Cellular heterogeneity in the developing forebrain masks transcriptional outcomes and principles ofEvf2enhancer lncRNA-Dlx5/6UCE-gene guidance

Abstract

AbstractDuring mouse embryonic brain development, theEvf2ultraconserved enhancer (UCE) lncRNA guides the Dlx5/6UCE to ∼129 sites across chr6. However, previous work identified only 4 transcriptionally regulated targets associated withEvf2-Dlx5/6UCE enhancer-gene guided sites (EGGs), raising questions about the significance of the majority ofEvf2-EGGs. Here, single cell transcriptomics (scRNAseq) shows thatEvf2-EGGs on chr6 coincide with subpopulation-specificEvf2transcriptional targets, revealing far greater alignment between EGGs and transcriptional targets than previously reported. Surprisingly, subpopulation-specificEvf2regulated gene networks in embryonic progenitors predict adult synaptic and seizure defects.Evf2regulation of EGGs to gene bodies (GB, Dlx5/6UCE locations within ±5kb of the target gene) divides chr6 into short-range (<10Mb distant), highly activated genes, and long/super-long-range (10-129Mb), moderately repressed genes. Clustering ofEvf2transcriptionally regulated chr6 targets in populations whereEvf2is first activated supports thatEvf2-EGG transcriptional effects can occur from EGG shifts as far as 3Mb, uncoupling enhancer shift distance and transcriptional direction from transcriptional outcome.Evf2RNA binding sites (RBSs) divide chr6 into 4 major regions, consistent with a role for RBS chromosomal spacing in long-and super-long-range EGG selectivity. Surprisingly, 95% of 147 RBSs genome-wide potentially form inter-chromosomal DNA loops.Evf2-regulated combinatorial recruitment ofEvf2-ribonucleoproteins at EGGs and RBSs, together with effects on homeodomain transcription factor DNA motif recognition, support a novel model of lncRNA directed multi-modal EGG selectivity during intra- and inter-chromosomal gene regulation.