Fig. 6: VGLL1 is necessary for sustaining the human TSC identity.

a Phase contrast images of 4CL H9 ESC-derived TSCs transduced with shLuc, shVGLL1-2 or shVGLL1-4. Scale bar, 100 µm. Representative of three independent experiments. b Analysis of cell numbers for 4CL H9 ESC-derived TSCs transduced with shLuc, shVGLL1-2 or shVGLL1-4. Data are presented as the mean ± SEM, n = 3 biological replicates. P value was calculated using a two-tailed unpaired Student’s t-test, ***P < 0.001, **P < 0.01, *P < 0.05. c Heatmap showing the expression of pluripotency, cell cycle and TSC genes for the indicated conditions. Example genes are shown for each cluster in the boxes. n = 2 biological replicates. d Enriched GO terms for downregulated genes in shVGLL1 compared to shLuc control in 4CL H9 ESC-derived TSCs. P value was calculated using a hypergeometric test (one-sided) and adjusted for multiple testing using the Benjamini-Hochberg correction. e Immunoprecipitation using 4CL H9 ESC-derived TSC lysates with anti-VGLL1 (left panel) or anti-TEAD4 (right panel) and subsequent Western blotting with anti-TEAD4, anti-VGLL1, anti-YAP and anti-WWTR1. Representative of three independent experiments. f Western blotting for the indicated histone marks for shVGLL1-2 and shVGLL1-4 compared to the shLuc control in 4CL H9 ESC-derived TSCs. Representative of three independent experiments. g Genome browser tracks showing VGLL1, TEAD4, H3K27ac genomic enrichment peaks (CUT&Tag) and chromatin accessibility peaks (ATAC-seq) for representative TSC genes loci in 4CL H9 ESC-derived TSCs. h Co-occupancy analysis by signal density pileups of VGLL1, TEAD4 binding in 4CL H9 ESC-derived TELC-D5 cells and TSCs. The three major groups are: group 1 (common between TELC-D5 and TSC), group 2 (TELC-specific) and group 3 (TSC-specific). For clarity, other groups are not labelled. i Enriched GO terms for groups 1, 2 and 3 of panel h. P value was calculated using a hypergeometric test (one-sided) and adjusted for multiple testing using the Benjamini-Hochberg correction. j Genome browser tracks showing VGLL1, TEAD4 and H3K27ac genomic enrichment peaks (CUT&Tag) in 4CL H9 ESC-derived TELC-D5 cells and TSCs for representative gene loci from group 2 (upper panel) and group 3 (lower panel).