Repressilator

Circadian circuits in plants feature a transcriptional regulatory feedback loop called the repressilator. In the core oscillator loop (outlined in gray) in A. thaliana, light is first sensed by two cryptochromes and five phytochromes. Two transcription factors, Circadian Clock Associated 1 (CCA1) and Late Elongated Hypocotyl (LHY), repress genes associated with evening expression like Timing of CAB expression 1 (TOC1) and activate genes associated with morning expression by binding to their promoters. TOC1, an evening gene, positively regulates CCA1 and LHY via an unknown mechanism.[3] Evening-phased transcription factor CCA1 Hiking Expedition (CHE) and histone demethylase jumonji C domain-containing 5 (JMJD5) directly repress CCA1. Other components have been found to be expressed throughout the day and either directly or indirectly inhibit or activate a consequent element in the circadian circuit, thereby creating a complex, robust and flexible network of feedback loops.[3]

The morning-phase expression loop refers to the genes and proteins that regulate rhythms during the day in A. thaliana. The two main genes are LHY and CCA1, which encode LHY and CCA1 transcription factors.[4] These proteins form heterodimers that enter the nucleus and bind to the TOC1 gene promoter, repressing the production of TOC1 protein. When TOC1 protein is expressed, it serves to regulate LHY and CCA1 by inhibition of their transcription. This was later supported in 2012 by Dr. Alexandra Pokhilo, who used computational analyses to show that TOC1 served this role as an inhibitor of LHY and CCA1 expression.[5] The morning loop serves to inhibit hypocotyl elongation, in contrast with the evening-phase loop which promotes hypocotyl elongation. The morning phase loop has shown to be incapable of supporting circadian oscillation when evening-phase expression genes have been mutated,[5] suggesting the interdependency of each component in this naturally-occurring repressilator.    

Early Flowering 3 (ELF3), Early Flowering 4 (ELF4) and Phytoclock1 (LUX) are the key elements in evening-phased clock gene expression in A. thaliana. They form the evening complex, in which LUX binds to the promoters of Phytochrome Interacting Factor 4 (PIF4) and Phytochrome Interacting Factor 5 (PIF5) and inhibits them.[3] As a result, hypocotyl elongation is repressed in the early-evening. When the inhibition is alleviated late at night, the hypocotyl elongates. Photoperiod flowering is controlled by output gene Gigantea (GI). GI is activated at night and activates the expression of Constans (CO), which activates the expression of Flowering Locus T (FT). FT then causes flowering in long-days.[3]

Mammals evolved an endogenous timing mechanism to coordinate both physiology and behavior to the 24 hour period.[6] In 2016, researchers identified a sequence of three subsequent inhibitions within this mechanism that they identified as a repressilator, which is now believed to serve as a major core element of this circadian network. The necessity of this system was established through a series of gene knockouts amongst cryptochrome (Cry), period (Per), and Rev-erb -- core mammalian clock genes whose knockouts lead to arrhythmicity.[6] The model that these researchers generated includes Bmal1 as a driver of E-box mediated transcription, Per2 and Cry1 as early and late E-box repressors, respectively, as well as the D-box regulator Dbp and the nuclear receptor Rev-erb-α. The sequential inhibitions by Rev-erb, Per and Cry1 can generate sustained oscillations, and by clamping all other components except for this repressilator oscillations persisted with similar amplitudes and periods.[6] All oscillating networks seem to involve any combination of these three core genes, as demonstrated in various schematics released by researchers.

Artificial repressilators were discovered by implanting a synthetic inhibition loop into E. coli.  This represented the first implementation of synthetic oscillations into an organism. Further implications of this include the possibility of rescuing mutated components of oscillations synthetically in model organisms.[7]

The artificial repressilator is a milestone of synthetic biology which shows that genetic regulatory networks can be designed and implemented to perform novel functions. However, it was found that the cells' oscillations drifted out of phase after a period of time and the artificial repressilator's activity was influenced by cell growth. The initial experiment[7] therefore gave new appreciation to the circadian clock found in many organisms, as endogenous repressilators are significantly more robust than implanted artificial repressilators. New investigations at the RIKEN Quantitative Biology Center have found that chemical modifications to a single protein molecule could form a temperature independent, self-sustainable oscillator .[12]

Artificial repressilators could potentially aid research and treatments in fields ranging from circadian biology to endocrinology. They are increasingly able to demonstrate the synchronization inherent to natural biological systems and the factors that affect them.[13]

A better understanding of the naturally-occurring repressilator in model organisms with endogenous, circadian timings, like A. thaliana, has applications in agriculture, especially in regards to plant rearing and livestock management.[14]