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.

中文翻译：

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植物增强剂在其功能元件之间表现出协同和加性相互作用

增强子是顺式调控元件，可响应众多发育和环境线索而塑造基因表达。在动物中，已经提出了几种模型来解释增强子如何整合多个转录因子的活性。然而，目前尚不清楚植物增强子如何整合转录因子活性。在这里，我们使用 Plant STARR-seq 来表征三种光响应植物增强子——AB80、Cab-1 和 rbcS-E9——源自与光合作用相关的基因。饱和诱变揭示了突变，其中许多突变聚集在较短的区域中，在光照、黑暗或两种条件下都强烈降低增强子活性。当在光下测试时，这些突变敏感区域不能自行发挥作用；相反，要充分开展活动，就需要与其他此类区域进行合作互动。相邻突变敏感区的突变之间发生上位相互作用，合成增强子中突变敏感区的间距和顺序影响增强子活性。相反，当在黑暗中进行测试时，突变敏感区域在赋予增强子活性方面独立且相加地发挥作用。总而言之，这项工作表明植物增强剂显示出其功能元素之间的合作和加性相互作用的证据。这些知识可以用来设计强效的、针对特定条件的合成增强剂。