title: Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes.
abstract: Transcriptional enhancers are critical for maintaining cell-type-specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Sakthi D
authors.initials: SD
authors.lastname: Moorthy
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Scott
authors.initials: S
authors.lastname: Davidson
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Virlana M
authors.initials: VM
authors.lastname: Shchuka
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Gurdeep
authors.initials: G
authors.lastname: Singh
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Nakisa
authors.initials: N
authors.lastname: Malek-Gilani
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Lida
authors.initials: L
authors.lastname: Langroudi
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Martchenko
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: So
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Neil N
authors.initials: NN
authors.lastname: Macpherson
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.|Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario M5S 3G5, Canada.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Mitchell
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism*; D014158:Transcription, Genetic