A Novel Nodal Enhancer Dependent on Pluripotency Factors and Smad2/3 Signaling Conditions a Regulatory Switch During Epiblast Maturation
Figure 6
HBE is required to activate ASE during ESC to EpiSC differentiation.
(A) RT-qPCR analysis of YFP expression during 10 d of ESC to EpiSC differentiation of NodalcondHBE-YFP(HBE+) and NodalΔHBE-YFP(HBE−) ESCs. One representative experiment. (B) Percentage of difference of YFP mRNA levels between NodalΔHBE-YFP and NodalcondHBE-YFP cells during 10 d of ESC to EpiSC differentiation. Error bars represent the mean + SD of triplicates and two independent experiments. (C–D″) Expression of Oct4 (C, D) and Venus-YFP (C′, D′) in NodalcondHBE-YFP (C–C″) and NodalΔHBE-YFP (D–D″) ESCs after 10 d of differentiation into EpiSC single confocal sections. n is the number of YFP-positive (C) or YFP-negative (D) samples on the total number of analyzed samples. Scale bar, 25 µm. (E) Part of the Nodal locus in the WT and the recombinant alleles comprising HBE, the first Nodal exon, and ASE and showing the position of regions 1–5 amplified in the ChIP experiments shown in (F) and (G). (F–G) ChIP with anti-H3K27me3 (F), anti-H3K27ac (G), or anti-GFP (F–G) antibodies on material from NodalcondHBE-YFP ESCs (green bars) and NodalΔHBE-YFP ESCs (red bars). The position in the locus of amplified regions 1–5 is shown in (E). An asterisk denotes significant differences between NodalcondHBE-YFP and NodalΔHBE-YFP ESCs (p<0.01).