Construction and Modelling of an Inducible Positive Feedback Loop Stably Integrated in a Mammalian Cell-Line

Abstract

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.

Automatic Control of Gene Expression in Mammalian Cells

“…The response of this monoclonal population to varying concentrations of Doxycycline (from 1 ng/ml to 1 µg/ml) has been previously characterised (8 ). However, in our preliminary experiments we observed an unexpected eect of Doxycycline, i.e.…”

Neurogenin3 Cooperates with Foxa2 to Autoactivate Its Own Expression

“…In addition, because autoactivation can release these factors from control by external stimuli, it may also ensure cell fate commitment (40). Another suggested function of positive autoregulatory loops is to raise the expression levels of cell fate-determining factors over a threshold necessary for initiation of their respective developmental programs (41).…”

Erratum: TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop

“…TFEB activity is regulated transcriptionally and post-transcriptionally by nutrients, and is required TFEB mRNA expression is induced by starvation by a post-transcriptional switch that controls TFEB nuclear translocation [29][30][31] , which allows TFEB to rapidly respond to nutrient availability, and a positive transcriptional autoregulatory component for a sustained response. Autoregulatory feedback circuits are used by eukaryotic cells to convert a graded input into a binary (ON/OFF) response in eukaryotic gene circuits [32][33][34] . Interestingly, the PGC1 gene, a direct target of TFEB, is also subject to an autoregulatory loop 35 .…”