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A gene signal amplifier platform for monitoring the unfolded protein response

Abstract

Gene expression in mammalian cells results from coordinated protein-driven processes guided by diverse mechanisms of regulation, including protein–protein interactions, protein localization, DNA modifications and chromatin rearrangement. Regulation of gene expression is particularly important in stress-response pathways. To address the need to monitor chromosomal gene expression generating a readily detectable signal output that recapitulates gene expression dynamics, we developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal target gene. We generated a multiplex reporter system for monitoring markers of the unfolded protein response, a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum. By recapitulating the transcriptional and translational control mechanisms underlying the expression of a target gene with high sensitivity, this platform provides a technology for monitoring gene expression with superior sensitivity and dynamic resolution.

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Fig. 1: Monitoring UPR signaling pathway-specific target genes through chromosomal integration of a reporter gene.
Fig. 2: Design and implementation of the gene signal amplifier platform.
Fig. 3: Characterization of MCL/BIP-tTA cells.
Fig. 4: Characterization of the multiplex UPR reporter system.
Fig. 5: Development of a predictive model to adapt the gene signal amplifier for the detection of any cellular target.
Fig. 6: Translation of the gene signal amplifier to H4 neuroglioma cells.

Data availability

The authors declare that data supporting the finding of this study are available within the article and its Supplementary Information. Additional data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was funded by the National Science Foundation (grant no. MCB-1615562, grant no. CBET-1805317 and grant no. CBET-1930149) and the Welch Foundation (grant no. C-1824), and was conducted in part using resources of the Shared Equipment Authority at Rice University. This project was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine, with funding from the CPRIT Core Facility Support Award (grant no. CPRIT-RP180672), and by the NIH (grant no. P30 CA125123 and grant no. S10 RR024574).

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C.A.O. and L.S. conceived the projected. C.A.O., B.B. and S.D.P. performed the experiments and C.A.O. and B.B. analyzed the data. C.A.O. and A.L.Y. generated the mathematical model. C.A.O. and L.S. wrote the manuscript.

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Correspondence to Laura Segatori.

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Origel Marmolejo, C.A., Bachhav, B., Patibandla, S.D. et al. A gene signal amplifier platform for monitoring the unfolded protein response. Nat Chem Biol 16, 520–528 (2020). https://doi.org/10.1038/s41589-020-0497-x

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