A tunable dual-input system for on-demand dynamic gene expression regulation

• Published in: Nature communications 2019-10, Vol.10 (1), p.4481-13, Article 4481
• Main Authors: Pedone, Elisa, Postiglione, Lorena, Aulicino, Francesco, Rocca, Dan L., Montes-Olivas, Sandra, Khazim, Mahmoud, di Bernardo, Diego, Pia Cosma, Maria, Marucci, Lucia
• Format: Article
• Language: English
• Subjects: 13; 13/1; 13/100; 13/109; 13/31; 13/62; 14/34; 14/35; 14/63; 38/90; 45/77; 49/22; 631/114/2397; 631/1647/277; 631/61/338/552; Animals; Anti-Infective Agents - pharmacology; Biological evolution; Control stability; Culture Media, Conditioned - pharmacology; Doxycycline - pharmacology; Dynamic stability; Embryo cells; Environmental effects; Feedback control; Flow Cytometry; Gene expression; Gene Expression Regulation - drug effects; Gene Expression Regulation - genetics; Gene regulation; HEK293 Cells; HeLa Cells; Humanities and Social Sciences; Humans; Level (quantity); Localization; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mammalian cells; Mice; Microscopy, Confocal; Mouse Embryonic Stem Cells - metabolism; multidisciplinary; Post-translation; Proteins; Red Fluorescent Protein; Science; Science (multidisciplinary); Signal transduction; Stem cell transplantation; Stem cells; Transcription; Trimethoprim - pharmacology
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-12329-9

Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells. Cellular systems have numerous mechanisms to control gene expression. Here the authors build a Tet-On system with conditional destablising elements to regulate gene expression and protein stability, allowing fine modulation of mESC signalling pathways.