Rapid Identification and Quantitative Assessment of Plant Enhancers in Full-Genome Libraries

Plants respond to environmental stimuli by tightly controlled changes in gene expression, requiring a dynamic regulatory network. Despite this need, a key mode of dynamic regulation, long-range activation or repression, appears to be absent from chromatin interaction datasets (3C, Hi-C) in Arabidopsis thaliana. The apparent lack of distal physical interactions among chromosomes suggests that Arabidopsis may not use typical enhancers, though they are frequent among other higher eukaryotes. We describe the use of an alternative method to identify and functionally characterize enhancers, STARR-seq. This method relies on simultaneously measuring the activity of a library of gene constructs, each containing a fragment of genomic DNA inserted into a transcribed sequence. If a fragment does not possess an enhancer element, the construct is expressed at low level due only to the minimal promoter driving transcription. If a fragment is able to confer distal activation, the gene is expressed at higher level and the functional enhancer sequence, codified in the transcript, increases in abundance. We show the efficacy of the plant-optimized method by testing a known enhancer in the experimental context, and demonstrate the scale to which the method can leverage next-generation sequencing to pinpoint functional enhancers in Arabidopsis and maize. We foresee this method being useful for addressing biological questions of gene regulation, as well as for application to emerging challenges in crop design.