Fig. 6: G4 structure stabilisation delays hESC differentiation.
From: G-quadruplex DNA structures in human stem cells and differentiation
a Schematic of G4-ligand treatment experiment: OCT4-EGFP expressing hESCs were differentiated into CNCCs using a 2-day pulse using GSK3β (CHIR99021) and ROCK inhibition (Y-27632)59. Samples were taken at the indicated times to determine the percentage of cells positive for the pluripotency marker OCT4 (by flow cytometry and immunofluorescence microscopy (IF)) or SOX10 (by IF; lineage marker for CNCC) or RNA-seq. Insert chemical structure of the G4-specific ligand PhenDC3. b–d Proportion of b OCT4 and d SOX10 positive cells determined in IF studies. N = 52 fields of view per sample. One biological replicate (see Source Data for detailed cell counts). *: p < 0.05, one-sided Pearson’s χ2 test for proportions (see Source Data for exact p-values and number of cells analysed). See Supplementary Fig. 17 for additional biological replicates. c Representative confocal IF images of differentiating OCT4-EGFP hESCs treated with either DMSO or PhenDC3. SOX10 = red, OCT4 = green and DAPI (nuclear stain) = blue. Scale bar = 100 μm. e Heatmap showing gene expression (Log10 median gene expression (TPM + 0.1)) of the indicated pluripotency and cranial neural crest genes in hESCs and hESCs treated with cranial neural crest induction media alone (Control) or with DMSO or 2 μM PhenDC3 at 3 and 5 days of differentiation. f Schematic of the main findings of our study: G4 abundance correlates with stem cell plasticity (top left); G4 presence is associated with particular promoter histone landscapes in differentiation (top right); and maintenance of a promoter G4 from hESCs to differentiated daughter cells preserves the transcriptional output of a gene independent of transcription levels (bottom).
