thrown for a loop. Curr. Opin. Genet. Dev. 16, 490–495
Plant Science Conferences in 2007 Gordon Research Conference – Epigenetics 5–10 August 2007 Holderness School, Plymouth, New Hampshire, USA http://www.grc.org/programs.aspx?year=2007&program=epigen Fourth International Symposium on Dynamics of Physiological Processes in Roots of Woody Plants 16–19 September 2007 Bangor, UK http://www.joensuu.fi/metsatdk/gsforest/documents/Roots_Bangor.pdf 16th Biennial Australasian Plant Pathology Society Conference 24–27 September 2007 Adelaide, Australia www.plevin.com.au/apps2007 www.sciencedirect.com
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Synthetic promoters: genetic control through cis engineering Mauritz Venter Institute for Plant Biotechnology, Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
Technological advances in plant genetics integrated with systems biology and bioinformatics has yielded a myriad of novel biological data and insights into plant metabolism. This unprecedented advance has provided a platform for targeted manipulation of transcriptional activity through synthetic promoter engineering, and holds great promise as a way to further our understanding of regulatory complexity. The challenge and strategy for predictive experimental gene expression is the accurate design and use of molecular ‘switches’ and modules that will regulate single or multiple plant transgenes in direct response to specific environmental, physiological and chemical cues. In particular, focusing on cis-motif rearrangement, future plant biotechnology applications and the elucidation of cis- and trans-regulatory mechanisms could greatly benefit from using plant synthetic promoters. Modifying promoters – the way forward? One of the major challenges in a plant genetic engineering program is to design a transformation-cassette that would enable the precise control of transgene activity. The choice of promoter, to confer constitutive, spatial and/or temporal transgene expression, is one of the key determinants used in plant biotechnology applications. In recent years, a wide range of different promoters from plant, viral and bacterial origin have been characterized and used extensively in regulated transgene expression systems in plant cells [1– 3]. Several plant genetic engineering strategies have incorporated the use of strong constitutive promoters in the study of gene and transcription factor (TF) function as well as for conferring transgene expression for crop improvement and bio-pharmaceutical applications. The well-described cauliflower mosaic virus (CaMV) 35S promoter [4–7] confers high-level gene activity and has been used most commonly in plant transgene expression studies. The way forward for the study and design of inducible transgene expression cassettes was laid by research investigating the modification of the 35S promoter using the core-region (essential to initiate transcription), combined with dissected cis-regulatory elements, as well as early analysis of other dicot- and monocot-promoters [8–15]. From these initial attempts it has become apparent that the use of a synthetic and regulatory module that can be tuned, that is suited for a specific application and driven by the core-transcriptional initiation region of a constitutive promoter will prove invaluable in Corresponding author: Venter, M. ([email protected]). Available online 9 February 2007. www.sciencedirect.com
future genetic engineering programs. A few exciting studies have described how changes in promoter architecture, and the targeted design of cis-motif context can improve the control of spatial and temporal gene activity, regulate multiple transgenes, and overcome drawbacks such as homology-dependent gene silencing in plant cells [16–20]. Results from these research efforts underscore the value of using synthetic promoters to assist in elucidating synergistic regulatory interactions, the role of individual cis-motifs and in biotechnological applications. The scope of several other important factors such as gene silencing and RNA interference (RNAi), introns and splicing, transgene selection, different inducible systems and transformation technologies is too large to be covered in this review although they all play major roles in plant inducible gene expression studies. There is a considerable amount of data reporting on the use of chimeric inducible systems including promoters and transcription factors. In an attempt not to summarize all the relevant literature, this review will focus specifically on key examples highlighting previous and current strategies for the accurate design of an inducible plant synthetic promoter. An integrative design strategy for analysis and putative prediction of gene expression is proposed that incorporates transcriptomics, conserved cis-motif organization and the use of intricate bioinformatic software. Re-designing the regulatory code Refined and targeted modification of promoter architecture necessary for coordinated manipulation of gene activity requires accurate deciphering of the regulatory framework. The basics of cellular processes on a nucleotide and protein level have been studied and defined, resulting in a unified theory of eukaryotic gene expression and regulation. Even with the significant understanding gained from this fundamental research, major overlaps and synergistic interactions at different levels of regulation are still poorly understood. Studies in model organisms focusing on the principles of transcriptional control, promoters, gene expression and regulatory networks [21– 28] have emphasized the importance of combining regulatory data (cis-motifs and transcription factors) and gene expression profiles to elucidate the underlying mechanisms governing genetic control. The construction of sophisticated in silico promoter models [29,30] has enabled more accurate prediction of gene expression and/or association (but not necessarily function). To predict desired gene expression patterns in response to specific cues it is necessary to understand combinatorial regulatory principles. A ground-breaking study conducted in the model eukaryotic
