Dynamically Reshaping Signaling Networks to Program Cell Fate via Genetic Controllers

Introduction Engineering of cell fate through synthetic gene circuits requires methods to precisely implement control around native decision-making pathways and offers the potential to direct developmental programs and redirect aberrantly activated cell processes. We set out to develop molecular network diverters, a class of genetic control systems, to activate or attenuate signaling through a mitogen-activated protein kinase (MAPK) pathway, the yeast mating pathway, to conditionally route cells to one of three distinct fates. The molecular network diverter (controller) interfaces with a native signaling pathway to conditionally route cells to one of three fates in response to distinct environmental signals. Signal A (left) and signal B (right) trigger the positive and negative elements of the diverter via their cognate switches to activate or inhibit, respectively, signaling through the yeast mating pathway. Methods We used a combination of genetic elements—including pathway regulators, RNA-based transducers, and constitutive and pathway-responsive promoters—to build modular network diverters. We measured the impact of these genetic control systems on pathway activity by monitoring fluorescence from a transcriptional pathway reporter. Cell fate determination was measured through halo assays, in which mating-associated cell cycle arrest above a certain concentration of pheromone from wild-type cells results in a “halo” or cleared region around a disk saturated in pheromone. A phenomenological model of our system was built to elucidate design principles for dual diverters that integrate opposing functions while supporting independent routing to alternative fates. Results We identified titratable positive (Ste4) and negative (Msg5) regulators of pathway activity that result in divergent cell fate decisions when controlled from network diverters. A positive diverter, controlling Ste4 through a feedback architecture, routed cells to the mating fate, characterized by pathway activation in the absence of pheromone. A negative diverter, controlling Msg5 through a nonfeedback architecture, routed cells to the nonmating fate, characterized by pathway inhibition in the presence of pheromone. When integrated into a dual-diverter architecture, the opposing functions of these positive and negative diverters resulted in antagonism, which prevented independent routing to the alternative fates. However, a modified architecture that incorporated both constitutive and feedback regulation over the pathway regulators enabled conditional routing of cells to one of three fates (wild type, mating, or nonmating) in response to specified environmental signals. Discussion Our work identified design principles for networks that induce differentiation of cells in response to environmental signals and that enhance the robust performance of integrated mutually antagonistic genetic programs. For example, integrated negative regulators can buffer a system against noise amplification mediated through positive-feedback loops by providing a resistance to amplification. Negative feedback can play an important role by reducing population heterogeneity and mediating robust, long-term cell fate decisions. The dual-diverter configuration enables routing to alternative fates and minimizes impact on the opposing diverter by integrating differential regulatory strategies on functionally redundant genes. Molecular network diverters provide a foundation for robustly programming spatial and temporal control over cell fate. Toward Synthetic Biology The detection of an appropriate point to intervene in a cellular pathway and minimize off-target effects on other cellular processes present problems for the design of circuits that control cellular signaling pathways and thus direct cell function. Galloway et al. (p. 1358, published online 15 August; see the Perspective by Sarkar) report progress on these challenges in the yeast Saccharomyces cerevisiae. A molecular control system was developed to direct the yeast cells to one of three cell fates. To avoid disruption of other cellular controls, exogenous ribozyme-based controllers that interfaced with the endogenous control circuits were used, which avoided genetic alteration to the cells. After enhancing the control circuits with feedback loops to make their behavior more reliable, the circuits were used to modulate the abundance of particular components that acted as critical regulators of yeast cell-fate decisions. This allowed direction of cell fate in response to a chosen chemical stimulus. These strategies may be adaptable to allow similar direction of the physiological state of mammalian cells, for example, to allow therapeutic applications of synthetic biology. A synthetic control module inserted into yeast cells allows control of cell fate in response to an environmental signal. [Also see Perspective by Sarkar] Engineering of cell fate through synthetic gene circuits requires methods to precisely implement control around native decision-making pathways and offers the potential to direct cell processes. We demonstrate a class of genetic control systems, molecular network diverters, that interface with a native signaling pathway to route cells to divergent fates in response to environmental signals without modification of native genetic material. A method for identifying control points within natural networks is described that enables the construction of synthetic control systems that activate or attenuate native pathways to direct cell fate. We integrate opposing genetic programs by developing network architectures for reduced antagonism and demonstrate rational tuning of performance. Extension of these control strategies to mammalian systems should facilitate the engineering of complex cellular signaling systems.