Fig 2: Neural crest-like cells derived from induced pluripotent stem cells (iPSCs) (iNCLCs) contribute to vascularized dentin–pulp complex regeneration in vivo. (A) iPSCs formed neural spheres under suspension culture. (B) The spheres attached to fibronectin-coated dish, and the cells migrated out from the spheres. (C) The morphology of the migrated cells after one time passage. (D,E) Reverse transcription PCR (RT-PCR) and immunocytochemistry were performed to evaluate the expression of neural crest-specific markers including p75 and AP2-a in iNCLCs. (F) Multilineage differentiation of iNCLCs. Osteogenic differentiation, positive staining with Alizarin Red S; adipogenic differentiation, positive staining with Oil Red O; chondrogenic differentiation, positive staining with Safranin O. (G) HE staining images of each group (the right two panels are the magnification of the boxed region; black arrowheads indicate odontoblast-like cells, yellow dash line area indicates newly formed dentin, and green arrowheads indicate blood vessel). (H) Histomorphometric analysis of newly formed vessel area; (I) Dentin sialophosphoprotein (DSPP) immunohistochemistry of each group (the right two panels are the magnification of the boxed region; dash blue line area indicates newly formed dentin); mouse femur bone tissue was treated as negative control group. (J) CD31 immunohistochemistry of each group (green arrowheads indicate blood vessels). (K) Transplanted tooth scaffold/iNCLCs complex formed teratoma, containing three germ-derived tissues including keratin-containing epidermal tissues (ectoderm), cartilage (mesoderm), and gut-like epithelial tissues (endoderm). *P < 0.05.

Fig 3: Overexpression of Tfap2a promoted GVBD but inhibited extrusion of the first polar body in mouse oocytes. (A) Images of Myc and Myc-Tfap2a mRNA-injected oocytes at different times after maturation in vitro. The arrowheads indicate the GVBD oocytes 3 h after IVM, whereas the red arrows indicate the oocytes with PB1, and the numbers in the images indicate the ratios of GVBD or PB1 extrusion to the total number of oocytes. Scale bar = 100 µm. (B) The effects of Tfap2a overexpression on the rates of GVBD and PB1 oocytes at different times after IVM. Data are represented as the mean ± SEM of at least three independent experiments. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Fig 4: TFAP2 and MITF co-regulate pigmentation and cell differentiation genes in SK-MEL-28 cell lines.(A) Venn diagram representing the overlap of genes apparently directly activated by MITF (i.e., expression lower in MITF-KO than in WT cells, FDR < 0.05, and an MITF peak within 100kb of the TSS) and genes directly activated by TFAP2 (i.e., expression lower in TFAP2-KO than in WT cells, FDR < 0.05, and an TFAP2-activated enhancer of any category within 100kb of the TSS). The number of overlapping genes with TFAP2-dependent MITF peaks are also shown (*). (B) Gene ontology (GO) biological process analysis enriched among MITF- and TFAP2-activated genes are shown (Top 5 hits). (C) A curated list of pigment-associated genes [118] was intersected with directly MITF-activated genes, overlapping genes of directly MITF- and TFAP2-activated genes, and directly TFAP2-acitvated genes and represented by gene list. (D) Venn diagram representing directly MITF inhibited genes (MITF peak within 100kb of a TSS), based on RNA-seq, in MITF-KO versus WT cells (FDR < 0.05) and directly TFAP2 inhibited genes (TFAP2-inhibited enhancers, of any category, within 100kb of a TSS), based on RNA-seq, in TFAP2-KO versus WT cells (FDR < 0.05). (E) Gene ontology (GO) biological process analysis enriched among MITF- and TFAP2-inhibited genes are shown (Top 5 most enriched GO terms). GO analysis was performed using PANTHER. (F) Dot plot of enrichment analysis showing the enrichment of melanoma gene signatures from the literature in directly TFAP2-activated and TFAP2-inhibted genes. P value is red lowest to blue highest; gene ratio is the fraction of all genes in the gene signature category that are included in the set identified here. TFAP2-activated genes associated with TFAP2-dependent MITF peaks are shown (*).

Fig 5: Categories of TFAP2-regulated enhancers.Box 1: TFAP2A peaks at open and active chromatin. (1A) TFAP2-pioneered-and-activated enhancers show reduced nucleosome accessibility (ATAC-seq) and reduced levels of active chromatin marks (H3K27Ac and H3K4Me3) in TFAP2-KO cells compared to in WT cells. We infer that TFAP2 paralogs pioneer chromatin access for transcriptional co-activators, like MITF and SOX10 (purple box), that in turn recruit chromatin remodelling enzymes and histone modifying enzymes. (1B) Non-pioneered TFAP2-activated enhancers show loss of active chromatin marks but unchanged nucleosome accessibility in TFAP2-KO cells compared to in WT cells. At these enhancers, we infer that TFAP2 paralogs recruit the binding of transcription factors that, in turn, recruit histone modifying enzymes. TFAP2 paralogs also may recruit such enzymes. It is possible that these elements are stably pioneered by TFAP2 paralogs [94]. (1C) At TFAP2-independent elements, neither the nucleosome accessibility nor active histone marks are altered in TFAP2-KO cells relative to in WT cells. Both types of TFAP2-activated enhancer are significantly enriched near genes whose expression is reduced in TFAP2-KO cells relative to in WT cells (i.e., TFAP2-activated genes). Such genes are associated with the gene ontology (GO) terms cell proliferation and pigmentation. TFAP2-independent elements are associated with neither TFAP2-activated nor TFAP2-inhibited genes. Box 2: TFAP2A peaks at closed and inactive chromatin. (2A) TFAP2-pioneered-and-inhibited enhancers show increased nucleosome accessibility and increased levels active chromatin marks in TFAP2-KO cells compared to in WT cells. At these sites we infer that TFAP2 paralogs recruit or stabilize the binding of enzymes that condense chromatin and that inhibit the binding of transcriptional activators that are otherwise inclined to bind at them. These elements are significantly enriched near genes whose expression is elevated in TFAP2-KO cells relative to in WT cells (i.e., TFAP2-inhibited genes). Such genes were associated with the GO terms cell-cell adhesion and cell migration. (2B) At TFAP2-independent elements, neither the nucleosome accessibility nor active histone marks are altered in TFAP2-KO cells relative to in WT cells. These elements were associated with neither TFAP2-activated nor TFAP2-inhibited genes.