A Platform Technology for Monitoring the Unfolded Protein Response.

Abstract

The unfolded protein response (UPR) is a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER) and is linked to the development of a range of diseases, including Alzheimer's disease, diabetes, and several types of cancer. UPR induction is typically monitored by measuring the expression level of UPR marker genes. Most tools for quantifying gene expression, including DNA microarrays and quantitative PCR with reverse transcription (RT-PCR), produce snapshots of the cell transcriptome, but are not ideal for measurements requiring temporal resolution of gene expression dynamics. Reporter assays for indirect detection of the UPR typically rely on extrachromosomal expression of reporters under the control of minimal or synthetic regulatory sequences that do not recapitulate the native chromosomal context of the UPR target genes. To address the need for tools to monitor chromosomal gene expression that recapitulate gene expression dynamics from the native chromosomal context and generate a readily detectable signal output, we developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal gene marker of the UPR. The platform is based on a genetic circuit that amplifies the output signal with high sensitivity and dynamic resolution and is implemented through chromosomal integration of the gene encoding the main control element of the genetic circuit to link its expression to that of the target gene, thereby generating a platform that can be easily adapted to monitor any UPR target through integration of the main control element at the appropriate chromosomal locus. By recapitulating the transcriptional and translational control mechanisms underlying the expression of UPR targets with high sensitivity, this platform provides a novel technology for monitoring the UPR with superior sensitivity and dynamic resolution.