Mesoderm to hemangioblast transition is accompanied by increased Eng expression and chromatin accessibility at hematoendothelial regulatory elements. (A) Schematic representation of the ENG locus. The transcription start site is marked with an arrow. The −8-kb, +7-kb, and +9-kb enhancers and the promoter (P) are marked in orange; exons are marked in brown, and the 5′untranslated region is marked in cyan. (B) Schematic representation of Bry-GFP ES cell differentiation. At day 3 of EB differentiation, Bry-GFP+/FLK1− (G+F−) and Bry-GFP+/FLK1+ (G+F+) cells were sorted and analyzed by RT-PCR and ChIP. (C) Bar graph shows Eng mRNA expression levels in sorted FLK1+ve and –ve mesodermal cell populations in day 3 EBs generated from Bry-GFP ES cells. (D) Bar graph shows levels of enrichment of the active chromatin mark, H3K9Ac at Eng −8, P, +7, and +9 hematoendothelial enhancers relative to immunoglobulin G in prehemangioblast mesoderm (G+F−; black) and in hemangioblast mesoderm (G+F+; gray). Eng −4 was included as a negative control region. **P < .01, ***P < .001.