Development of a gene signal amplifier platform for monitoring the Unfolded Protein Response
Origel Marmolejo, Carlos Alberto
Doctor of Philosophy
Sophisticated mechanisms controlling regulation of gene expression in mammalian cells mediate the translation of instructions stored in the genome into molecular components that define cellular physiology. Gene expression results from coordinated protein-driven processes guided by regulatory mechanisms that rely on protein-protein interactions, protein localization, and chromatin rearrangement. Characterizing gene expression profiles is critical to understand stress-response signaling pathways that rely on regulation of gene expression to integrate information about the nature, duration, and intensity of stress stimuli. Current technologies to monitor gene expression are mainly based on transcriptomic analyses, such as DNA microarrays, RNA-seq, and quantitative RT-PCR, which do not provide temporal resolution of gene expression dynamics. Reporter gene assays based on minimal or synthetic regulatory sequences enable facile detection of gene expression, but often fail to recapitulate the innate chromosomal regulatory mechanisms. To address the need to monitor gene expression from the native chromosomal context and thus encompassing the complexity of mammalian regulatory systems while generating a readily detectable signal output that recapitulates gene expression dynamics, I developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a target gene. Specifically, I generated a HEK293 master cell line containing a genetic circuit for signal amplification and developed derivatives cell lines engineered to link the expression of chromosomal genes to the genetic circuit for signal amplification using CRISPR-Cas9 technology. Iterations of mathematical modeling and experimental tests led to the development of a cell-based reporter platform that can detect changes in gene expression with high sensitivity, dynamic range, and dynamic resolution. To validate this reporter platform, I generated a multiplex reporter system for monitoring markers of the unfolded protein response (UPR), a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum. I implemented the gene reporter platform to profile the temporal pattern of regulation of genes markers of different branches of the UPR which determines the outcome of UPR activation, namely stress attenuation or apoptosis. By recapitulating the transcriptional and translational control mechanisms underlying the expression of a target gene, this platform provides a novel technology for monitoring gene expression with high sensitivity and dynamic resolution.
Synthetic biology; Unfolded Protein Response; Genetic circuit