Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters

Authors: JORES, TOBIAS - University Of Washington; TONNIES, JACKSON - University Of Washington; WRIGHTSMAN, TRAVIS - Cornell University; Buckler, Edward - Ed; CUPERUS, JOSH - University Of Washington; FIELDS, STANLEY - University Of Washington; QUEITSCH, CHRISTINE - University Of Washington

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 1/8/2021
Publication Date: 1/8/2021
Citation: Jores, T., Tonnies, J., Wrightsman, T., Buckler IV, E.S., Cuperus, J.T., Fields, S., Queitsch, C. 2021. Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters. bioRxiv. https://doi.org/10.1101/2021.01.07.425784.
DOI: https://doi.org/10.1101/2021.01.07.425784

Interpretive Summary: We know that turning genes on or off or regulating their activity levels controls the vast majority of how a plant looks at the end of the growing season. We don’t yet know the effects of most of the DNA changes around these genes on their activity level. By changing the DNA around a single, easily-visible gene and observing how it modulates the activity, we can begin to filter out the changes that are most important. We created an assay to efficiently assess how the DNA around a given gene changes its activity in plants. We assayed tens of thousands of different sets of DNA changes around a single consistent gene to see how they affect its activity levels in a plant cell. We showed that there are some strong consistent changes or groups of changes that dramatically affect activities in all species tested and some that are more species-specific. We also created computational models that are able to accurately predict the effect on activity of any DNA change requested, even those not tested in our experiments. When we mutate the DNA into new sets of yet unobserved changes that will produce the greatest activity differences according to our models, the activity levels also change in actual plant cells, validating the utility of our models. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.

Technical Abstract: Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements in order to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements – notably the TATA-box – as well as promoter GC content and promoter-proximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detected species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.