Review
Synthetic promoters: genetic control through cis engineering

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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.

Section snippets

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

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

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 core-promoter 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

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

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, 60, 61, 62. A few key

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 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).

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 cis-regulatory 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

Acknowledgements

I thank Sue Bosch for constructive comments and critical review of this manuscript.

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