Supplementary Figure 5: Whsc1 binds specifically to the enhancers of mesendoderm TFs.

a, Top panel: HiC contact maps at 10kb resolution from ESCs around Foxa2, Gata4, Sox1 and Pax6 loci (GSE96611). TADs are marked by solid black lines and subTAD are indicated by dashed black lines. The regions analysed using H3K27ac ChIP-seq data are indicated as black boxes; Bottom panel: H3K27ac ChIP-seq profiles on the loci of Foxa2, Gata4, Sox1 and Pax6 loci in neural progenitor cells (NPC) (GSE35496), Eomes+ mesendodermal progenitors (GSE103262), Activin A-induced mesendodermal precursors (MP) (GSE38596) and Flk1+ mesendodermal progenitors (GSE47082). The black arrows below each panel correspond to putative regulatory regions up or downstream of the respective gene. b, ChIP-qPCR quantification of Whsc1 occupancy on the same enhancers as shown in Fig. 5a (above) in D6 EBs from Whsc1 +/+ and Whsc1 −/− cells with numbers indicating distance to the TSS in kb. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. c, Replicated UMI-4C profiles for baits located on the Foxa2 (left) and Gata4 (right) promoters assayed in Whsc1 +/+ and Whsc1 −/− ESCs and D6 EBs. Top panel, contact profiles generated from the average of two independent biological replicates; bottom panel: average contact fold change D6 EBs versus D0 ESCs from two independent biological replicates. d, H3K27ac enrichments on putative regulatory regions of Foxa2, Gata4, Sox1 and Pax6 were quantified by ChIP-qPCR in Day 6 Whsc1 +/+ and Whsc1 −/− EBs. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. e, ChIP-qPCR quantification of H3K4me2 occupancies on putative regulatory regions of T, Gata6, Foxa2, Gata4, Sox1 and Pax6 in D6 Whsc1 +/+ and Whsc1 −/− EBs. Data represent mean±s.d. from n=3 independent experiments and p-values were calculated by two-tailed unpaired t-test. f, ChIP-qPCR quantification of Whsc1 (left) and H3K27ac (right) occupancies on putative regulatory regions of T, Gata6, Gata4 and Foxa2 in Whsc1 +/+ and Whsc1 −/− ESCs. These regulatory regions were bound by Whsc1 in D6 with increase H3K27ac enrichments (Fig. 5b, d, Supplementary Fig. 5b and d). Data represent mean±s.d. from n=3 independent experiments.