miRNAs are small non-coding RNAs able to modulate target-gene expression. It has been postulated that miRNAs confer robustness to biological processes, but a clear experimental evidence is still missing. Using a synthetic biology approach, we demonstrate that microRNAs provide phenotypic robustness to transcriptional regulatory networks by buffering fluctuations in protein levels. Here we construct a network motif in mammalian cells exhibiting a “toggle - switch” phenotype in which two alternative protein expression levels define its ON and OFF states. The motif consists of an inducible transcription factor that self-regulates its own transcription and that of a miRNA against the transcription factor itself. We confirm, using mathematical modeling and experimental approaches, that the microRNA confers robustness to the toggle-switch by enabling the cell to maintain and transmit its state. When absent, a dramatic increase in protein noise level occurs, causing the cell to randomly switch between the two states.

Highlights d Islets contain a minority of ''extreme'' beta cells with elevated insulin mRNA levels d Elevated proinsulin yet lower insulin protein suggests these are basal secretors d Extreme cells exhibit discordant apical-basal mRNA and protein localization d The proportion of extreme cells increases in insulin-resistant animals

Understanding the relationship between topology and dynamics of transcriptional regulatory networks in mammalian cells is essential to elucidate the biology of complex regulatory and signaling pathways. Here, we characterised, via a synthetic biology approach, a transcriptional positive feedback loop (PFL) by generating a clonal population of mammalian cells (CHO) carrying a stable integration of the construct. The PFL network consists of the Tetracycline-controlled transactivator (tTA), whose expression is regulated by a tTA responsive promoter (CMV-TET), thus giving rise to a positive feedback. The same CMV-TET promoter drives also the expression of a destabilised yellow fluorescent protein (d2EYFP), thus the dynamic behaviour can be followed by time-lapse microscopy. The PFL network was compared to an engineered version of the network lacking the positive feedback loop (NOPFL), by expressing the tTA mRNA from a constitutive promoter. Doxycycline was used to repress tTA activation (switch off), and the resulting changes in fluorescence intensity for both the PFL and NOPFL networks were followed for up to 43 h. We observed a striking difference in the dynamics of the PFL and NOPFL networks. Using non-linear dynamical models, able to recapitulate experimental observations, we demonstrated a link between network topology and network dynamics. Namely, transcriptional positive autoregulation can significantly slow down the “switch off” times, as comparared to the nonautoregulatated system. Doxycycline concentration can modulate the response times of the PFL, whereas the NOPFL always switches off with the same dynamics. Moreover, the PFL can exhibit bistability for a range of Doxycycline concentrations. Since the PFL motif is often found in naturally occurring transcriptional and signaling pathways, we believe our work can be instrumental to characterise their behaviour.

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.

Mammalian glucose homeostasis is controlled by the antagonistic hormones insulin and glucagon, secreted by pancreatic beta and alpha cells respectively. These two cell types are adjacently located in the islets of Langerhans and affect each others’ secretions in a paradoxical manner: while insulin inhibits glucagon secretion from alpha cells, glucagon seems to stimulate insulin secretion from beta cells. Here we ask what are the design principles of this negative feedback loop. We systematically simulate the dynamics of all possible islet inter-cellular connectivity patterns and analyze different performance criteria. We find that the observed circuit dampens overshoots of blood glucose levels after reversion of glucose drops. This feature is related to the temporal delay in the rise of insulin concentrations in peripheral tissues, compared to the immediate hormone action on the liver. In addition, we find that the circuit facilitates coordinate secretion of both hormones in response to protein meals. Our study highlights the advantages of a paradoxical paracrine feedback loop in maintaining metabolic homeostasis.