Manipulation of Gene Expression in Plants Through Artificially Made Zinc Fingers

Transcription factors vary in their composition and mode of action. An important class of transcription factor molecules fold around zinc atoms and contact DNA in a “finger-like” fashion, and hence are named zinc finger proteins (TFszf). They modulate gene expression at the terminus of cellular signal transduction. Like all other transcription factors, they bind to DNA April 2003, Pages 163168and other regulatory bodies to turn on or off transcription of the coding regions of a gene.

In the April  2003 issue of Current Opinion of Plant Biology (vol. 6: 163-168), CF Barbas III at the Scripps Research Institute, CA and his associates affiliated with the University of Arizona and Diversa Corporation, CA, review the progress of this relatively new field.  This review describes how predefined and artificially made zinc-finger proteins can be applied to regulate virtually any gene of interest.

Gene regulation with transcription factors: Transcription factors are small molecules that attach to specific sites on a DNA molecule in order to activate or deactivate expression of certain genes. The authors state that they are modular proteins that have a DNA-binding domain and an effector domain. The latter regulates the type of reactions that occur at the site. These factors have evolved over a long period of time under selection pressure allowing them to exercise precise control over particular genes. Determination of base sequencing of the whole genome of Arabidopsis in 2000 and two years later of the indica and japonica rice led to the identification of a large number of theoretically possible transcription factors. However, the pace of relating them to molecular targets has fallen far behind.  As such, they cannot, at the moment, be used as gene-specific tools for regulating endogenous gene expression.

Adaptability of zinc-finger proteins: The advantage of zinc-finger proteins is that they recognize specific DNA sequences. The zinc-finger motif is the most common DNA-binding motif in nature. In the human genome, it is by far the most commonly encoded type of protein, and certain plant species also contain this motif, presumably for gene recognition and control.

Construction of artificial zinc fingers: The authors point out that several methods are currently available to construct artificial zinc finger proteins. The most commonly used technique consists of putting together previously selected operable zinc-finger modules. Such modules are constructed starting with the middle finger in a 3-finger protein. Using phage-display technology, artificially made modules are selected to recognize a new 3-bp binding site. The optimal binding capacity is further enhanced by site-directed mutagenesis. Assembly of modules can be achieved in any order to form new three- or six-finger proteins. The authors call attention to the fact that a number of zinc-finger-based artificial transcription factors have already been assembled and applied in the model plant Arabidopsis. Furthermore, they point out that a population of only 64 modules is necessary to recognize all possible 3 base pair codon sites. Thus, even though the human genome consists of 3.2 billion base pairs of DNA, it has been shown that the newly constructed six-finger proteins have the capacity of binding to a majority of these sites. Each one of them recognizes a unique site in the human genome. Furthermore, using this technology combined with PCR-based assembly of known zinc-fingers, it has been possible to bioengineer new proteins. Researchers in this area are confronted with one big challenge as to how a character altered by means of artificial zinc-finger proteins can be faithfully transferred to its progeny.

How do zinc-finger proteins modify floral organ traits? In wild-type flowers of Arabidopsis, all the four whorls, namely, the sepals, petals, stamen and carpel are fully developed. According to the floral-identity model, AP1 is expressed in the sepals and petals, whereas AP3 is expressed in the petals and stamen. In this connection, the authors refer to the study of Guan and his associates; they designed TFszf to target the Apetalla3 (AP3) promoter in Arabidopsis for activation or repression. By means of a diagram, the authors have explained how partial transformation of the sepals into petals can be brought about by expressing an Ap3-specific activator
(VP64::AP3) with the Ap1 promoter. On the other hand, partial transformation of the petals into sepals can be achieved by expressing an Ap3-specific repressor (SID::AP3) with the help of the same promoter. Thus by modifying the structure of the zinc-finger and binding it to the specific site of the gene, the expression of members of a floral whorl can be partially altered.

Modular construction of artificial transcription factors. By means of a series of diagrams, the authors describe the 18-bp long binding site of the AP3 (Apetalla3) gene. Existing zinc-finger domains as shown in the diagram recognize each of the 3-bp subsites in the target sequence, preceding the coding region. Depending upon whether an activation or repression domain is fused with the zinc-finger protein, which is, in this case SID (Sin3A interaction domain), it is possible to either activate or deactivate the AP3 gene. In other words, an artificial transcription factor, TFszf, can be created to control the function of an endogenous gene.

Conclusions: In their concluding remarks, the authors describe that artificial transcription factor technology such as the one used in the production of TFszf will prove to be a powerful tool for regulating specific genes of interest. There are already positive indications that by using artificial transcription factors such as laboratory-made zinc-finger proteins, it may be possible to either up- or downregulate specific plant genes. Once this technology is more fully developed, it could potentially be used to manipulate the expression of any given plant gene. However, a good deal of research efforts need to be undertaken in order to ensure that characters, altered by artificially made zinc-finger protein technology, remain stable and are inherited to subsequent generations without reversion.

 

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