Fig. 3. Enforced GATA2 results in an increase in mesodermal gene expression. (A) iGATA2 ES cells were differentiated for 2 days in SR and treated with 0.3 µg/ml Dox for an additional 2 days. RNA was then utilized for qRT-PCR to examine the genes indicated. Genes were normalized against Gapdh and then the ratio of the gene quantity (+Dox) to gene quantity (-Dox) was determined to yield normalized fold change. (B) Upper portion depicts the mouse Bmp4 locus. Gray boxes are non-translated exons and the open box indicates the translated exon. Coordinates and arrows indicate conserved, consensus GATA-factor-binding sites. Enlarged below is a section containing the TGE of a 5' to 3' alignment of highly conserved, intronic regions of homologs of the Bmp4 gene. GATA sites 1 and 2 are indicated by brackets; arrows above the alignment indicate GATA site orientation. (C) ChIP analysis of GATA2 occupancy of the Bmp4 locus in D4 EBs. A2Lox ES Cells were differentiated for 4 days in serum, crosslinked with 1% formaldehyde and processed to examine GATA2 occupancy at the three conserved consensus GATA-factor-binding sites. (D) Bmp4 enrichment in Gata2-expressing cells. iGATA2 ES cells were differentiated in SR medium for 4 days and Dox added on D2. At D4, cells were stained for hCD4 expression and sorted into hCD4^- and hCD4⁺ and then RNA generated for qRT-PCR to examine co-enrichment of Gata2 and Bmp4 in hCD4⁺ cells. (E) FACS analyses of Flk-1⁺ cell generation by GATA2 and BMP4. Cells were differentiated in SR medium for 2 days and then treated with combinations of Dox (0.3 µg/ml), BMP4 (5 ng/ml) and noggin (10 ng/ml). Cells were utilized for FACS analyses on D3. Numbers indicate percentage of Flk-1⁺ cells generated. (E') Summary data of four independent sets of FACS experiments showing the percentage of Flk-1⁺ cells generated in each condition. Values indicate mean±s.e.m.; ^*, P<0.01 versus untreated.