Figure 1.
Schematic for the imprinting mechanism of IGF2 and KCNQ1.
In (A), the rectangle represents each gene, and the black circle is methylated DNA. In (B), the green arrow in the lower panel represents lncRNAs KCNQ1OT1, and the hexagon represents KvDMR1 that is methylated or not.
Figure 2.
Enhanced mRNA levels of IGF2 in the spinal cord and brains of human fetuses with NTDs.
(A) In the spinal cord and (B) brains of human fetuses with NTDs, IGF2 mRNA levels were dramatically upregulated. *: P<0.05; **: P<0.01; ***: P<0.0001 (Student's t-test).
Figure 3.
Hypermethylated H19 DMR1 and stably methylated IGF2 DMR0 in human fetuses with NTDs.
The DNA methylation levels of the H19 DMR1 (A) and IGF2 DMR0 (B) were examined through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In the upper panel in (A) and right panel in (B), the methylation status of all detected CpG sites was visualized. Each solid circle represents a “CG” site. (A)The number in upper panel refers to the CG site(s) in the lower histogram. (B) The number in the left panel refers to the site in the right histogram. ***: P<0.0001 (Student's t-test).
Figure 4.
Chromatin status in the promoter region of the IGF2 gene is negatively associated with gene expression.
(A) and (D): The genomic profile of genes displayed in UCSC (hg19), the genomic locus of target regions in ChIP assay (black block on the gene schematic) and the CpG island (green block underneath the gene schematic) assayed by the MASSARRAY platform are shown. The capital letter beside the block indicates the corresponding panel below. The number beside the block indicates the genomic location that was targeted. In the ChIP assay results (B and E), the enrichment in the y-axis represents the relative enrichment fold in which the higher enrichment in case or in control is designated as 1. C) and F) indicate the DNA methylation level. Notably, in F, the DNA methylation status of KvDMR1 is shown.*: P<0.05; **: P<0.01 (Student's t-test). TSS: transcription start site.
Figure 5.
Wide ectopic mRNA levels of imprinted genes in mouse fetuses with RA-induced spina bifida.
(A and B) The real-time qPCR assay results indicate the fold change in imprinted genes on each chromosome in the spinal cord tissue of E18 RA-induced mice with NTDs compared with control. The x-axis represents the chromosome and the relative genomic position of each gene. The y-axis represents the relative mRNA abundance in NTDs compared with the control. The red column indicates a maternally expressed gene, while the blue column indicates a paternally expressed gene. *: P<0.05; **: P<0.01; ***: P<0.0001 (Student's t-test).
Figure 6.
More open chromatin structure in mice fetuses with RA-induced spina bifida.
(A) The methylation level of H19 DMR1 is stable in RA-treated mice. For details, see the legend in Fig. 3. (B) and (F): The genomic profile of genes displayed in UCSC (mm9), the genomic locus of target regions in ChIP assay (black block in the gene schematic) (corresponding results shown in C and D or G and H), and the CpG island (green block underneath the gene schematic) (corresponding results shown in E and I) detected by MASSARRAY assay are shown. The number beside the block indicates the genomic location that was targeted. In the ChIP assay results (C and D or G and H), the enrichment in the y-axis represents the relative fold enrichment in which the higher enrichment in case or control is designated as 1. *: P<0.05; **: P<0.01; ***: P<0.0001 (Student's t-test). TSS: transcription start site.