Super-enhancers include classical enhancers and facilitators to fully activate gene expression.

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact 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 target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains 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 upregulate 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 multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes. [Display omitted] • Large synthetic alleles allow de novo assembly of multipartite enhancers • Mouse α-globin super-enhancer contains classical enhancers and facilitators • Facilitators have no inherent enhancer activity but potentiate classical enhancers • Newly identified facilitators act in a position-dependent manner De novo construction of a multipartite enhancer unveils a new class of regulatory element referred to as "facilitators." Facilitators lack intrinsic enhancer activity but enhance the activity of classical enhancers in a position-dependent manner. [ABSTRACT FROM AUTHOR]