Identification of plant enhancers using an in vitro transcription system
Final Report Abstract
Understanding plant gene regulation has been a priority for generations of plant scientists. However, due to its complex nature, the regulatory code governing plant gene expression has yet to be deciphered comprehensively. Self-transcribing active regulatory region sequencing (STARR-seq), a high-throughput assay for the genome-wide identification of functional enhancers, has led to large gains in our understanding of gene regulation in animals. However, in plants, the assay suffers from a low signal-to-noise ratio and is further hindered by the relatively low transformation efficiency of plant cells. To circumvent bottlenecks caused by the commonly low transformation efficiency of plant cells, I originally proposed to perform STARR-seq in a previously descried plant-based in vitro transcription system. However, my attempts to produce transcription-competent extracts from plant cells following published procedures failed. Instead, I focused on using Agrobacterium-mediated transient transformation of tobacco leaves and established a high-efficiency transformation method that can yield up to 25 million transformed cells in a single experiment. In parallel, I developed a version of STARR-seq optimized for use in plants. This Plant STARR- seq assay possesses a greatly improved signal-to-noise ratio and a large dynamic range. With Plant STARR-seq, condition-specific enhancer activity can be measured and enhancers can be characterized at single-nucleotide resolution. To demonstrate the high-throughput capabilities of Plant STARR-seq, I used the assay to measure the activity of over 75,000 core promoters in the genomes of Arabidopsis, maize and sorghum. This study revealed to what extent core promoter elements—like the TATA-box—as well as GC content and promoter-proximal transcription factor binding sites influence promoter strength. Based on these results, I was able to design strong, synthetic promoters comparable in activity to the viral 35S minimal promoter. Furthermore, I built computational models that accurately predict promoter strength and can be used to optimize core promoter activity through in silico evolution. The approach developed in this core promoter study can be followed to characterize additional cis-regulatory elements and to generate synthetic elements with desirable features. In the future, I will continue to study plant gene regulation using Plant STARR-seq and complementary approaches as an Emmy Noether group leader at the Heinrich Heine University of Düsseldorf.
Publications
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Identification of Plant Enhancers and Their Constituent Elements by STARR-seq in Tobacco Leaves. The Plant Cell, 32(7), 2120-2131.
Jores, Tobias; Tonnies, Jackson; Dorrity, Michael W.; Cuperus, Josh T.; Fields, Stanley & Queitsch, Christine
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Plant genome engineering from lab to field—a Keystone Symposia report. Annals of the New York Academy of Sciences, 1506(1), 35-54.
Cable, Jennifer; Ronald, Pamela C.; Voytas, Daniel; Zhang, Feng; Levy, Avraham A.; Takatsuka, Ayumu; Arimura, Shin‐ichi; Jacobsen, Steven E.; Toki, Seiichi; Toda, Erika; Gao, Caixia; Zhu, Jian‐Kang; Boch, Jens; Van Eck, Joyce; Mahfouz, Magdy; Andersson, Mariette; Fridman, Eyal; Weiss, Trevor; Wang, Kan ... & Bagchi, Rammyani
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Synthetic promoter designs enabled by a comprehensive analysis of plant core promoters. Nature Plants, 7(6), 842-855.
Jores, Tobias; Tonnies, Jackson; Wrightsman, Travis; Buckler, Edward S.; Cuperus, Josh T.; Fields, Stanley & Queitsch, Christine
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EUGENe: A Python toolkit for predictive analyses of regulatory sequences.
Klie, Adam; Stites, Hayden; Jores, Tobias; Solvason, Joe J.; Farley, Emma K. & Carter, Hannah
