ENGINEERING COMBINATORIAL AND DYNAMIC DECODERS USING SYNTHETIC IMMEDIATE-EARLY GENES

Abstract

Mammalian cells use signaling pathways to process information about the outside world; this pathway activity must be decoded into downstream gene expression. Some hallmarks of this decoding process are dynamic control, where only certain time-varying signals trigger a response, and combinatorial control, where two or more signals are interpreted together through a logic gate. Although there have been many studies of signaling pathways and many target genes have been identified, it is not clear how decoding of specific dynamic and combinatorial signals is accomplished. A common design principle of downstream gene decoding is regulation at multiple steps throughout the central dogma. In addition to transcriptional activation, signaling pathway outputs can modulate mRNA stability, protein translation, or protein stability. However, the signal-processing properties of this multi-step regulation are still poorly understood. Here, we have developed a flexible platform to produce synthetic immediate-early genes (synIEGs) that contain user-defined transcriptional and post-transcriptional regulation, as well as live-cell biosensors throughout the central dogma. We find that the genome is largely uniform in accepting synIEGs in terms of transcriptional potential; randomly integrated synIEGs all start transcription with similar dynamics to one another as well as endogenous IEGs. We demonstrate the utility of this approach by engineering synIEGs with both dynamic and combinatorial control in mammalian cells. Finally, we construct a novel AND gate that triggers apoptosis in response to the combined delivery of growth factor and DNA damage stimuli. Our synIEGs demonstrate that regulation at multiple nodes through the central dogma, along with the modular plasticity of mRNA/protein domains, can be used to decode both dynamic and combinatorial information.