Calcium signaling represents an attractive regulatory mechanism for use in engineered biological circuits, as the influx of calcium ions into cells can be triggered by a plethora of chemical and physical signals. Engineered calcium-dependent transcription factors, presented in this work, are based on the family of transcription factors NFAT. Their activity is regulated by the calcium/calmodulin-dependent phosphatase calcineurin which controls the translocation of NFAT proteins from the cytoplasm to the nucleus. Since tight regulation of each component of a synthetic cellular circuit is crucial for robust functioning of such systems, we prepared engineered NFAT variants with modified and controlled subcellular localization properties. This enabled potent NFAT activity while maintaining robust activation control. Undesired NFAT activity in the absence of stimulation was shown to be reduced both by insertion of additional nuclear export signal (NES) or via fusion of the synthetic membrane anchor peptide KRɸ to the transcription factor.
Most synthetic calcium-dependent gene circuits use the endogenous NFAT signaling pathway for signal transduction. For incorporation of NFAT signaling into a controllable synthetic genetic circuit, it is crucial for the system to function independently of endogenous cell processes. For this reason, we prepared artificial transcription factors in which the native human or murine NFAT DNA-binding domain was replaced with a designed TALE DNA-binding domain, allowing targeting of specific DNA regulatory regions, and the VP16 or VP64 activation domain, allowing transcription upregulation of downstream genes. Replacement of the DNA-binding domain allows targeting of selected target genes while at the same time eliminates off-target activation of NFAT promoter-driven gene expression. This replacement ensured efficient calcium-dependent activation of target transgene expression in several mammalian cell lines.
The possibility of external control of cell-signaling pathways through various physicochemical signals is an important feature of synthetic biological circuits, as it allows non-invasive control of cellular processes. Ultrasound represents a signal which can be highly spatio-temporally controlled and has been previously shown to stimulate mechanosensitive ion channels. In the present work, we developed a method for stimulation of mammalian cells with ultrasound and showed that ultrasonic waves can trigger the activation of engineered Ca2+-dependent transcription factors in cell lines as well as in experimental animals. The developed method of ultrasound stimulation represents an innovative approach for control of engineered biologic circuits due to its non-invasive properties and the ability to penetrate deep into tissues. In vivo applicability of the designed synthetic system underlines important potential use in therapeutic applications.
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