Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach

Abstract

The Notch effector gene Hes1 is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations in vivo, the mechanism driving oscillations has never been proven. Here, we applied a "build it to understand it" synthetic biology approach to construct simplified "hybrid" versions of the Hes1 ultradian oscillator combining synthetic and natural parts. We successfully constructed a simplified synthetic version of the Hes1 promoter matching the endogenous regulation logic. By mathematical modeling and single-cell real-time imaging, we were able to demonstrate that Hes1 is indeed able to generate stable oscillations by a delayed negative feedback loop. Moreover, we proved that introns in Hes1 contribute to the transcriptional delay but may not be strictly necessary for oscillations to occur. We also developed a novel reporter of endogenous Hes1 oscillations able to amplify the bioluminescence signal 5-fold. Our results have implications also for other ultradian oscillators.

Episodic Ultradian Events—Ultradian Rhythms

“…As such, Hes1 and Hes7 are unlikely to underlie the coordinated physiological EUEs that are observed in adulthood. However, their rhythms are considered essential in cell fate and vertebrate segmentation respectively [56].…”

Regulation of Gene Expression and Signaling Pathway Activity in Mammalian Cells by Automated Microfluidics Feedback Control

“…We assessed the feasibility of precisely regulating the expression of a gene of interest from an inducible promoter by means of automated microfluidics feedback control. We stably integrated in Chinese Hamster Ovary (CHO) cells ( Figure 1b) a tetracycline responsive promoter 18 driving the expression of a fluorescent reporter protein (Ub V76 GFP) destabilised both at the mRNA and protein level 19 . These cells constitutively express the artificial tetracycline-responsive transcriptional activator tTA.…”

“…We used a destabilised mRNA, in addition to a destabilised protein, to maximally speed up system dynamics, as these are dictated by both mRNA and protein degradation rates. 19 Indeed, in the absence of the mRNA degradation tag, the dynamics of the system are dominated by the Ub V76 GFP mRNA, whose half-life is about 130 min 19 , even though the Ub V76 GFP protein half-life is just 20 min 19 . On the contrary, when the 3'UTR degradation tag was added, the mRNA half-life became 18 min 19 , thus considerably speeding up the system.…”

“…19 Indeed, in the absence of the mRNA degradation tag, the dynamics of the system are dominated by the Ub V76 GFP mRNA, whose half-life is about 130 min 19 , even though the Ub V76 GFP protein half-life is just 20 min 19 . On the contrary, when the 3'UTR degradation tag was added, the mRNA half-life became 18 min 19 , thus considerably speeding up the system. In our previous proof-of-principle study 15 , we used a slower degrading protein with an half life of 2 hours (d2YFP) with no mRNA destabilisation.…”

Synthetic tissue engineering: Programming multicellular self-organization by designing customized cell-cell communication

“…This artificial genetic oscillator shows that we can design and build functional biological systems, but the circuit behavior was not perfect with increasing amplitude and inconsistent periods. Then synthetic biologists have been using the genetic oscillator as a model of synthetic biological system and engineering genetic circuits to improve the robustness and tunability of the oscillation [ 4 – 9 ]. The engineering-based synthetic approach provides a powerful way to explore possible molecular networks by rewiring new signaling pathways and changing parameters such as protein expression level and strength of molecular linkages, leading to understanding basic biological logic of how molecular networks are organized with current regulations to output cellular functions [ 10 ].…”