The Evf2 Ultraconserved Enhancer lncRNA Functionally and Spatially Organizes Megabase Distant Genes in the Developing Forebrain

Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5′UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3′ end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3′-activator and 5′UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility. Cajigas et al. provide evidence for RNA-dependent mechanisms that regulate both gene selection and gene activity over unprecedented distances in the genome. These studies reveal that RNA-dependent precise spatial control of genes synergizes with a key DNA regulatory element, contributing to interneuron diversity during development and, ultimately, adult seizure susceptibility.