HBE is an enhancer active in pluripotent cells.

<p>(A) HBE is a hotspot for the binding of pluripotency factors and Smad3. <i>Nodal</i> regulatory elements are represented by green boxes and <i>Nodal</i> exons by blue boxes. Binding peaks of Nanog, Sox2, Klf4, Oct4, and Smad3 at the <i>Nodal</i> locus in ESCs are represented by black bars that represent either the summit of the peak of ChIP-seq data or its center for ChIP-chip data aligned to UCSC Genome Browser on Mouse Feb. 2006 (NCBI36/mm8) Assembly (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>). (B and C) Luciferase reporter assays for early <i>Nodal</i> enhancers using either a minimal (E1b) or the endogenous promoter (NIS), in ESCs (B), or in EpiSCs (C). Luciferase activities are shown relative to HBE construct. An asterisk indicates significant differences from the control (ctrl) (<i>p</i><0.01).</p

HBE is required for activation of <i>Nodal</i> in the early mouse embryo.

<p>Detection of mCherry (A, B, C, and D) and YFP (A′, B′, C′, and D′) in E4.5 (A–B″) or E6.5 (C–D″) aggregation chimeras generated from <i>Nodal^condHBE-YFP</i> (A–A″ and C–C″) or <i>Nodal^ΔHBE-YFP</i> (B–B″ and D–D″) ES cells and WT embryos. Images are single confocal sections. Cortical actin in blue. <i>n</i> is the number of representative embryos on the total number of embryos analyzed. Scale bar, 25 µm for E4.5 embryos and 50 µm for E6.5 embryos.</p

HBE is required for <i>Nodal</i> expression in ESCs but not in EpiSCs.

<p>(A) Depiction of the two <i>Nodal</i> alleles (WT on top and recombinant at the bottom) before and after Cre recombination. (B–C″) Expression of Oct4 (B, C) and YFP (B′, C′) in recombinant ESCs before (B–B″) and after (C–C″) Cre recombination. (D–E″) Expression of Oct4 (D, E) and YFP (D′, E′) in recombinant EpiSCs, 6 d after transfection with a control plasmid (D–D″) or with Cre recombinase (E–E″). Single confocal sections. <i>n</i> is the number of YFP-positive colonies. Scale bar, 25 µm.</p

HBE is required to activate ASE during ESC to EpiSC differentiation.

<p>(A) RT-qPCR analysis of <i>YFP</i> expression during 10 d of ESC to EpiSC differentiation of <i>Nodal</i>^condHBE-YFP(HBE+) and <i>Nodal</i>^ΔHBE-YFP(HBE−) ESCs. One representative experiment. (B) Percentage of difference of <i>YFP</i> mRNA levels between <i>Nodal</i>^ΔHBE-YFP and <i>Nodal</i>^condHBE-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 <i>Nodal</i>^condHBE-YFP (C–C″) and <i>Nodal</i>^ΔHBE-YFP (D–D″) ESCs after 10 d of differentiation into EpiSC single confocal sections. <i>n</i> 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 <i>Nodal</i> locus in the WT and the recombinant alleles comprising HBE, the first <i>Nodal</i> 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 <i>Nodal</i>^condHBE-YFP ESCs (green bars) and <i>Nodal</i>^ΔHBE-YFP ESCs (red bars). The position in the locus of amplified regions 1–5 is shown in (E). An asterisk denotes significant differences between <i>Nodal</i>^condHBE-YFP and <i>Nodal</i>^ΔHBE-YFP ESCs (<i>p</i><0.01).</p

HBE-YFP expression in the blastocyst is dependent on Activin/Nodal signaling.

<p>(A–B) Detection of Oct4 (A, B) and HBE-YFP (A′, B′) in transgenic mouse blastocysts cultured either in DMSO (A–A″) or SB431542 (B–B″). Scale bar, 25 µm. Single confocal sections. Cortical actin in blue. (C) Percentage of YFP-positive embryos after 24 h culture in DMSO or in SB431542. (D) Percentage of YFP positive ICM nuclei in embryos after 24 h culture in DMSO or SB431542. An asterisk indicates significant difference from the control (ctrl) (<i>p</i><0.01). (E) Number of Oct4-positive ICM cells per embryo after 24 h culture in DMSO or in SB431542.</p