Bryk et al. 2017

Citation

Bryk, J., Reeves, R. G., Reed, F. A., & Denton, J. A. (2017). Transcriptional effects of a positive feedback circuit in Drosophila melanogaster. BMC Genomics, 18(1), 990.

Links

• https://doi.org/10.1186/s12864-017-4385-z
• https://scholar.google.com/scholar?cluster=1000773242820105337
• http://hawaiireedlab.com/pdf/b/bryketal2017.pdf (internal lab link only)

Published Abstract

Background

Synthetic systems that use positive feedback have been developed to control human disease vectors and crop pests. The tTAV system, which has been deployed in several insect species, relies on a positive feedback circuit that can be inhibited via dietary tetracycline. Although insects carrying tTAV fail to survive until adulthood in the absence of tetracycline, the exact reason for its lethality, as well as the transcriptomic effects of an active positive feedback circuit, remain unknown.

Results

We engineered the tTAV system in Drosophila melanogaster and investigated the effects of tTAV genome integration locus on the whole fly transcriptome during larval and adult life stages in four transgenic fly strains using gene expression microarrays. We found that while there were widespread effects on the transcriptome, the gene expression differences after removal of tetracycline were not consistent between integration sites. No specific region of the genome was affected, no common set of genes or pathways, nor did the integration site affect the transcripts in cis.

Conclusion

Although the positive feedback tTAV system is effective at killing insect larvae regardless of where it is inserted in the genome, it does not exhibit a specific, consistent transcriptional signature. Instead, each insertion site is associated with broad, but different, transcriptional effects. Our results suggest that lethality may not be caused by a direct effect on transcription of a set of key genes or pathways. Instead, we propose that rather than a specific action of a tTAV protein, it is the stochastic transcriptional effects specific to each insertion site that contribute to the tTAV-induced mortality.

Synopsis

We wanted to determine the genome-wide transcriptional response to a runaway tTAV positive feedback loop. The tTAV insert works as expected in D. melanogaster resulting in lethality when larvae are raised without tetracycline. While we did see thousands of genes across the Drosophila genome that significantly changed expression, there was no clear pattern that emerged in the types or location of genes with altered expression or even in the direction of altered expression. The tTAV system was inserted at different points in the genome and these different lines gave different groups of genes with altered expression. If these results can be generalized the insertion site of a highly expressed transgene has a profoundly idiosyncratic effect on genome-wide transcription patterns.

Background

The tTAV gene expression system was developed into a genetic pest management tool by various groups (e.g., Horn & Wimmer 2002; Alphey 2015). A positive feedback form of the tTAV system was being marketed by Oxitec as "RIDL." In this system a transgene promotes its own expression by binding to DNA. If left unchecked this causes a runaway positive feedback and is toxic to the organism. However, the protein produced also binds to tetracycline and if tetracycline is added to the insect's diet then the lethal effect is masked (tetracycline bound protein does not bind to DNA and drive gene expression).

In a nutshell, in applications large amounts of insects can be reared with tetracycline. Then released into the wild to mate with wild individuals. The toxic effect is dominant and kills off the offspring of the wild individuals that inherit it (who presumably do not have tetracycline in their diet). If enough individuals can be released relative to the wild population this can cause a reduction in wild population numbers over the following generations. This is similar to classical SIT (Sterile Insect Technique) for population suppression.

Importance

The cause of this lethality is unknown. One can imagine that a positive feedback loop of gene expression and protein production may over-use transcriptional and translational resources and/or overload protein degradation pathways, or disrupt off target gene expression. These hypotheses are not mutually exclusive. Looking at the shift in expression of other genes in the genome in response to tTAV runaway expression could be informative and give us clues as to what process results in lethality.

If the transcriptional response to a single insert were studied we would have found a significant response, with many genes affected, and may have been misled by the resulting pattern (if for example a number of these genes were in a certain pathway), overgeneralizing the particular signal seen.

Critiques

This project was started many years ago and used microarrays designed for Drosophila. If we were to do it again we would likely use RNA-seq. This would also allow us to quantify tTAV mRNA abundance, which was the biggest factor missing in this study.

Follow-Up

Does this widespread idiosyncratic effect extends to other genetic modifications or is specific to the tTAV system. For example, what is the genome-wide translational response, if any, to Actin5c driven expression of EGFP? Importantly, does any effect seen vary widely by insertion site?

See Also

Horn C, Wimmer EA. A transgene-based, embryo-specific lethality system for insect pest management. Nat Biotechnol. 2002;21:64–70.

Alphey, Luke. "Expression system for insect pest control." U.S. Patent 9,121,036, issued September 1, 2015.