Elucidating Mechanisms and Design Principles for Chemically Inducible Expression Modulation - PROJECT SUMMARY Molecular tools that can precisely modulate gene expression provide unique opportunities to both study the relationships between genotype and phenotype, as well as correct the pathologies caused by dysregulated gene expression. However, restricting the activity of these tools to the appropriate times or cell types remains a major hurdle to their effectiveness at interrogating biology and their safety in therapeutic settings. Our research program’s main goal is to elucidate the design principles for synthetic signaling systems that enable precision control of gene expression and, in doing so, to create fundamental insights into the mechanistic basis of natural signal transduction. Our work spans 3 methods of modulating expression and in each case seeks to overcome a major engineering challenge by generating novel fundamental insights into the transduction mechanism through a combination of high-throughput screening and machine learning. The first are Cas9-based synthetic transcription factors, which enable targeted changes to the transcription of a gene. We plan to restrict their activity via fusion to nuclear receptors that will make their nuclear localization, and hence regulation, conditional on a chemical inducer. We aim to elucidate how the structure of nuclear receptors encodes their nuclear trafficking kinetics and dynamic range, and then use these insights to design controls systems that can rapidly implement strong regulation in response to a non-toxic chemical cue. The second are ribozyme-based tools that regulate expression at the RNA level through splicing or trans- cleavage. We plan to make their activity contingent on the presence of either native mRNAs, through template dependent splicing, or chemicals, using aptazymes. We aim to understand how changes to the sequence, and resulting structure, of these RNA devices alter their capacity to transduce their triggers into catalysis. The resulting insights will be used to identify a combination of mutations that can overcome the low catalytic efficiencies often associated with these tools. The third are chemicals that activate human G-protein coupled receptors (GPCRs) to modulate expression of the genes they regulate. We plan to identify plant metabolites that act as selective agonists by developing a high-throughput screen that enables massively parallel characterization of GPCR-ligand interactions. We aim to elucidate the design principles for functional expression of human GPCRs in yeast and use the resulting biosensors to reveal the ligand features necessary for selective activation of GPCRs. Overall, this research will create novel tools to enable precision control of gene expression and generate fundamental insights into how molecular architecture, structure, and ligand specificity impact signal transduction. Thus, it both aligns with the NIGMS mission and fills the need for “transcriptional control tools” recently identified in the NIGMS NOSI: Synthetic Biology for Biomedical Applications (NOT-EB-23-002).
