Conserved genomic organization of mouse and human Ddc/DDC. The mouse Ddc genomic region was used as the base sequence for construction of a VISTA plot on the human genome. For simplicity, only the exon1-exon2 interval is shown. A 75% sequence similarity threshold was applied and is shown on the plot by a thin bar. Conserved coding exons are purple, conserved noncoding exons are light blue, and conserved noncoding nonexonic sequences are pink.

Progressive silencing of Ddc_exon1a during mouse postnatal development. (A) Northern blot analysis of mouse Ddc expression in total RNA extracted from e13.5 embryo, newborn heart, 4-week heart, and 3-month heart, showing a 2-kb transcript. (B) RT-PCR analysis of Ddc_exon1a expression in newborn heart and 3-month heart cDNAs using the primers EXON1A-F and EXON6-R (see Materials and Methods), which amplify a 786-bp product. The 500-bp marker is indicated for a reference (arrows). RNA integrity was verified by amplification of β-actin sequences from each sample, and samples treated with (+) and without (-) reverse transcriptase are indicated.

UpDp proximal chromosome 11 microarray tissue expression profiles. (A) Ddc expression levels in T65H proximal MatDp11 versus PatDp11 mouse tissues as quantified using an Affymetrix U74v2 array probe set 160074_at (newborn heart, liver, carcass, and e13.5 placenta) and 430v2 array probe set 1426215_at (newborn brain, e13.5 embryo). Both probe sets are complementary to the same target sequence in exon 15. UpDp tissue expression profiles recovered for the two known imprinted genes Grb10 (B) and U2af1-rs1 (C) contained within the same UpDp region are shown for comparison. Where present, asterisks indicate statistically significant differential expression (GCOS change P ≤ 0.003) between MatDp11 and PatDp11 tissues.

Immunohistochemical localization of Ddc expression during mouse heart development. Sites of positive Ddc immunoreactivity are marked by the brown signals. The hematoxylin counterstain appears as blue nuclear staining. (A) Parasagittal view of the e10.5 heart with positive Ddc staining in a small number of cardiomyocytes. Magnification, ×200. (B) Higher-magnification view of the e10.5 heart. Magnification, ×400. (C) Parasagittal view of the e14.5 heart, showing widespread Ddc staining in scattered trabecular cardiomyocytes. Magnification, ×200. (D) Higher-magnification view of the e14.5 heart. Magnification, ×400. (E) Parasagittal view of the newborn heart, showing persistent Ddc staining in trabecular cardiomyocytes. Magnification, ×10. (F) Higher-magnification view of the newborn heart. Magnification, ×400. (G) Low-magnification view of the 3-month heart showing an absence of Ddc staining. Magnification, ×10. (H) Higher-magnification view of the 3-month heart. Magnification, ×400. (I and J) Other sites of Ddc expression in e14.5 embryos: adrenal gland with Ddc staining in the secretory adrenomedullary cells (I) and Ddc expression in differentiating pancreatic acinar cells (J). Magnification (both panels), ×200. Control sections incubated with anti-rabbit immunoglobulin G secondary antibody alone did not produce signals (not shown).

Dnmt3L-dependent regulation of Ddc expression. (A) Relative gene expression levels were compared in Dnmt3L^mat/+ mutant e8.5 embryos and wild-type controls using the Affymetrix mouse 430v2 GeneChip system. Hybridization intensity signals are shown for Ddc, Grb10, and the nonimprinted control gene Fignl1. Where present, asterisks indicate statistically significant differential expression (GCOS change P ≤ 0.0003 or ≥ 0.997) between Dnmt3L^mat−/+ embryos and wild-type controls. (B) qPCR shows overexpression of Ddc in e8.5 maternal imprint-free embryos (Dnmt3L) relative to wild-type (WT) controls. For each genotype, expression of Ddc is shown as a ratio of the mean Ct value in relation to the mean Ct value for β-actin. Ct values were transformed as described elsewhere (53), and the relative Ddc/β-actin expression ratio in WT embryos was set to 1. n = 3 for each sample type. The 95% confidence intervals are shown.

Tissue- and transcript-specific imprinting of mouse Ddc. (A) Map of the ∼84-kb genomic region containing the Ddc locus. Coding exons are shown as open boxes, noncoding exons are shown as filled boxes, and open triangles indicate the positions of primers used in allele-specific RT-PCR assays. The location of a G→A SNP in exon 6 between the Mus mus musculus (B6) and Mus mus castaneus (CAST) subspecies is shown by a vertical arrow. (B) Deduced structures of the Ddc_exon1a transcript variant and the related Ddc transcript in their respective genomic contexts. (C) Exon-specific RT-PCR in newborn mouse tissues. The primers EXON1A-F and EXON6-R amplify the Ddc_ exon1a transcript (786-bp product); EXON1-F and EXON6-R amplify the related Ddc transcript (762-bp product). The 500-bp marker is indicated for a reference (arrows). (D) Allele-specific RT-PCR assays in newborn mouse tissues. cDNA fragments were recovered from reciprocal B6 × CAST intersubspecies hybrid tissues by RT-PCR and directly sequenced using the exon-specific primer combinations described above. The G→A SNP (arrows) in exon 6 was used to infer the parental origin of the expressed allele.

DNA methylation analysis of the Ddc_exon1a promoter region. (A) Schematic of the Ddc/Grb10 genomic interval, showing relevant sequence features. Genes are shown as open boxes, CpG islands are shown as shaded boxes, and the CpG island allelic methylation status (where known) is indicated by solid (methylated) or open (unmethylated) circles. The position of the maternally methylated Grb10 CGI2 region (gDMR) is shown (5). (B) Enlarged view of the ∼400-bp Ddc_exon1a promoter region. Relevant CpG dinucleotides (numbered 1 to 5) are shown as filled lollipops. A C→A SNP between B6 and CAST strains was used to infer parental origin of the strands (vertical arrow). (C) Bisulfite DNA sequencing profiles of the Ddc_exon1a promoter region in newborn heart, newborn brain, and adult heart tissues derived from reciprocal B6 (B) × CAST (C) intersubspecies hybrids with the maternal (Mat) and paternal (Pat) origin of the strands indicated. Methylated and unmethylated CpGs are represented as solid and open circles, respectively.