Fig. 3

Efflux of neRNAs alters higher-order chromatin topology. (A) Mean signal plots and heatmaps show density of mapped ATAC-seq reads 2 kb up- and downstream of ENSEMBL-annotated transcriptional start sites (TSS) in GRCm39. For every time point (n = 5 time points), data were partitioned into 4 clusters based on a k-means algorithm. Bottom micrographs show polyadenylated neRNAs identified using a FISH probe in control (T0) and differentiating NPCs in vitro (time points correspond to those of the ATAC-seq profile) (Scale bars: 6 μm). Right panels show global functional mapping of ATAC-seq profile (see methods) into compacted and de-compacted genomic loci in NPCs at t = 1 h after induction of differentiation. (B) Micrographs shows the accessible negatively supercoiled chromatin in differentiating NPCs identified by a psoralen probe (see methods). Scale bar top: 10 μm, bottom: 4 μm. Sc⁺: positively supercoiled, Sc⁻: negatively supercoiled. (C) FISH probe shows presence of polyadenylated neRNA in cultured nuclei (t = 20 min) isolated from NPCs (Scale bar: 8 μm). Electron microscopy photomicrographs demonstrate condensation of chromatin after RNAse-A-assisted degradation of neRNAs. Scale bars: 1 μm. (D) Application of Psoralen/UVA (Pso⁺) to stabilise higher order chromatin topology prior to induction of neuronal differentiation. Expression of pro-neural transcription factors in Pso⁺ NPCs 24 h after induction of neuronal differentiation is significantly higher than control NPCs (left graph) and almost equivalent to the level that is achieved in control differentiating NPCs after 7 days (right graph). * two-tailed p-value < 0.0001, n = 3 biological replicates