Fig 2: Dynamic features of MRG identify cell fate transitions during SE lineage commitment.(A) Phase contrast images of the differentiating hESCs during 7 days of culture. Scale bar, 100 µm. (B) Heatmap of MRG expression changes during SE differentiation. Hierarchical clustering yields three clusters of genes and four major groups of samples. The color bar shows the relative expression value [z score of TPM (transcripts per kilobase of exon model per million mapped reads)] from the RNA sequencing (RNA-seq). (C) The trend of expression changes of the three clusters identified from RNA-seq. (D) Gene ontology (molecular function) analysis of MRGs at each time point. (E) Immunofluorescence staining of KRT7 and KRT19 in the differentiated cells on D0, D2, and D7. Scale bar, 100 µm. (F) K-means clustering analysis of differential open chromatin regions during the first 2 days of SE differentiation. The color bar shows the relative assay for transposase accessible chromatin with high-throughput sequencing(ATAC-seq) signal (z score of normalized read counts) as indicated. (G) Snapshots of genome browser showing chromatin accessibility at KRT7, KRT8, and KRT18 loci. Gene expression is also displayed in heatmaps (log2 TPM). The genome browser view scales were adjusted on the basis of the global data range. (H) Representative gene ontology terms (biological process) identified from the differentially expressed genes in D2 differentiated cells. (I) Schematic diagram of SE differentiation.

Fig 3: GRHL3 is required to activate SE identity genes through opening their chromatin accessibilities.(A) Wild-type (WT) and GRHL3-knockout hESCs during SE differentiation. Top: Phase contrast images. Bottom: Immunofluorescence staining of GRHL3 (green) and KRT18 (red). Scale bars, 100 µm. (B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of representative genes in wild-type and GRHL3-knockout hESCs after 2 days of differentiation. qRT-PCR values were normalized to the values in wild-type group. Values are presented as means ± SD (n = 3 biological replicates; *P < 0.05; **P < 0.01; ***P < 0.001; t test). (C) Scatterplot of differential accessibility in wild-type versus GRHL3-knockout (KO) hESCs after 2 days of differentiation. Sites identified as significantly differentially bound [log2 fold change > 1 or < -1 and false discovery rate (FDR) < 0.05] are shown in color (red, peaks increased; blue, peaks decreased). (D) Metaplots of average ATAC-seq density around the SE identity genes in wild-type and GRHL3-knockout hESCs after 2 days of differentiation. (E) Gene set enrichment analysis of the SE identity gene set in the gene expression matrix of wild-type and GRHL3-knockout hESCs after 2 days of differentiation. NES, normalized enrichment score. (F) Enrichment of transcription factor motifs identified by HOMER at GRHL3 peaks. (G) Heatmaps of the binding signals of TFAP2C and GRHL2 at the center of GRHL3 peaks. (H) Heatmaps of H3K27ac, H3K4me1, H3K4me3, and ATAC-seq signals at GRHL3 peaks in hESCs and SE-initiating cells. (I) Gene ontology (biological process) analysis for the GRHL3 putative target genes. The GRHL3 peaks were annotated as follows: The intergenic peaks were assigned to the closest genes, and the intragenic peaks were assigned to those genes. (J) Pie chart of the percentages of SE identity genes with or without GRHL3 binding in SE-initiating cells. (K) Genome browser view of H3K27ac, H3K4me3, H3K4me1, GRHL3, ATAC-seq, and RNA-seq signal at TFAP2C, KRT7, and KRT8/18 loci.