THE CELLS ARE ALL-RIGHT: Regulation of theLeftygenes by separate enhancers in mouse embryonic stem cells

Abstract

ABSTRACTEnhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding theLefty1andLefty2paralogs in mouse embryonic stem cells. TheLeftygenes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between theLeftygenes, we reveal a distance-dependent dosage effect of theLefty2enhancer onLefty1expression. These findings indicate a potential role for both discrete CTCF bound regions of the genome and chromatin distance in insulating gene expression domains in theLeftylocus in the absence of architectural insulation.