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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.
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