1. Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach
- Author
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Lorena Postiglione, Immacolata Garzilli, Marco Santorelli, Daniela Perna, Francesco Annunziata, Barbara Tumaini, Akihiro Isomura, Diego di Bernardo, Ryoichiro Kageyama, Santorelli, M., Perna, D., Isomura, A., Garzilli, I., Annunziata, F., Postiglione, L., Tumaini, B., Kageyama, R., and Di Bernardo, D.
- Subjects
0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,endocrine system ,Period (gene) ,Green Fluorescent Proteins ,Biomedical Engineering ,Endogeny ,CHO Cells ,Computational biology ,Biology ,Protein Engineering ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Mice ,03 medical and health sciences ,Synthetic biology ,Cricetulus ,0302 clinical medicine ,Bacterial Proteins ,Biological Clocks ,Genes, Reporter ,Negative feedback ,Animals ,HES1 ,Promoter Regions, Genetic ,Ultradian rhythm ,Feedback, Physiological ,Genetics ,Binding Sites ,Mechanism (biology) ,Effector ,General Medicine ,Models, Theoretical ,Introns ,Sp3 Transcription Factor ,030104 developmental biology ,Doxycycline ,embryonic structures ,Transcription Factor HES-1 ,Synthetic Biology ,Single-Cell Analysis ,Carrier Proteins ,030217 neurology & neurosurgery - Abstract
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.
- Published
- 2018
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