title: 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 compared to the non-autoregulated 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.
authors.affiliation: Telethon Institute of Genetics and Medicine, Naples, Italy.
authors.forename: Velia
authors.initials: V
authors.lastname: Siciliano
authors.forename: Filippo
authors.initials: F
authors.lastname: Menolascina
authors.forename: Lucia
authors.initials: L
authors.lastname: Marucci
authors.forename: Chiara
authors.initials: C
authors.lastname: Fracassi
authors.forename: Immacolata
authors.initials: I
authors.lastname: Garzilli
authors.forename: Maria Nicoletta
authors.initials: MN
authors.lastname: Moretti
authors.forename: Diego
authors.initials: D
authors.lastname: di Bernardo
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D025461:Feedback, Physiological / Q000502:physiology*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006706:Homeostasis; D006801:Humans; D008957:Models, Genetic; D017711:Nonlinear Dynamics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D058615:Synthetic Biology; D049490:Systems Biology; D014158:Transcription, Genetic
chemical_list: D004247:DNA; D004318:Doxycycline