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organism Saccharomyces cerevisiae (yeast) revealed how conserved cis-motif-logic can be used for relatively accurate prediction (73%) of a distinct expression pattern during a specific condition [31]. Results of this study suggest that it should be possible to design a synthetic promoter model that could confer a particular expression pattern in a biotechnological application dependent on the availability of regulatory information. The strategy is simple. However, the complexity and synergistic interplay of a multitude of regulatory mechanisms (although individually well characterized) poses a significant challenge for future promoter design. Given this complexity it is evident that the integration of an improved understanding of genome-wide regulatory mechanisms of complex, multi-cellular eukaryotes and the power of systems biology, will serve as the basis for synthetic and predictive biology [32,33]. In silico predictions of gene regulatory events must ultimately be validated experimentally. Synthetic promoters play a major role in elucidating cis-motif logic (orientation, strength–weight, position) to study gene regulation in vivo [34,35] as well as advancing intricate design strategies of expression cassettes to be used for metabolic pathway engineering [16,36–39] and gene therapy applications [40–43]. With the focus on plants, the accessibility of a vast amount of genetic data that were produced as a result of sequencing the whole-genomes of model organisms such as Arabidopsis thaliana (thale cress) and Oryza sativa (rice) have enabled the rapid identification of large sets of promoter sequences. Recent years have also witnessed much progress in understanding plant promoter architecture and general TF assembly [44–47]. As a result of the preceding factors, several promoters and TFs have been selected for inducible transgene expression studies. Promoters used in these studies include unmodified wild-type, synthetic (with new combinations of cis-motifs from various sources) and truncated (reduced to cis-motifs essential for desired expression profile) modules, and are divided into the following categories: (i) constitutive, (ii) inducible, (iii) tissue- and (iv) developmental-stage specific. Most promoters have a core- or minimal-region necessary to initiate transcription. Established strategies for the isolation of promoters using approaches for the large-scale identification of candidate genes, expressed under a certain condition are effective but time-consuming. By contrast, the development of high-throughput technologies for extensive identification of TF-binding sites (such as chromatin immunoprecipitation assays – ChiPs) and advances in plant TF-database assistance has proved invaluable in characterizing native plant promoters [48]. ChiP is a strategy that enables the rapid identification of large sets of promoter regions using a specific TF as a probe [49,50]. However, until now these strategies have not been optimized for a wide variety of plant species. In addition to fundamental cis-motif elucidation, other factors considered to be stumbling blocks in applied plant genetic engineering include expression of multiple transgenes, specific inducibility of gene expression and homology-dependent gene silencing. The inability of conventional wild-type plant promoters to address these hurdles has renewed interest in the development of synthetic promoters. www.sciencedirect.com
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Even better than the real wild-type thing A synthetic promoter designed to initiate transcription of protein-encoding genes should adhere to the universal requirements necessary for eukaryotic gene transcription. Such a promoter is a stretch of DNA comprising a corepromoter region and multiple repeats or combinations of heterologous upstream regulatory elements (cis-motifs or TF-binding sites). The core-promoter region (also known as the minimal-region) usually contains a TATA-box necessary for recruiting RNA polymerase II and the orchestrated assembly of general transcription factors to form the preinitiation complex (PIC) [51]. The CaMV 35S core-promoter is ideal for transcription initiation and has been used in several plant promoter engineering strategies. The two most frequently used strategies are (i) combinatorial engineering of cis-motifs upstream of the core-promoter and/or (ii) combined with bidirectionalization of a unidirectional promoter (Figure 1). Synthetically organized motifs include enhancers, activators and/or repressors that bind to TFs expressed in response to specific conditions. Promoters that respond to inducers can be regulated by (i) environmental cues (i.e. light, cold and heat stress); (ii) biotic and abiotic stress (pathogens, wounding, insects, drought and salinity); (iii) hormones (i.e. ethylene, auxin, abscisic and salicylic acid); and (iv) chemicals (i.e. tetracycline, copper, estradiol and dexamethasone). The use of synthetic promoters that allow for targeted inducibility is of considerable interest for plant engineering strategies. Non-plant transactivated- and chemical-inducible systems (using transformation cassettes incorporating a core-promoter and multimers of the upstream activation sequences) have gained much attention, although not emphasized in this discussion. Some of these systems have proven highly flexible and could be used to either repress or activate plant transgene expression. Comparative and indepth analyses of these systems have recently been reviewed [52,53]. cis-Motif engineering An in-depth study using synthetic promoters illustrated the usefulness of combining TF knowledge to ‘cut and paste’ pathogen-inducible cis-motifs [17,54]. Results from this study showed that promoter inducibility and strength varied depending on motif copy number and, more specifically, spacing of motifs (with the same core-sequence) relative to the TATA-box. Moreover, one of the main observations in this detailed study revealed that promoter activity was not necessarily enhanced with an increase in motif copy number [17] and, in several instances, it was shown that a single copy of a specific cis-motif was sufficient for a pathogen-induced response. The functionality of defense-related plant TF binding sites appears to be conserved among different plant species [17]. This shared regulatory function will assist in the future design and application of pathogen-inducible promoters across the borders of plant species [54]. There are still several combinatorial mechanisms of regulatory context and signaling that are largely unknown, which prevents the optimal design of a synthetic pathogen-inducible promoter. We are still in the early stages of understanding plant– pathogen interactions and it is evident that the use of
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Figure 1. Simplified representation of a plant synthetic promoter. Core-region containing TATA-box (green block arrow) of a wild-type constitutive promoter, such as CaMV 35S, is used to drive transcription. A uni- or bidirectional synthetic promoter comprises the stretch of DNA containing multiple copies of a specific cis-motif (blue ellipses) upstream of 35S core-promoter to bind transcription factors (red block arc) expressed in response to specific stimuli.
synthetic promoters will not only be valuable for genetic engineering of disease resistant plants, but also contribute to elucidating pathogen-induced motif function out of native context [17,54]. Homology-dependent gene silencing (HDGS) is of great concern in plant genetic engineering strategies and is thought to be caused by multiple copies of homologous transgene and promoter sequences. Transgene silencing can occur on a transcriptional and posttranscriptional level [55,56]. Investigations in Nicotiana tabacum (tobacco) have shown that the use of synthetic promoters with minimal sequence similarity could serve as a valuable tool to overcome HDGS in plant transgenic strategies [18]. Comparison was made between the ‘reshuffling’ (or swapping) of previously described functional domains of 35S [7] and the incorporation of cis-motifs in a synthetic stretch of DNA, both driven by 35S core-promoter sequence [18]. Both strategies were evaluated in comparison to high-level expression of the wild-type CaMV 35S promoter. Results from this study showed that domain swapping led to a less efficient promoter activity compared with insertion of cis-elements in a synthetic DNA-sequence [18]. However, it is suggested that repetitive use of ciselements with identical core-sequences and homologous intervening regions (within a functional domain) might cause depletion of TFs, consequently reducing endogenous gene expression [18]. Therefore, design strategies using multimers of cis-motifs need to be optimized to achieve the desirable inducibility of the transgene without compromising endogenous ‘house-keeping’ regulation in the plant cell. An important study investigated individual and combinatorial functions of light responding elements (LREs) in a synthetic stretch of DNA driven by the core-promoters of www.sciencedirect.com
the nopaline synthase (NOS) gene and CaMV 35S [15]. The NOS core-promoter confers no basal transcriptional activity, enabling an ‘ON or OFF’ phenotype to be used and proved to be ideal for engineering inducible synthetic systems [15]. Owing to limitations in our understanding and shortfalls in current design strategies only a limited number of studies have shed light on multimeric cis-motif design. Inducible transgene expression in both directions In many plant biotechnological applications (e.g. metabolic engineering or pathogen resistance) the advantages of expressing multiple transgenes are not only advantageous, but necessary. Gene-stacking, or pyramiding, remains a challenge for crop improvement and several different systems and strategies have been reviewed [57,58]. There are unique reports describing naturally occurring bidirectional promoters in different organisms, expressing two genes simultaneously [59–62]. A few key studies have focused on the usefulness of modifying a unidirectional promoter to a bidirectional promoter for specific biotechnological applications in plants (e.g. simultaneously expressing sense and antisense transcripts to mediate gene silencing) [16,63]. Comparative analysis of bi-directional promoters in Vitis vinifera (grape) and Nicotiana tabacum (tobacco), demonstrated higher reporter-gene expression efficiency compared with a unidirectional expression system incorporating similar enhancer and core-promoter complexes [64]. Furthermore, this study supported previous views on how the formation of transcription machinery is more efficient in a bidirectional mode compared with that in a unidirectional regulatory structure. In addition to the
