FIGURE 4.

H3K27me3 is specifically reduced during ME initiation. A, H1 cells were treated with 25 ng/ml AW in B27 medium for the indicated times before they were harvested for histone extraction, followed by immunoblotting with the indicated antibodies. B and C, H1 cells were treated with FLyA (20 ng/ml bFGF, 10 μm Ly294002, and 20 ng/ml activin A) for the indicated times before they were harvested for qPCR (B) or for histone extraction followed anti-H3K27me3 immunoblotting (C). D and E, H1 cells treated with BF (5 ng/ml BMP4 and 40 ng/ml bFGF) for the indicated times before they were harvested for qPCR (D) or for histone extraction and anti-H3K27me3 immunoblotting (E). F and G, H1 cells treated with 5 ng/ml BMP4 in B27 medium for the indicated times before they were harvested for qPCR (F) or for histone extraction and then anti-H3K27me3 immunoblotting (G). H, IGV showed H3K27me3 enrichment at 2 and 6 h on the specific trophoblast genes CGA and GCM1, as revealed by H3K27me3 ChIP-seq upon AW treatment. I and J, H1 cells were treated with 10 μm SB431542 and 100 ng/ml Noggin in B27 medium for the indicated times before they were harvested for qPCR (I) or for histone extraction and then anti-H3K27me3 immunoblotting (J). K, IGV showed H3K27me3 enrichment at 2 and 6 h on the specific neuroectoderm genes OTX2 and SOX1, as revealed by H3K27me3 ChIP-seq upon AW treatment. H3K27me3 bands were quantified, and the relative levels were shown after normalization to histone H3, and the statistical data are shown as mean ± S.E. (error bars) (n = 9, including 3 biological replicates and 3 technical replicates for A, C, E, G, and J). A Friedman test was performed in A, C, E, G, and J.