Extended Data Fig. 6: Eomes- and Brachyury-rescue constructs restore ME program activation and repression of pluripotency and NE programs.
a, ChIP-seq coverage tracks of Eo-GFP, Eo-FL, and Eo-VP16 for indicated genes show identical binding of GFP and VP16 fusion constructs to the full-length (FL) EOMES. Counts normalized to RPKM are indicated in a-c. b, RNA-seq coverage tracks of mesoderm (Mesp1, Msgn1, Mixl1, Myocd and Nkx2-5) and definitive endoderm (Foxa2 and Hhex) markers that are rescued by FL and VP16 activator constructs (Eo-FL, Bra-FL, Eo-VP16 and Bra-VP16) but not by repressor constructs (Eo-EnR and Bra-EnR) or Eo-TBX. c, RNA-seq coverage tracks of pluripotency (Pou5f1, Sox2, Lefty2 and Nkx1-2) and NE (Sox3, Brsk2 and Nefm) markers show selectivity of transcriptional repression by Eomes- or Brachyury- FL, VP16, and EnR constructs. Pluripotency genes are predominantly repressed by Eomes, and NE markers by Brachyury in a direct manner as demonstrated by EnR-mediated repression, and indirectly by VP16 activator-mediated mechanisms. Expression of Eo-TBX does not rescue the expression of EPI genes. d, e, Expression levels indicated by centred scaled counts of mesoderm (Mes) and definitive endoderm (DE) marker genes downregulated in dKO cells are rescued after induced expression of Eomes- (d) or Brachyury- (e) FL, VP16-activator, but not of EnR-repressor constructs. f, g, Expression levels indicated by scaled centred counts of EPI and NE marker genes after induced expression of Eomes- (f) or Brachyury- (g) rescue constructs show reduced expression by FL, VP16 activator, and EnR repressor constructs, but not by Eo-TBX. Bars represent centred scaled counts from n=3 biologically independent RNA-seq experiments in d-g. Error bars indicate s.e.m.