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above, recent investigations shed light on how the synthetic cis-motif context (of a synthetically designed activation module) and combinatorial interactions regulate gene expression and influence the stability of transcriptional assembly on the TATA- box of the corepromoter [19]. It appears that there is a fine balance between complete occupancy of synthetic cis-motifs and success-rate of complete transcriptional activation and stability of the initiation complex assembled on the TATA-box. This suggests that depletion of endogenous TFs because of an excess of synthetic TF-binding sites can cause transcriptional inactivation [18,19]. This is one of the major challenges that could greatly limit future promoter engineering strategies. Nevertheless, advances in bioinformatics (incorporating plant TF-databases) [65] combined with plant TF-function and accurate in silico promoter models could assist in a more refined strategy for designing synthetic promoters. A recent investigation successfully integrated computational assistance and bidirectionalization to construct a synthetic transcriptional unit for high-level reporter-gene expression in response to specific elicitors, which yielded exciting results [20]. The complete synthetic stretch of DNA, known as the transcription activation module (TAM), was constructed from regulatory sequences compiled from a database of highly expressed plant genes [66]. Stepwise construction of this TAM consisted of: (i) screening a computational database for genes with high expression levels in plants and (ii) identification of conserved TATA-box proximal regions and motifs 500 base pairs upstream from the transcriptional start site. Initial analysis showed that this TAM could express the b-glucuronidase (GUS) reporter gene in stably transformed tobacco (Nicotiana tabacum) at higher levels compared with those expressed by the wild-type CaMV 35S promoter [66]. As already discussed in this review, it has been shown that the TAM could facilitate stable enhancer complex (enhanceosome) assembly for accurate transcriptional initiation [19]. The TAM was modified in a more recent study to confer bidirectional expression of reporter genes, b-glucuronidase (GUS) and green fluorescence protein (GFP), and evaluated in transient and stably transformed tobacco (Nicotiana tabacum) leaf discs [20]. Expression of both reporter genes was up-regulated in a bidirectional mode in response to specific elicitors, suggesting that enhanceosome assembly to form a stable PIC on both TATA-boxes (modulated by a DNA-bending sequence within the TAM) could initiate transcription in both directions [20]. Specific design strategies for the development of synthetic promoters are in their infancy. The investigations described here, which were performed by several different research groups, are some of the major examples of the strategies being used in plant promoter engineering, and could serve as important technical contributions for future biotechnological applications as well as in the elucidation of synergistic motif-TF interplay. In silico analysis to assist in the grand design The combination of high-throughput gene expression profiles with promoter architecture and bioinformatics is a powerful approach to compile data for the possible prediction of coordinated gene expression during a specific www.sciencedirect.com
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condition [31,67,68]. In this section, a simplified representation will be used to illustrate how this strategy could facilitate a practical and refined design of a plant synthetic promoter able to confer differential gene expression during defined conditions (Figure 2). Promoter regions of gene-clusters co-expressed during a specific condition contain conserved cis-motifs. The biggest challenge is to dissect which conserved motif is associated directly with the specific condition under investigation and which are associated with co-expression [21]. The emergence of plant TF-binding site database-assistance [65] has greatly facilitated large-scale plant promoter analysis. The three major plant TF-binding site databases, PLACE [69], PlantCARE [70] and TRANSFAC [71], are updated constantly and provide a plethora of possible cis-motif combinations. To analyze and/or construct accurate in silico promoter models, it is essential to distinguish between overrepresented motifs and background ‘noise’. Several computational methods have been developed for this purpose [72–74], and assessments of the most prominent cis-motif detection and/or classification methods and statistical tools have been reviewed [75]. Identification of cis-regulatory codes remain complex, particularly given that within a promoter sequence of 500 to 5000 base pairs the core sequence of a cis-regulatory motif can range between 4 to 10 base pairs. Gibbs sampling [76] and expectation maximization (MEME) [77] are powerful methods for predicting over-represented