Uncoupling Antisense-Mediated Silencing and DNA Methylation in the Imprinted Gnas Cluster
Figure 3
Nesp is expressed and truncated from the paternal allele in +/Nesp-Tint2.
(A) Loss of methylation at the Nesp DMR in +/Nesp-Tint2. (A Left) Schematic summary of the transcriptional status of Nespas and the methylation status of Nespas and Nesp on the paternal allele of wild-type and +/Nesp-Tint2. Dashed line indicates low level of transcript. Row of filled circles, methylated allele; row of open circles, unmethylated allele. (A Top Right) Southern analysis showing promoter methylation at the Nesp DMR is lost in +/Nesp-Tint2. Genomic DNA from newborn liver was digested with EcoRI (-), EcoRI and HpaII (H), or EcoRI and MspI (M). (A Bottom Right) Bisulphite profile of the paternal allele of the Nesp DMR in newborn brain from +SD2/+ and +SD2/Nesp-Tint2. Sequence variants allowed the maternal and paternal alleles to be discriminated; see Table S1 for primers. Each row of circles represented a clone derived from the paternal allele and each circle corresponded to a separate CpG (filled circles, methylated CpGs; open circles, unmethylated CpGs). Each block of circles represented the data from an individual mouse. (B) Low level of truncated Nesp from the paternal allele in +/Nesp-Tint2. (B Top Left) RNA blot analysis showing expression of Nesp in poly(A)+ RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C. The full length transcript was expressed from the maternal allele. (B Bottom Left)The smaller truncated transcript was expressed from the targeted paternal allele as summarised in the schematic. (B Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR, measured using a TaqMan assay that detects exon 1 spliced onto 2. The reference gene was Gapdh. Error bars (RQmin/RQmax) were based on a 95% confidence level.