ABSTRACT
How enhancers activate their distal target promoters remains incompletely understood. Here we dissect how CTCF-mediated loops facilitate and restrict such regulatory interactions. Using an allelic series of mouse mutants, we show that CTCF is neither required for the interaction of the Sox2 gene with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs, between Sox2 and its distal enhancers, generated boundaries with varying degrees of insulation that directly correlated with reduced transcriptional output. However, in both epiblast and neural tissues, enhancer contacts and transcriptional induction could not be fully abolished, and insertions failed to disrupt implantation and neurogenesis. In contrast, Sox2 expression was undetectable in the anterior foregut of mutants carrying the strongest boundaries, and these animals fully phenocopied loss of SOX2 in this tissue. We propose that enhancer clusters with a high density of regulatory activity can better overcome physical barriers to maintain faithful gene expression and phenotypic robustness.
Subject(s)
Chromatin , Enhancer Elements, Genetic , Mice , Animals , Enhancer Elements, Genetic/genetics , Promoter Regions, Genetic/genetics , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolismABSTRACT
Precisely controlled gene regulatory networks are required during embryonic development to give rise to various structures, including those of the cardiovascular system. Long non-coding RNA (lncRNA) loci are known to be important regulators of these genetic programs. We have identified a novel and essential lncRNA locus Handsdown (Hdn), active in early heart cells, and show by genetic inactivation that it is essential for murine development. Hdn displays haploinsufficiency for cardiac development as Hdn-heterozygous adult mice exhibit hyperplasia in the right ventricular wall. Transcriptional activity of the Hdn locus, independent of its RNA, suppresses its neighboring gene Hand2. We reveal a switch in a topologically associated domain in differentiation of the cardiac lineage, allowing the Hdn locus to directly interact with regulatory elements of the Hand2 locus.
