Detailed temporal dissection of an enhancer cluster reveals two distinct roles for individual elements

Abstract

14Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. Yet, 15how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the 16 functions and interdependencies of five enhancer elements that form a previously identified enhancer 17 cluster and activate the Fgf5 locus during exit from naïve murine pluripotency. Four elements are located 18 downstream of the Fgf5 gene and form a super-enhancer. Each of these elements contributes to Fgf5 19 induction at a distinct time point of differentiation. The fifth element is located in the first intron of the 20 Fgf5 gene and contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression 21 levels. This amplifier element strongly accumulates paused RNA Polymerase II but does not give rise to a 22 mature Fgf5 mRNA. By transplanting the amplifier to a different genomic position, we demonstrate that it 23 enriches for high levels of paused RNA Polymerase II autonomously. Based on our data, we propose a 24 model for a mechanism by which RNA Polymerase II accumulation at a novel type of enhancer element, 25 the amplifier, contributes to enhancer collaboration. 26

High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale

“…The finding that interacting with more than three enhancers provided little additional increase in expression may also provide insights into super-enhancers. Although it is clear at least some constituent parts of super-enhancers act in a simple additive manner 19 , this result suggests additivity is not the entire picture, and may be consistent with enhancers having distinct mechanistic roles or heirachy 58,59 . Therefore, high levels of transcription driven by multiple enhancers, and/or the effect of superenhancers could be as a result of combining multiple enhancer functions.…”

Parallel Characterization ofcis-Regulatory Elements for Multiple Genes Using CRISPRpath

“…Genomic studies of chromatin marks have revealed hundreds of thousands candidate CREs in the human genome but with very little quantitative information regarding how CREs contribute to gene regulation 35, 36 . Using CRISPRpath, we can systematically classify enhancers based on their effect sizes on transcription.…”

“…This observation can be explained by the fact that enhancer-like promoters will not only form chromatin loops with their distal target genes, but also with CREs for controlling the expression of their own genes. Genomic studies of chromatin marks have revealed hundreds of thousands candidate CREs in the human genome but with very little quantitative information regarding how CREs contribute to gene regulation (36,37). Using CRISPRpath, we can systematically classify enhancers based on their effect sizes on transcription.…”

Super-Enhancers and CTCF in Early Embryonic Cell Fate Decisions

“…SEs also exhibit relatively higher levels of H3K27Ac and are larger in size overall compared with enhancers ( Parker et al, 2013 ; Pulakanti et al, 2013 ). Several studies show that SE differ from classical enhancers due to their stronger ability to drive gene expression than classical enhancers ( Hnisz et al, 2015 ; Huang et al, 2016 ; Shin et al, 2016 ; Thomas et al, 2021 ). SEs are also highly cell-type specific and are often found near key lineage determining genes, implying they are critical to the establishment and/or maintenance of cell identity.…”