Fli1, Pu.1, and Gata2 bind the endoglin hematopoietic enhancers in vivo. (A) Variation in gene expression during in vitro differentiation of GFP-Bry ES cells to embryoid bodies. (i) Levels of endoglin expression in hemangioblast containing DP (Bry⁺/Flk⁺) cells relative to prehemangioblast SP (Bry⁺/Flk⁻) cells. (ii) TF levels in ES cells, cell fractions sorted from day 3.5 (DN, SP, and DP) and unsorted day 4.5 EBs. (B) In situ hybridization for Fli1, Gata2, Scl, and Pu.1 transcripts in fetal livers of E11.5 mouse embryos (magnified ×10). (C) ChIP-chip profiles of TF binding across the Eng locus of HPC-7 hematopoietic progenitor cells. Vista plots of sequence alignments of mouse and human Eng loci are shown in the top panel with ChIP-chip profiles shown below. The gray bars indicate the genomic positions of the conserved −8, P, +7 and +9 segments. ChIP assays were performed in HPC-7 hematopoietic progenitor cells with anti-Fli1, -Pu.1, -Gata2, and -Scl antibodies and MS1 endothelial cells with anti-Fli1 and Gata2 antibodies. Samples were hybridized in triplicate and fold enrichment over nonenriched input (normalized to the median of values across the Eng locus) is plotted (log₂) against genomic position in kilobases. The width of each bar represents the width of each spotted oligonucleotide on the array. The HPC-7 plots show enrichment of Fli1 and Pu.1 at the Eng promoter and the Eng −8, Eng +7 and Eng +9 enhancers, enrichment of Gata2 at the Eng +7 enhancer but no enrichment of Scl at the Eng locus. The enrichment profile of Fli1 in MS1 cells is similar to that of HPC-7 cells but Gata2 is not enriched at the Eng locus in endothelial cells. EB, Embryoid Body; DN, Double Negative; SP, Single Positive; DP, Double Positive.