Fig. 2

ZIC3 is required for enhancer activity in SL-ESCs, but not in 2iL-ECSs. a Top: Barplot that depicts the number of C1 STARR-seq peaks (n = 18,544) with significantly higher (FC ≥ 2.5, p < 0.05, DESeq2) enrichment in 2iL (red; n = 1442) or SL (green; n = 3688). Bottom: Scatterplot of the STARR-seq enrichment in 2iL or SL for C1 STARR-seq peaks. Differential peaks elevated in 2iL (red) or SL (green) are annotated. Non-differential peaks are gray. b TF motifs enriched at differential C1 STARR-seq peaks in 2iL- (red) and SL-ESCs (green). P values were derived using a binomial test with all C1 STARR-seq peaks as background set (Homer2). c STARR-seq peaks (highlighted in yellow) near the Tcfcp2l1 gene that is higher expressed in 2iL-ESCs [50]. A STARR-seq enhancer near the TSS is repressed by TCF3 in SL-ESCs [PD]. d Western blot analysis of ZIC3 and a GAPDH control in 2iL- and SL-WT ESCs (left) and two Zic3^−/− ESC clones cultured in SL (right). e Cell morphology of Zic3^−/− and WT ESCs cultured in 2iL or SL. Zic3^−/− ESCs drastically change morphology. f Examples of STARR-seq peaks with elevated signal in SL-ESCs. Sites are more accessible in SL-ESCs and have slightly higher H3K27ac, although this signal is relatively low (see, e.g., Figure S1I). P300 ChIP-seq signal is similar between 2iL- and SL-ESCs [PD]. g Luciferase signal (Firefly/Renilla) scaled to F/R of a control region in WT and Zic3^−/− ESCs for the three genomic locations shown in f. See Table S3 for genomic location and primer sequences. h PCA plot using the 1000 most variable genes in Zic3 WT vs Zic3^−/− RNA-seq. Loss of Zic3 barely affects the transcriptome of 2iL-ESCs, but alters that of SL-ESCs considerably. i Differentially expressed genes in Zic3^−/− vs WT ESCs cultured in SL. Strongly upregulated genes are enriched for endodermal markers like Sox17, Gata4, and Dab2 and Cubn. j RNA-seq expression of selected pluripotency factors and endodermal marker genes. The color scale denotes DEseq2 normalized reads (log₂) per gene. Biological duplicates are shown for Zic3^−/− ESCs. k UMAP clustering of Zic3^−/− and WT ESC cultured in 2iL and SL. Top: annotation of genotype and culture condition. Bottom: cluster assignment using shared nearest neighbors on the first 12 principal components and resolution = 0.1. l Heatmap of selected marker genes for the clusters shown in k. Rows are genes, columns are cells. Cells originating from clusters 1–4 are annotated with their respective colors in the boxes in the top. Color gradient depicts the expression Z-score relative to the average cell (unclustered). For the genes colored in blue or red to the left of the heatmap, a browser screenshot of their genomic locus is shown in Figure S2I. Some of the panels in these figures contain public data. These panels are annotated with [PD]. The accession numbers of public data and their corresponding panels are annotated in Additional file 2: Table S1