Engineering dynamical control of cell fate switching using synthetic phospho-regulons

Significance Many long-term cellular decisions in development, synaptic plasticity, and immunity require cells to recognize input dynamics such as pulse duration or frequency. In dynamically controlled cells, incoming stimuli are often processed and filtered by a rapid-acting signaling layer, and then passed to a downstream slow-acting layer that locks in a longer-term cellular response. Directly testing how such dual-timescale networks control dynamical regulation has been challenging because most tools in synthetic biology allow rewiring of slow gene expression circuits, but not of rapid signaling circuits. In this work, we developed modular peptide tags for engineering synthetic phosphorylation circuits. We used these phospho-regulons to build synthetic dual-timescale networks in which the dynamic responsiveness of a cell fate decision can be selectively tuned. Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although long-term fate decisions are ultimately controlled by a slow-timescale switch (e.g., gene expression), input signals are first processed by a fast-timescale signaling layer, which is hypothesized to filter what dynamic information is efficiently relayed downstream. Directly testing the design principles of how dual-timescale circuits control dynamic sensing, however, has been challenging, because most synthetic biology methods have focused solely on rewiring transcriptional circuits, which operate at a single slow timescale. Here, we report the development of a modular approach for flexibly engineering phosphorylation circuits using designed phospho-regulon motifs. By then linking rapid phospho-feedback with slower downstream transcription-based bistable switches, we can construct synthetic dual-timescale circuits in yeast in which the triggering dynamics and the end-state properties of the ON state can be selectively tuned. These phospho-regulon tools thus open up the possibility to engineer cells with customized dynamical control.