Fig 1: 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 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: SOX2 inhibits DNMT1-dependent maintenance of DNA methylation during replication.a In vitro methylation assay to measure DNMT1 methyltransferase activity using hemi-methylated probes containing WT or Sc PF motifs in the presence or absence of the corresponding PF. Relative DNMT1 activity is represented as scintillation counts in WT probes, corrected for the amount of recovered DNA probes and compared relative to the Sc probe. Results are shown as mean + SEM of n = 3 biologically independent replicates. SOX2, ETS1 and OTX2 significantly reduce DNMT1 activity. p values: ETS1 p = 0.0018, OTX2 p = 0.0238, SOX2 p = 0.026 (two-tailed unpaired t test; *p < 0.05, **p < 0.01). b In vitro methylation assay using the probe containing the OCT4SOX2 motif in the presence of OCT4, SOX2 or SOX2 + OCT4 proteins. Results are shown as mean + SEM of n = 3 biologically independent replicates. p values: SOX2 + OCT4 p = 0.018, SOX2 p = 0.003, (two-tailed unpaired t test, *p < 0.05, **p < 0.01). c, d In vitro replication assay to assess the effect of SOX2 and OCT4 binding on the maintenance of DNA methylation during replication. Two probes containing the OCT4SOX2 motif were used: FR1 (c) and FR2 (d). The indicated concentrations represent those of active hOCT4 and hSOX2 recombinant proteins that were used. Methylation levels following replication are measured based on the integration of radioactively labelled methyl group during replication and compared to “no_protein” control. Results are presented as mean + SD of n = 5 (hSOX2 samples) or n = 3 biologically independent replicates (hOCT4 and hOCT4 + hSOX2 samples) and analysed as radioactive signal in the presence of the protein relative to the signal in the absence of protein. p values: FR1_600nM_SOX2 p = 0.0092, FR1_600nM_OCT4 + SOX2 p = 0.0068, FR2_450nM_SOX2 p = 0.0071, FR2_600nM_SOX2 p = 0.0021, FR2_450nM_OCT4 + SOX2 p = 0.0026, FR2_600nM_OCT4 + SOX2 p = 0.0047 (two-tailed unpaired t test, **p < 0.01). For all panels, source data are provided as a Source data file.
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