Extended Data Fig. 9: Zic2 and Zic3 redundantly regulate Mesp1 activity.
a, Expression of three core pluripotency genes in Mesp1 WT and two independent Zic2/3 KO PSC cell lines cultured in Lif/2i medium. Data from two independent experiments, b, FACS profiles of EBs at day 4 of differentiation from Mesp1 WT and Zic2/3 dKO cell lines, illustrating the decrease in Flk1 and PDGFRa expression in Zic2/3 dKO cell lines both in no dox and dox conditions. c, Table shows the distribution of genes that were downregulated in Zic3 KO and Zic2/3 dKO cells within the temporal categories of Mesp1 direct upregulated target genes. There was no particular enrichment for early, constant and late genes within the Zic2/3-dependent fraction of Mesp1 target. d, Barplot illustrating the proportion of early, constant and late Mesp1 binding sites within Mesp1 ChIP-seq peaks conserved in Zic2/3 dKO cell lines. *** for late genes, z = -5.845, p < 0.00001. These values were calculated through a two-tailored Z-test. Data shown represent two biologically independent replicates. e, Representation of the proportion of Mesp1 ChIP-seq peaks as well as ATAC-seq peaks that are opened (UP) or closed (DOWN- upon Mesp1 induction in WT cells which are preferentially closed in Zic2/3 dKO cells after Mesp1 induction. n = 2 independent experiments for Mesp1 ChIP-seq and ATAC-seq in WT cells; n = 3 independent experiments for ATAC-seq in Zic2/3 dKO cell lines. *** all three comparisons were significant with p < 0.00001 and respectively z = 34.9 (Mesp1 ChIP-seq), z = 54.2 (ATAC-seq UP) and z = -20.1 (ATAC-seq DOWN). These values were calculated through a two-tailored Z-test. f, Motif enrichment analysis of ATAC-seq peaks that were preferentially closed in Zic2/3 dKO cells in comparison to WT cells. p-values are calculated through a binomial test.
