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 α-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. 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.