The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals
Figure 6
Methylation Analysis of DLK1 Exon 5 and DIO3 Promoter in Wallaby and Platypus
(A) Hypermethylation was observed in both the wallaby and platypus DLK1 exon 5 regions. Wallaby genomic DNA from d23 RPY fetus (gDNA) and wallaby sperm gDNA was digested with XbaI (Xb), further digested with HpyCH4IV (Hy) and analysed by Southern blot hybridisation using MeDLK1Ex5 as a probe. Platypus gDNA was digested with StuI (St) and further with MspI (Ms), HpaII (Hp), and HhaI (Hh) and analysed by Southern blot hybridisation using OaDLK1Ex5 as a probe.
(B) A map depicting the HpaII and HhaI sites and methylation status in Dlk1 exon 5. Black circles indicate methylated sites, white circles unmethylated sites, and half black circles indicate partial methylation. CpG islands in the region are shown as grey boxes.
(C) The Dio3 promoter region is unmethylated in both wallaby and platypus. Wallaby fetal head gDNA was digested with HindIII and further with MspI (Ms), HpaII (Hp), and HhaI (Hh) and hydridised with MeDIO3CpG. The methylation-sensitive HpaII and HhaI tracks exhibited full digestion indicating the region is unmethylated. Platypus gDNA was digested with XbaI (Xb) then with MspI (Ms), HpaII (Hp), HhaI (Hh), or SmaI (Sm) and hydridised with OaDIO3CpG. High CG content results in many HpaII and HhaI fragments, which are unmethylated and too small to be resolved on this filter. The smallest SmaI site expected was identified, showing the platypus Dio3 promoter is unmethylated. Control hybridisation with a OaDIO3 promoter proximal probe identified a fully methylated XbaI fragment of >3 kb in the HpaII, HhaI, and SmaI tracks, confirming the integrity of the genomic DNA in these tracks (unpublished data).