Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.

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 loo...

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Autores principales:	Velia Siciliano, Filippo Menolascina, Lucia Marucci, Chiara Fracassi, Immacolata Garzilli, Maria Nicoletta Moretti, Diego di Bernardo
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/c58c4d0a15284675958c260275386728
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spelling	oai:doaj.org-article:c58c4d0a15284675958c2602753867282021-11-18T05:50:26ZConstruction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.1553-734X1553-735810.1371/journal.pcbi.1002074https://doaj.org/article/c58c4d0a15284675958c2602753867282011-06-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21765813/pdf/?tool=EBIhttps://doaj.org/toc/1553-734Xhttps://doaj.org/toc/1553-7358Understanding 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.Velia SicilianoFilippo MenolascinaLucia MarucciChiara FracassiImmacolata GarzilliMaria Nicoletta MorettiDiego di BernardoPublic Library of Science (PLoS)articleBiology (General)QH301-705.5ENPLoS Computational Biology, Vol 7, Iss 6, p e1002074 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biology (General) QH301-705.5
spellingShingle	Biology (General) QH301-705.5 Velia Siciliano Filippo Menolascina Lucia Marucci Chiara Fracassi Immacolata Garzilli Maria Nicoletta Moretti Diego di Bernardo Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.
description	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.
format	article
author	Velia Siciliano Filippo Menolascina Lucia Marucci Chiara Fracassi Immacolata Garzilli Maria Nicoletta Moretti Diego di Bernardo
author_facet	Velia Siciliano Filippo Menolascina Lucia Marucci Chiara Fracassi Immacolata Garzilli Maria Nicoletta Moretti Diego di Bernardo
author_sort	Velia Siciliano
title	Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.
title_short	Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.
title_full	Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.
title_fullStr	Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.
title_full_unstemmed	Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.
title_sort	construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/c58c4d0a15284675958c260275386728
work_keys_str_mv	AT veliasiciliano constructionandmodellingofaninduciblepositivefeedbackloopstablyintegratedinamammaliancellline AT filippomenolascina constructionandmodellingofaninduciblepositivefeedbackloopstablyintegratedinamammaliancellline AT luciamarucci constructionandmodellingofaninduciblepositivefeedbackloopstablyintegratedinamammaliancellline AT chiarafracassi constructionandmodellingofaninduciblepositivefeedbackloopstablyintegratedinamammaliancellline AT immacolatagarzilli constructionandmodellingofaninduciblepositivefeedbackloopstablyintegratedinamammaliancellline AT marianicolettamoretti constructionandmodellingofaninduciblepositivefeedbackloopstablyintegratedinamammaliancellline AT diegodibernardo constructionandmodellingofaninduciblepositivefeedbackloopstablyintegratedinamammaliancellline
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Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.

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