Super-enhancers require enhancers and facilitators to fully activate gene expression [ATAC-seq]

Super-enhancers are compound regulatory elements which control expression of key cell-identity genes. They recruit high levels of tissue-specific transcription factors, co-activators such as the mediator complex, and they contact their target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating expression of their cognate genes. Here, through comprehensively rebuilding the endogenous a-globin super-enhancer, we show that super-enhancers comprise bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully up-regulate their target genes. Without facilitators, classical enhancers exhibit reduced mediator recruitment, enhancer RNA transcription and enhancer-promoter interactions. Facilitators are interchangeable, but display functional hierarchy based on their position within a super-enhancer. Facilitators thus play an important role in potentiating super-enhancer activity and ensuring robust activation of target genes. Overall design: Characterisation of a mouse model lacking four out of the five alpha-globin super-enhancer constituents demonstrates that super-enhancer constituents rely on each other to achieve maximal target gene activation. Furthermore, the strongest alpha-globin super-enhancer constituent requires the other four constituents (or a subset thereof) for maximal enhancer-promoter interaction frequency, coactivator recruitment and eRNA expression. Rebuilding the alpha-globin super-enhancer further demonstrates the cooperation between its constituents. The super-enhancer is made up of two functionally distinct element types: active enhancers, and facilitators. Facilitators have little or no ability to activate gene expression directly, but they potentiate the activity of enhancers within the same cluster.

超级增强子（super-enhancer）是一类复合调控元件，负责调控关键细胞身份基因的表达。其可招募大量组织特异性转录因子及中介体复合物（mediator complex）等辅激活因子，并以高频率与靶基因启动子发生相互作用。多数超级增强子包含多个组成型调控元件，但目前尚不清楚这些元件在激活其对应靶基因表达时是否具有独特功能。本研究通过对内源性α-珠蛋白超级增强子进行系统性重构，证实超级增强子由两类生物信息学特征相似但功能截然不同的元件组成：经典增强子与辅助元件（facilitator elements）。辅助元件本身不具备内在增强子活性，但在缺失辅助元件的情况下，经典增强子无法完全上调其靶基因的表达。当缺乏辅助元件时，经典增强子的中介体招募能力、增强子RNA（enhancer RNA，eRNA）转录水平及增强子-启动子相互作用频率均会下降。辅助元件具有可互换性，但会根据其在超级增强子中的位置呈现功能层级性。由此可见，辅助元件在增强超级增强子活性、保障靶基因高效激活方面发挥着关键作用。

整体实验设计：构建缺失α-珠蛋白超级增强子5个组成元件中4个的小鼠模型，实验结果表明，超级增强子的各组成元件彼此依赖，方可实现靶基因的最大程度激活。此外，功能最强的α-珠蛋白超级增强子组成元件需要其余4个组成元件（或其任意子集）的协同，才能达到最高的增强子-启动子相互作用频率、辅激活因子招募水平及增强子RNA表达量。对α-珠蛋白超级增强子的重构实验进一步验证了其各组成元件之间的协同效应。该超级增强子由两类功能截然不同的元件组成：经典增强子与辅助元件。辅助元件几乎无法直接激活基因表达，但可增强同一簇内增强子的活性。