Fig 2: Effect of tag multimerisation on the efficiency of IP from crude cell lysates. (A) Infrared fluorescent Western blot detection of the FLAG-tagged Sox3 proteins. The Sox3 protein tagged with FLAG either in monomeric (a) or trimeric (b) form in IP samples and the dilution series of input lysates were detected using anti-Sox3 antibody. IP was performed in duplicate and under two IP conditions, i.e., 1 µg or 0.1 µg of anti-FLAG (IE6) antibody was incubated with the embryo lysates in 1 mL of IP solution. The recovery rates (in percentages) of FLAG-tagged Sox3 are shown at the bottom of each blot. The two blots in panels (a,b) were processed in parallel and scanned simultaneously. (B) HiBiT detection of input dilutions of FLAGx3-tagged Sox3 on the same membrane shown in (Ab) and 0.5 ng (15 fmol) of FLAGx3-tagged GST, which was used as the standard for absolute protein quantification. (C) Comparison of the experimental and theoretical IP recovery values. The averaged recovery rates of duplicated IP experiments and theoretical recovery rates are shown. The process used for the theoretical recovery calculation is given in the Materials and Methods section.

Fig 3: Disrupted pathway of 17ß-E2 synthesis and up-regulated apoptosis in the sox3-/- ovaries. (A) The expression levels of cyp19a1a in both sox3+/+ and sox3-/- ovaries based on RNA-seq data. (B) Quantitative real-time PCR analysis of cyp19a1a expression levels in both sox3+/+ and sox3-/- ovaries. The transcript levels were related to ß-actin expression. Relative level, 2-??Ct. T-test was performed. *P < 0.05. (C) The standard curve of 17ß-E2 concentration at OD450. (D) Down-regulated 17ß-E2 in sox3-/- gonads in comparison with the wild type gonads. Data represented means ± SEM. T-test was performed. *P < 0.05; **P < 0.01. (E) Immunofluorescence analysis of Sox3 protein in adult ovary. Anti-Sox3 and FITC-conjugated goat anti-rabbit IgG (H + L) antibodies were used to detect Sox3 (green). The nuclei were stained by Hoechst (blue). Preimmune serum was used as a negative control. The white square area in the inset was enlarged and showed on the right. Sox3 positive signals were observed in somatic cells (theca cells and granulosa cells) (white arrowheads) in ovary. Scale bar, 50 µm. (F) Immunofluorescence analysis of Cyp19a1a protein in adult ovary. Anti-Cyp19a1a and FITC-conjugated goat anti-rabbit IgG (H + L) antibodies were used to detect Cyp19a1a (green). The nuclei were stained by Hoechst (blue). Preimmune serum was used as a negative control. The white square area in the inset was enlarged and showed on the right. Cyp19a1a positive signals were observed in somatic cells (theca cells and granulosa cells) (white arrowheads) in ovary. Scale bar, 50 µm. (G) Assessment of apoptosis using Annexin V-FITC/PI and flow cytometry. CHO cells were treated with 17ß-E2 (500 ng/mL) or DMEM (control) for 36 h, and then treated with etoposide (3 µg/mL) for 12 h and assayed by flow cytometry. Viable cells exhibited Annexin V-/PI- (symbol 3 in the plot); early apoptotic cells exhibited Annexin V+/PI-(symbol 4 in the plot); late apoptotic cells exhibited Annexin V+/PI+ (symbol 1 in the plot); necrotic cells and some late apoptotic cells exhibited Annexin V-/PI+ (symbol 2 in the plot). (H) Percentages of both early and late apoptotic cells based on the apoptosis assessment by flow cytometry and Annexin V/PI in (G). T-test was performed. *P < 0.05

Fig 4: p63 regulates Sox3 binding to neural enhancers and the neural program. a Differential analysis of Sox3 binding between WT and tp63-/- at 36 hpf, calculated in a set of 147,588 Sox3 BSs (FDR < 0.05) from ChIPmentation (n = 2 biological replicates per condition). The log2 base mean Sox3 binding levels in the WT versus the log2 fold-change of expression are plotted. Peaks showing a statistically significant differential expression (p < 0.01) are highlighted in blue (downregulated) or red (upregulated). b Heatmaps and average profiles of the Sox3 BSs at 36 hpf from (a) in WT and tp63-/- embryos. Peaks were separated according to the differential analysis in up-regulated (Up; n = 2,976) or down-regulated (Down; n = 941) in tp63-/- mutant compared to WT. c Bar plot with the mean expression of sox3 gene in WT vs. tp63-/- embryos at 36 hpf as measured by RNA-seq (n = 3, for each condition). Error bars represent s.d. Individual values are shown as red dots. Non-corrected p-value from the differential expression analysis is shown. d Box plots showing the expression fold-change in tp63-/- embryos of the transcripts associated to Up- (n = 222), Down-regulated (n = 75) and not changed (NC; n = 1,255) Sox3 BSs from (a) compared to the total de-regulated transcripts (n = 2,344) in that mutant. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. Statistical significance was assessed using the Wilcoxon’s rank sum test. For (c) and (d), source data are provided as a Source Data file. e Genome tracks of p63 ChIP-seq, Sox3 ChIP-seq, and RNA-seq in the indicated developmental stages showing signal intensities in a region of chromosome 8. An up-regulated Sox3 BS in tp63-/- mutant and the associated up-regulated gene (cldn5a) are highlighted in light orange. The Genes track represents ENSEMBL annotated genes. See also Supplementary Fig. 3

Fig 5: p63 acts as a pioneer TF to promote epidermal gene expression. a Heatmaps (left) and average profiles (right) of ATAC-seq signal from WT and tp63−/− embryos in 8 somites stage (n = 2 biological replicates per condition) for the groups resulting from the alluvial plot transitions from Fig. 5a. A dotted red line represents the WT profile peak in each stage. Groups 2 and 3 were omitted from the analyses due to their small number of peaks. b Genome tracks of p63 ChIP-seq, ATAC-seq and RNA-seq in the indicated developmental stages showing signal intensities in a region of chromosome 9. A down-regulated ATAC-seq peak in tp63−/− mutant and the associated down-regulated gene (col18a1a) are highlighted in light orange. The Genes track represents ENSEMBL annotated genes. c Box plots showing the expression fold-change in tp63−/− embryos of the transcripts associated to group 4 ATAC-seq peaks (n = 227) compared to the remaining de-regulated transcripts in that mutant (n = 2,117). Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. Statistical significance was assessed using the Wilcoxon’s rank sum test. Source data are provided as a Source Data file. d Proportion of up- and down-regulated transcripts in tp63−/− embryos associated to ATAC-seq peaks from group 4 compared to the remaining de-regulated transcripts in that mutant. Statistical significance was assessed using the Fisher’s exact test. e Model proposed to illustrate the dual role of p63 during ectoderm specification. During gastrulation (top), p63 prevents Sox3 binding to enhancers associated to neural genes probably in the neural plate border, where they are co-expressed, resulting in the inhibition of neural expression. During segmentation and pharyngula (bottom), non-accessible chromatin at enhancers associated to epidermal genes and bound by p63 during gastrulation becomes open, triggering an epidermal transcriptomic program that results in the differentiation of epidermis and related structures. NP neural plate, SE surface ectoderm, NPB neural plate border, Epi epidermis, FB fin bud, PA pharyngeal arches. See also Supplementary Fig. 5