Engineering and Rewiring of a Calcium-Dependent Signaling Pathway.

Maja Meško,Roman Jerala,Tina Lebar,Mojca Benčina,Petra Dekleva

doi:10.1021/acssynbio.0c00133

Abstract

An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes. Calcium-dependent regulation is an attractive approach for achieving such control, as diverse stimuli induce calcium influx by activating membrane channel receptors. Most calcium-dependent gene circuits use the endogenous nuclear factor of activated T-cells (NFAT) signaling pathway. Here, we employed engineered NFAT transcription factors to induce the potent and robust activation of exogenous gene expression in HEK293T cells. Furthermore, we designed a calcium-dependent transcription factor that does not interfere with NFAT-regulated promoters and potently activates transcription in several mammalian cell types. Additionally, we demonstrate that coupling the circuit to a calcium-selective ion channel resulted in capsaicin- and temperature-controlled gene expression. This engineered calcium-dependent signaling pathway enables tightly controlled regulation of gene expression through different stimuli in mammalian cells and is versatile, adaptable, and useful for a wide range of therapeutic and diagnostic applications.

Highlights

An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes
Synthetic biology has advanced the design of gene circuits[1] that are responsive to various external signals, such as small molecules, light, radiowaves, and temperature.[2−5] Engineered transcription factors, based on designed DNA-binding domains such as transcription activator-like effectors (TALEs) or clustered regularly interspaced short palindromic repeats (CRISPR), can target an almost unlimited number of DNA targets, making them a powerful tool for the regulation of virtually any selected gene.[6−12] When coupled with inducible systems, designable transcription factors enable the external control of gene expression.[13−15]
The ionophore-mediated transcriptional activation was reduced to background levels by the calcineurin inhibitor cyclosporine A (CsA), confirming that the modest response did occur through the canonical calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway (Figure 1C)

Summary

Introduction

An important feature of synthetic biological circuits is their response to physicochemical signals, which enables the external control of cellular processes. Calcium-dependent transcription factors are attractive tools for synthetic biology applications, as many different physical and chemical stimuli can induce the cellular uptake of calcium ions by activating a variety of membrane receptors, such as Gprotein coupled receptors and calcium-selective ion channels.[16] Most calcium-dependent synthetic gene circuits engineered to date in eukaryotic cells harness the nuclear factor of activated T-cells (NFAT) signaling pathway.[3,4,17,18] NFAT is a central transcription factor in mammalian cells that is regulated by calcium influx via activation of the calcineurin phosphatase, which in turn dephosphorylates NFAT This modification results in the translocation of NFAT from the cytosol to the nucleus, where it regulates gene expression.[19,20] An optogenetic approach has been reported that exploited calcium signaling for the activation of NFAT-regulated transgenes and endogenous genes in mammalian cells,[21] while other successful attempts at external NFAT regulation include the use of stimuli such as fatty acids,[22] radio-waves,[3] ultrasound,[23] and menthol.[4] NFAT-regulated exogenous gene expression has been demonstrated in vivo in hypercalcemic mice.[24]. The engineered NFAT-based transcription factors characterized here are suitable for a range of therapeutic and diagnostic applications, as the calcium-dependent circuit can be coupled to diverse calcium signal transducing receptors

Objectives

Methods

Results

Conclusion