Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters
Adam M. Zahm,William S. Owens,Samuel R. Himes,Kathleen E. Rondem,Braden S. Fallon,Joshua S. Bloom,Sriram Kosuri,Justin G. English +7 more
TL;DR: In this article , the authors introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length, which can be used in luciferase reporter assays, eliciting 50-100 fold dynamic ranges in response to stimuli.
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Abstract: Cellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery. This technology has found extensive use in biological research for applications including reporter gene assays, biomarker development, and programming synthetic circuits in living cells. However, a reliable and precise method for selecting minimally-sized synthetic promoters with desired background, amplitude, and stimulation response profiles has been elusive. In this study, we introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length. This comprehensive library allows for rapid identification of promoters with optimal transcriptional output parameters across multiple cell lines and stimuli. We showcase this library’s utility to identify promoters activated in unique cell types, and in response to metabolites, mitogens, cellular toxins, and agonism of both aminergic and non-aminergic GPCRs. We further show these promoters can be used in luciferase reporter assays, eliciting 50-100 fold dynamic ranges in response to stimuli. Our platform is effective, easily implemented, and provides a solution for selecting short-length promoters with precise performance for a multitude of applications.
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Citations
Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.
Jean-Benoît Lalanne,Samuel G. Regalado,Silvia Domcke,D. Calderon,Beth Martin,Xiaoyi Li,Tony Li,Chase C. Suiter,Choli Lee,Cole Trapnell,J. Shendure +10 more
TL;DR: Researchers develop a dual RNA cassette for multiplex single-cell reporter assays, enabling accurate and precise measurement of developmental cis-regulatory elements in multicellular systems, with applications in characterizing native and synthetic CREs at scale.
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TL;DR: The approach integrating structure and unbiased functional interrogation defines a new framework for understanding the rich complexity of GPCR signaling, which is uncovered by integrative cryo-EM and deep mutational scanning.
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Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes
Adelaide Tovar,Yasuhiro Kyono,Kirsten Nishino,Maya Bose,Arushi Varshney,Stephen C. J. Parker,Jacob O. Kitzman +6 more
TL;DR: This study suggests that cell-specific regulatory activity is partially influenced by enhancer-promoter compatibility and indicates that careful attention should be paid when designing MPRA libraries to capture context- specific regulatory processes at disease-associated genetic signals.
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