Fig 2: SPF activity is not sufficient, in isolation, to generate chromatin accessibility.a, b Upper panels. Results of ATAC-qPCR experiments in FR1_OCT4SOX2 ESCs (a) and FR1_CTCF ESCs (b) containing WT and Sc motifs in -SssI and +SssI conditions. We measured very low levels of chromatin accessibility at the FR1 locus in all four conditions when compared to known accessible (Zfp345 large ATAC-peak; Kif3b medium ATAC-peak) and inaccessible (Intergenic small ATAC peak) regions of the chromatin in mESCs97. Results are shown as mean+SD of n = 3 biologically independent replicates. Source data are provided as a Source data file. Lower panels. Representative genome browser tracks of the chromatin accessibility landscape around the FR1 locus. ATAC-Seq experiments were performed in FR1-OCT4SOX2 ESCs containing WT/±SssI and Sc/±SssI motifs (a) and in FR1-CTCF ESCs containing WT/-SssI and Sc/-SssI motifs (b). The ATAC-Seq signal is low over the FR1 locus compared to neighbouring accessibility peaks, highlighting the low levels of chromatin accessibility, which remain unchanged despite SPF binding in the WT conditions. c OCT4 and SOX2 ChIP-Seq in FR1 OCT4SOX2 ESCs reveals binding of both TFs at the FR1 locus. Displayed are representative genome browser tracks of the ChIP-Seq data across the FR1 locus.

Fig 3: Hierarchy of transcription factor binding.SPFs engage their target sequences in closed chromatin and in the presence of DNA methylation. Upon binding, most SPFs drive DNA demethylation through active processes, mainly mediated by the TET enzymes, while SOX2 leads to passive DNA demethylation. Loss of DNA methylation allows the binding of methylation-sensitive PFs. Nucleosome remodelling and deposition of histone modifications associated with open chromatin regions, mediated by both SPFs and PFs, create a favourable environment for the binding of settler TFs.

Fig 4: Identification of super pioneer transcription factors (SPFs).a Heatmaps indicating methylation percentages of individual CpGs in the FR1 containing WT (left panel) and Sc (right panel) motifs in the +SssI condition. Each line represents FR1 containing the indicated motif. Each square within the line corresponds to one CpG. The methylation percentage of individual CpGs is represented by a colour code. CpGs’ distance from the 5’ end of the motifs is indicated below the heatmaps. CpGs within the motif, when present, are indicated as m1, m2 and m3. b Differential methylation between WT and Sc motifs in the FR1/+SssI condition. Differential methylation was calculated for each CpG as ?met = % met_WT - % met_Sc and represented by a colour code. Results were hierarchically clustered using the complete linkage method with Euclidian distance. CpGs’ distance from the 5’ end of the motifs is indicated below the heatmaps. The coordinates of statistically significant hypomethylated regions (HMRs) in WT condition are indicated on the side. c Differential methylation around OCT4SOX2 WT motif in the FR1/+SssI condition between cells that underwent SOX2 and/or OCT4 knockdown (siRNA) and untransfected cells (no_siRNA). Differential methylation was calculated for each CpG as ?met = % met_WT (siRNA) - % met_WT (no_siRNA) and represented by a colour code. CpGs’ distance from the 5’ end of the motifs is indicated below the heatmaps.

Fig 5: The dynamic expression of Mettl3 and m6A during the differentiation of aNSCsRepresentative immunostaining showing the expression of Mettl3 in Nestin+ (A) and Sox2+ (B) aNSCs. Immunofluorescence staining showing the expression of Mettl3 in Tuj1+ neuronal cells (C) and GFAP+ astrocytes (D). Western blot (E) and quantification (F) showing the increased expression of Mettl3 upon the differentiation of aNSCs (n = 3). Western blot (G) and quantification (H) showing the increased expression of Mettl14 upon the differentiation of aNSCs (n = 3). Tubulin and Gapdh were used as loading controls. Dot-blot assay (I) and quantification (J) revealing the increase in m6A levels upon the differentiation of aNSCs (n = 3). Data are presented as mean ± SEM. Unpaired t-test, **, P < 0.01; ***, P < 0.001. Scale bar, 50 µm. aNSC, adult neural stem cell; Tuj1, ß-III tubulin; GFAP, glial fibrillary acidic protein.