Fig. 4

Induction of R-loops drives transition of bistable Phase I transdifferentiating pericytes to monostable Phase II neural progenitors. a Quantification of γH2AX immunofluorescence signal in cultured pericytes after induction of transdifferentiation (y-axis: increased intensity (∆) of the fluorescent signal in transdifferentiating cells relative to control cells, *p < 0.01). Acridine orange staining shows depletion of RNA concurrent with induction of DNA damage at t = 15 min. b Gel shows DNA integrity of vegfa locus at various timepoints (T: 0–50 min) during transdifferentiation of pericytes, probed using four specific PCR primers (1–4: primers as per bottom schematic figure, see Additional file 1: Table S1). c Micrographs show immunohistochemical detection of RNA:DNA hybrids (R-loops), using S9.6 antibody, during transdifferentiation of pericytes. Scale bar 10 μm. d Transcriptional fingerprinting of RNASEH1, RNASEH2 subunits, and topoisomerase subunits in transdifferentiating pericytes (t = 0–50 min) [87]. e DRIP-seq profile of transdifferentiating pericytes shows chromosomal distribution of R-loops 10 min after induction of transdifferentiation. turquoise: R-loop⁺ loci shared by control cells in growth medium (GM) and transdifferentiating cells in neural induction medium (NI), purple: R-loop⁺ loci specific to transdifferentiating cells. Greyscale heatmap in the chromosome body shows γH2AX ChIP-seq profile of transdifferentiating cell. GO enrichment analysis (right network) revealed that the R-loop⁺ genes are predominantly involved in regulating smooth muscle vs. neuronal physiology. f The impact of R-loop formation on rewiring the existing functional network topology was predicted by analyzing the DRIP-seq profile of transdifferentiating pericytes. The bottom graph shows the number of R-loop⁺ genes in functional GO clusters (x-axis) plotted against the total number of network connections established by the R-loop⁺ genes in the cluster (y-axis). Signal transduction pathways accommodate the highest number of R-loop⁺ genes with extensive connectivity profile. Further analysis of the signalling pathways based on the same approach revealed that G-protein coupled receptor, Rho, and receptor tyrosine kinase signalling cascades are more likely to be disrupted by induction of R-loops during transdifferentiation of pericytes (top graph). g Gels demonstrate the impact of forced expression of N-cadherin, RNASEH1, and RNAHEH2 subunits on R-loop-mediated DNA cleavage that affects vegfa locus (b) upon induction of pericyte transdifferentiation (D: days post-induction). Note that amplified activity of RNaseH and N-cadherin prevents transdifferentiation-mediated cleavage of VEGF-1 and VEGFA-2. h Immunohistochemical profile of transdifferentiating pericytes (3 days post-induction) shows immature Nestin⁻/Dcx⁺ neurons and fewer β3-tubulin⁺ neurons (refer to Additional file 1: Fig. S4 for quantification). Forced expression of N-cadherin (ΔCDH2) and RNASEH2B (ΔRNASEH2B) blocks transdifferentiation evidenced by presence of Nestin⁺/Dcx⁻ population. Expression of ATOH1 and MASH1 upon overexpression of CDH2 and RNAHSEH subunits in pericytes cultured in growth medium (GM: grey) and after neural induction (t = 24 h) (Red: p < 0.01). Scale bar 40 μm