Plant enhancers exhibit both cooperative and additive interactions among their functional elements

Abstract

Enhancers are cis-regulatory elements that shape gene expression in response to numerous developmental and environmental cues. In animals, several models have been proposed to explain how enhancers integrate the activity of multiple transcription factors. However, it remains largely unclear how plant enhancers integrate transcription factor activity. Here, we use Plant STARR-seq to characterize three light-responsive plant enhancers—AB80, Cab-1, and rbcS-E9—derived from genes associated with photosynthesis. Saturation mutagenesis revealed mutations, many of which clustered in short regions, that strongly reduced enhancer activity in the light, in the dark or in both conditions. When tested in the light, these mutation-sensitive regions did not function on their own; rather, cooperative interactions with other such regions were required for full activity. Epistatic interactions occurred between mutations in adjacent mutation-sensitive regions, and the spacing and order of mutation-sensitive regions in synthetic enhancers affected enhancer activity. In contrast, when tested in the dark, mutation-sensitive regions acted independently and additively in conferring enhancer activity. Taken together, this work demonstrates that plant enhancers show evidence for both cooperative and additive interactions among their functional elements. This knowledge can be harnessed to design strong, condition-specific synthetic enhancers.

Dissecting and redesigning enhancers of photosynthesis genes

“…With notable advances in high-throughput sequencing technologies, this presents an exciting opportunity to delve into the study of cis-elements in plants. In this issue, Tobias Jores, Jackson Tonnies, and colleagues ( Jores et al 2024 ) have provided an in-depth understanding of how 3 light-responsive enhancers function, both cooperatively and additively.…”

“…They demonstrated that these 3 enhancers exhibit light-responsive activity. Here, the same group of researchers, Tobias Jores, Jackson Tonnies, and colleagues ( Jores et al 2024 ) , aimed to interrogate these plant enhancers at nucleotide resolution. They divided each enhancer sequence into 5′ and 3′ fragments ( Fig.…”

“…Mutation-sensitive regions in the 3′ enhancer segments are indicated by shaded rectangles labeled a–e. Reprinted from Jores et al (2024) , Figures 1A and 3A.…”