Being able to turn genes on and off as required is a significant goal in genetic manipulation. Synthetically constructed gene circuits which turn on and off in response to specific activators have previously been demonstrated and the ability knock out specific genes with such technology as CRISPR/Cas 9 has made gene manipulation easier and cheaper.
But the purpose of this particular article in the Plant Biolotechnology Journal (open-access = awesome) was focused on manipulating the mechanics of gene transcription repression to turn off gene repressors (and activate expression) and then reverse the process in subsequent generations as required.
Transcription factors, the proteins that form part of the machinery that transcribes DNA sequences into RNA, contain motifs that allow for them to be activated and deactivated. Regulation of gene transcription using the activation and deactivation of transcription factors is a necessity in all cells, allowing the expression of genes to be limited to particular moments within a cycle or at different stages of development.
The examples used in this study when demonstrating the novel bit of tech the researchers developed to force the repression and activation of different genes illustrate the need for this function well. One gene, MYB80, codes a transcription factor necessary for pollen development and the programmed cell death (PCD) of the tapetal in plant anthers. If allowed to transcribe one of its targeted genes unregulated, PCD occurs too early and the male flowers are rendered sterile, meaning the transcription must be regulated if the plant is to develop properly. This consequence was discovered in an earlier study when a mutant of the transcription factor was created which deleted part of the encoding gene which resulted in the loss of the regulatory motifs, likely meaning the the transcription factor was unhindered in transcribing its target genes.
Another transcription factor studied by the researchers was that encoded by WUSCHEL (WUS), required for the development of the organising centre beneath the stem cells in the meristem. Regulation of WUS activity is required to create distinct cell boundaries and a lack of regulation leads to disorganised growth and male sterility. Similar to MYB80, the transcription factor has a number of regulatory motifs at the C-terminal end of the protein which interact with corepressors.
Manipulating the function of transcription factors to regulate gene transcription is therefore another potential method of turning genes on and off to test the knock-on effects and potentially alter and improve plant characteristics.
Synthetically Regulating the Transcription Factors
How these regulatory motifs inhibit gene transcription, and how we might be able to cause the same inhibition, was the focus of this research. Using the MYB80 and WUS as targets, the researchers created what they dubbed “Conserved Sequence-guided Repressor Inhibition”, or “CoSRI”.
The name of the protein gives away how it works. This study and previous studies have found that within specific repressors are regions that are conserved across many species. Studies that have removed these conserved regions have resulted in the types of defects discussed above due to the unrestrained transcription of the genes targeted by the subject transcription factors. When the conserved sequence is reinstated, repression of transcription is reinstated and phenotypes return to match the wild-type plants.
Fig 1a from the article. The figure demonstrates how the CoSRI is targeted to the conserved section of the repressor gene and the section of the CoSRI interfering with the binding of the corepressor to the repressor.
So the researchers targeted these conserved sequences as being the sites to direct their newly constructed proteins, thereby building in some specificity to which repressors are targeted. Conjugated to this new protein is a repression-motif interacting region, a section of protein which interacts with the DNA binding section of the transcription factor, stopping the corepressors from interacting with the DNA binding domain of the transcript factor.
Testing the Constructs
Genes for the CoSRI proteins designed specific to the MYB80 and WUS transcription factors were transformed into Arabidopsis thaliana separately.
When the MYB80-specific CoSRI was introduced, the same silique abortion and male sterility were observed. Sterile lines analysed showed that the transcript levels of the CoSRI were at the same levels of greater than the levels of the endogenous transcription factors. Further, transcripts of the genes targeted by the endogenous genes were reduced in sterile lines.
Similarly, CoSRI genes specifically targeting the WUS transcription factor was transformed into A. thaliana, again resulting in altered phenotypes, specifically defective shoot meristems resulting in poor growth and failure of silique elongation leading to loss of fertility.
In short, the researchers had developed a synthetic construct that competed with corepressors for the ability to bind to the repression-binding motif of the transcription factor and blocked the active repression of gene transcription normally mediated by the transcription factors.
Restarting the Repression
Being able to stop the repression of gene transcription is great, but what about reversing it? The example of such a use given in the paper is forcing male sterility in, say, a hybrid plant species, but then being able to reverse the sterility later.
To achieve this, the researchers created what they termed a restorer construct which combined the first 188 amino acids at the N-terminus of the corepressor (the part required to repress transcription by the transcription factor) with a conserved region of the target transcript factor. The result is a protein that is directed to, in one example, the MYB80 transcription factor that carries with it the necessary part of the corepressor to repress the workings of the target transcription factor and restoring the wild-type phenotype.
Figure 1b from the article. The figure depicts the interaction of the CoSRI with the repressor to block corepressors from the repression binding site of the transcription factor, and the use of the restoring CoSRI to reverse the sterility caused by the first CoSRI.
The researchers tested whether the new construct interfered with the transcription levels of any other genes or the transcript levels of the MBY80 targets in wild-type A. thaliana that had not been previously transformed with a CoSRI, finding no interference.
In lines that had both the CoSRI and the restorer construct, sterility was reversed when the transcript levels of the restorer were similar to that of the CoSRI. Fertility was only partially restored or not restored at all when the restorer construct levels were significantly lower than their competition.
This new technique appears to provide an indirect and reversible method of altering the transcription of specific genes. Being able to stop and restart transcription factors for specific genes could be invaluable to plant (and other) research and the technique could find its way in the the toolbox of genetic engineering technology that is exploding at the moment.
The technology could also open up the possibility of being able to alter plant characteristics to suit seasonal requirements, particularly if we develop the ability to not just knockdown and re-initiate gene transcription but also to carefully calibrate gene transcript levels.