Regulation of Lineage Specific DNA Hypomethylation in Mouse Trophectoderm
Figure 5
Overexpression of the Np95 gene fails to restore somatic levels of DNA methylation in trophoblast differentiation.
(A) Immunostaining analysis of ZHBTc4-derived trophoblast cells (+Dx), Np95-KO ES cells, ZHBTc4 ES cells overexpressing exogenous Np95 (+MycNp95 ES) and ZHBTc4-derived trophoblast cells overexpressing exogenous Np95 (+MycNp95+Dx) using antibodies against Dnmt1, Np95 or Myc. DNA and replication sites were visualized with DAPI and EdU respectively. Scale bar, 10 µm. (B) mRNA expression of Np95, Oct3/4, Rhox6 and Plate 1 genes during ZHBTc4 trophoblast differentiation with (+Np95) or without (ZH) overexpression of exogenous Np95. A representative clone for Np95-overexpressing ZHBTc4 cells is shown in the figure. Values are means ± SD of technical replicates (n=3). Other independent clones also showed similar results for marker gene and Np95 expression. (C) Dnmt1 localization at replication site in Np95-overexpressing ZHBTc4-derived trophoblast cells at day 4 after differentiation. The control data in ZHBTc4-derived trophoblast cells (ZH+Dx) is identical to ZHBTc4+Dx of Figure 4C. Values are means ± SD of biological replicates (n=3). ***: p<0.001, **: p<0.01, ns: not significant; ANOVA and Bonferroni’s multiple comparison test. (D) DNA methylation analysis by Sequenom in ZHBTc4 ES (Day0) and ZHBTc4-derived trophoblast cells (Day4+Dx) with (+Np95) or without (ZH) overexpression of exogenous Np95. The control data (+emp) is identical to the data of +emp in Figure 3B. Values are means ± SD of biological replicates (n=3-5). ***: p<0.001, **: p<0.01; paired t-tests.