A shift of the hypomethylated region from 5′upstream to 3′downstream in adulthood. (A) IGB images of the 3 genes with 5′-upstream region E14Hypo-T-DMRs. Comparative MATscores of E14.5NSph and the AdBr to E11.5NSph as the control were plotted and displayed from 6 kb upstream to 4 kb (Ntng1) or 2.5 kb downstream of the TSS. Open arrowheads indicate hypomethylated T-DMRs. Regions analyzed by bisulfite sequencing (C) or COBRA are indicated by gray rectangles. (B) Relative positions of hypomethylated T-DMRs to TSS in NSphs (upper panel) and the adult brain (lower panel) are illustrated. (C) DNA methylation status of Ntng1 in E11.5NSph, E14.5NSph, and the AdBr was analyzed by bisulfite sequencing. Each open and filled circle represents unmethylated and methylated CpG, respectively. Percentages of methylated CpGs are indicated. (D) Expression of Ntng1 was analyzed by Q-RT-PCR using total RNA from undifferentiated (1, 2) and differentiated NSph (2d), E14.5 telencephalon (E14.5Br), and the AdBr. The levels of expression, which were normalized with those of internal control (Actb), are indicated by the ratios to the average of AdBr. Error bars indicate SE of the triplicated PCR.