Fig. 7: Late-stage TFs collaborate in regulating the EVT-specific gene expression program.

a Gene track view of late-stage TF binding loci in mature EVTs (EVT D8), near EVT factor loci. b Peak scores of late-stage TFs on class 1–4 genes. Box plots represent the median (black bar) and interquartile range (box boundaries), with whiskers indicating minimum to maximum values. Statistical significance by two-tailed Wilcoxon rank-sum test (NS not significant). All peak scores identified from ChIP-seq results were employed for statistical analysis. c Heatmaps of class 1–4 gene expression in EVTs after KD of individual late-stage TFs. Genes were ordered based on relative gene expression levels in the KD cells compared to the control (from lowest to highest). Occupancy signals plotted as a moving average (window size: 50). Red bars indicate enriched signals. d Relative mRNA expression of EVT marker genes in TSCs overexpressing individual late-stage TFs, induced by administrating Dox for 6 days. Error bars: mean ± SD (n = 3, independent repeats). Statistical significance was determined by two-sided Student’s t-test. e Venn diagram illustrating overlap of top 2000 target genes of DLX5, DLX6, and ASCL2. f Model: TFAP2C primes late-stage TFs during early stage of differentiation, while late-stage TFs activate themselves and repress TFAP2C, forming a transcriptional regulatory circuit to ensure proper EVT differentiation.