motifs. There are numerous other methods based on different operating principles [75] and some are modified and/or improved versions combined with statistical modeling techniques such as hidden Markov models (HMMs) [78]. Initial promoter sequence output, using plant TF-database assistance, can be refined by these probabilistic algorithms to select over-represented cis-motifs in a multiple set of sequences upstream of coexpressed genes. Furthermore, it should be possible to identify universal or common cis-motifs that are synergistically associated with cis-motifs of promoters that are induced during different conditions [21]. Probabilistic fine-tuning of promoter architecture is followed by construction of a motif synergy map using design ‘rules’ AND-NOT-OR cis-motif logic. This simplified approach accentuates a combination of previous studies conducted in S. cerevisiae [21,31] and Arabidopsis [79] that could assist in the design of one or several plant synthetic promoters to facilitate a more accurate prediction of gene expression in response to each specific condition (Figure 2). Single copies of a cis-motif in a synthetic stretch of DNA might be sufficient for a desired gene expression profile [17]; however, the operation principles of the current computational methods are more useful for identifying a cluster of over-represented cis motifs in a contiguous segment of DNA [75]. By contrast, conventional views regarding the nature of combinatorial control appears to be inconsistent with recent and exciting experimental evidence showing that quantitative transcriptional control of the Drosophila melanogaster (fruitfly) even skipped gene (eve) is operative outside a compact arrangement of cis-motifs [80]. It is evident that precise genetic control is governed by TF binding specificity [80] and a complex network of regulatory mechanisms on different levels (chromatin, histone and
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Figure 2. Combinatorial cis-motif engineering for the accurate design of synthetic promoters. (a) DNA microarrays enable large-scale identification of induced gene expression (indicated by blue lightning bolt) in response to a specific condition (e.g. stress, chemical induction or plant development or growth). Upstream promoter regions (black line and white block arrow) of gene clusters contain a multitude of conserved and non-conserved cis-motifs (black circles, triangles, rectangles and squares) [67]. (b) Probabilistic motif detection algorithms (MEME and Gibbs sampling) combined with plant TF-database assistance enable the identification of conserved motifs (indicated by blue circles, yellow rectangles, red triangles and green squares) within multiple promoter sequences associated with a particular condition [65,76,77]. (c) A cis-motif synergy map is constructed to gain a holistic view of: (i) motif occurrence in relation to other motifs associated with a specific condition [21,23] and (ii) motif spacing relative to the TATA-box of the corepromoter. Computational promoter modeling [29,30] based on the combined regulatory organization associated with different conditions and gene expression patterns enable the use of a more refined synthetic design strategy. (d) Plant synthetic promoters are engineered by dissection of cis-regulatory context (crossed out elements are removed) specific for induction of transgene activity in response to different conditions. Transcription is initiated by the 35S core-promoter sequence (indicated by green block arrow). www.sciencedirect.com
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nuclear organization) [24] and over long genomic distances [81,82]. Given that a single TF can bind to cis-motifs of different genes, permitting combinatorial control [45,46], the possibilities for future inducible expression systems are exciting and challenging. Concluding remarks The goal of this review was to prioritize specific past and more recent research investigations that have focused on design strategies and applications of plant synthetic promoters. It is evident that the complexity in dissecting cisregulatory architecture alone (where spacing, relative to the TATA-box and other motifs, orientation, copy number and function of the motif) poses a major challenge for synthetic promoter design. Advances in bioinformatics and a more holistic deciphering of regulatory processes including plant TF networks and cis- and/or trans-synergistic interactions, could greatly accelerate design strategies for the construction of a synthetic promoter with enhanced inducibility and selectivity. The well established examples described have been discussed in the context of relatively new strategies that serve as small ‘stepping stones’ in the burgeoning and exciting field of plant synthetic biology. Acknowledgements I thank Sue Bosch for constructive comments and critical review of this manuscript.
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Plant Science Conferences in 2007 Gordon Research Conference – Epigenetics 5–10 August 2007 Holderness School, Plymouth, New Hampshire, USA http://www.grc.org/programs.aspx?year=2007&program=epigen Fourth International Symposium on Dynamics of Physiological Processes in Roots of Woody Plants 16–19 September 2007 Bangor, UK http://www.joensuu.fi/metsatdk/gsforest/documents/Roots_Bangor.pdf 16th Biennial Australasian Plant Pathology Society Conference 24–27 September 2007 Adelaide, Australia www.plevin.com.au/apps2007 www.sciencedirect.com