ABSTRACT: During mouse embryonic brain development, the Evf2 ultraconserved enhancer lncRNA (e-lncRNA) guides the Dlx5/6UCE enhancer to ~129 sites across chr6. However, previous work identified only 4 transcriptionally regulated targets associated with Evf2 enhancer-gene guided sites (EGGs), raising questions about the significance of the majority of Evf2-EGGs. Here, single cell transcriptomics shows that Evf2-regulated EGGs on chr6 coincide with subpopulation-specific Evf2 transcriptional targets, revealing far greater alignment between EGGs and transcriptional effects than previously reported. Evf2 regulation of EGGs to gene bodies (GB ±5kb) divides chr6 into short-range (<10Mb), highly activated and long/super long-range (10-129Mb), moderately repressed genes. However, chr6 clustering of Evf2 transcriptionally regulated targets in populations where Evf2 is first activated, supports that transcriptional outcomes of Evf2-EGGs are not limited to gene body guidance, but may result from EGGs as far as 3Mb, uncoupling shift distance from transcriptional effects. Analysis of directly bound Evf2 RNA binding sites on chr6 (6 RBSs), divides chr6 into 4 regions, consistent with a role for RBS spacing in Evf2-EGG mediated, chromosome-spanning transcriptional regulation. In addition to chr6 Evf2 RBSs, 141 genome-wide RBSs are identified, 69 exactly aligning (>25bp) with at least one RBS, In addition to chr6 Evf2 RBSs, 141 genome-wide RBSs are identified, 69 exactly aligning (>25bp) with at least one RBS. RBS DNA identities are associated with transcription factor enrichment rather than DNA looping, as shown by HiCseq (chromosome capture of dual crosslinked) of E13.5GEs. While 80/147 RBSs are involved in inter-or intra-chr interactions, RBS chromosomal domains are differentially enriched for intra- and inter-chromosomal interactions, and domain specific Evf2-regulated RNP binding. Together with heterogeneous Evf2 regulation of protein pool/condensates (PPCs) of diverse Evf2 ribonucleoproteins (RNPs) (Smarcc2, an RNA binding chromatin remodeler, and Nono, an RNA paraspeckle organizer, Sox2 (a homeodomain transcription factor), RNP recruitment at RBSs and transcriptional targets, we propose a model whereby direct e-lncRNA binding, combinatorial Evf2-RNP recruitment, and homeodomain transcription factor DNA motif recognition/binding/activity, enables multi-modal EGG selectivity and transcriptional outcomes.
