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. 2021 Aug 26;12:5136. doi: 10.1038/s41467-021-25370-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Schematics of EpiSC differentiation towards PSM. The time-points selected for ChIP-seq/RNA-seq analyses are highlighted in black. b Top: overlap of downregulated genes in Pbx-DKO cells (RNA-seq) and genes associated with PBX-occupied regions at 24 h (ChIP-seq). GO-term enrichment analysis displays significant overrepresentation of terms associated with mesoderm and somite development (Binomial test with Bonferroni correction). Bottom: heatmap showing RNA-seq expression Z-scores computed for 125 downregulated PBX targets in Pbx-DKO (yellow) compared to WT cells (black). Representative markers are indicated. c Top: overlap of upregulated genes in Pbx-DKO cells (RNA-seq) associated with PBX-occupied regions (ChIP-seq) at 24 h. GO-term enrichment analysis shows correlation with terms associated with neurectoderm and embryonic development (Binomial test with Bonferroni correction). Bottom: heatmap displaying RNA-seq expression Z-scores computed for 126 upregulated PBX targets in Pbx-DKO (yellow) compared to WT cells (black). Representative markers are indicated. In b and c, significance was assessed by DEseq2 on the basis of two-sided Wald test with Benjamini–Hochberg adjusted P-values (P ≤ 0.05, fold-change ≥1.5). n = 3 biological replicates. d Schematics of EpiSC differentiation towards PSM illustrating the time-points selected for chromatin accessibility analysis (ATAC-seq). e Heatmap of chromatin accessibility of WT (black) and Pbx-DKO cells (yellow) at PBX-occupied regions that are associated with downregulated genes in Pbx-DKO along PSM differentiation, showing reduced accessibility in Pbx-DKO cells at 24 and 36 h. f Heatmap of chromatin accessibility at PBX-bound regions associated with upregulated genes in Pbx-DKO compared to WT cells. Chromatin accessibility is comparable between WT (black) and Pbx-DKO cells (yellow). The scale represents normalised counts (RPKM) for ATAC-seq peak signals ±500 bp around the centre of the peak in e and f. g Homer de novo motif analysis of 200 bp summit regions along differentiation reveals changing in binding sites enrichment at different time-points, highlighting the variety of PBX-binding partners during PSM differentiation. Only the most relevant binding motifs are shown for each time-point. See Supplementary Data 5 for the complete list.