DNA Methylation Status Of Meg8 Gene And IG-DMR Within Dlk1-Dio3 Imprinted Domain In Cloned Bovines

Somatic cell nuclear transfer(SCNT)has been successfully applied to many mammalian species, but the low efficiency of SCNT and organ abnormalities restricts it’s application. The incomplete reprogramming of donor cell nuclei leading to aberrant expression or lack of expression of some developmentally important genes has been implicated as a primary reason for the low efficiency of SCNT. Genomic imprinting is an epigenetic phenomenon depending on asymmetric epigenetic modifications. DNA methylation is a major epigenetic modification of the genome that regulates some crucial aspects of its function. There are differentially cattle methylated regions within imprinted genes, genomic imprinting and DNA methylation is the important content in study of reprogramming.Although Dlk1-Dio3 imprinted domain play an important role in embryo development and organogenesis, few studies about the imprinted domain in cattle were reported largely because of the lack of information on sequence and polymorphisms of Dlk1-Dio3 imprinted region In this study, we analyzed allele specific DNA methylation status of Meg8 and IG-DMR in the imprinted domain, These results are essential to analyze the molecular mechanism of this imprinted domain and to increase our understanding on nuclear reprogramming events.Meg8 gene is identified as a maternally expressed no coding RNA gene located in Dlk1-Dio3 imprinted domain.The analysis of Meg8 sequence revealed that no significant CpG island in 5’end of Meg8 while there is significant CpG island within intron 3. In order to identify the role of gene internal CpG island in the regulation of imprinting effect we analyzed allele specific DNA methylation status of Meg8 intron 3, The results demonstrated that C-strand showed significantly lower DNA methylation of 17 CpG in Meg8 intron 3 than T-strand, and Meg8 intron 3 exhibited hypermethylation in cloned bovines, which was similar to controls. However, the methylation status of Meg8 intron 3 in clones was significantly different from controls, and all exhibiting same complete methylation pattern. The expression of Meg8 gene in cloned bovines showed monoallelic expression, speculated that the methylation of Meg8 intron3 CpG island didn’t play a role in regulating of Meg8 imprinting expression.IG-DMR is identified as ICR, which located between Dlk1 and Gtl2 genes in Dlk1-Dio3 imprinted domain. In order to identify the role of IG-DMR in regulating imprinting of Dlk1-Dio3 imprinted domain, we analyzed the DNA methylation status of the 9 CpG sites within IG-DMR. The result showed that C-strand and G-strand both exhibited hypermethylation in normal bovine. But in clones, G-strand showed significantly lower methylation level than C-strand. The methylation level of clones was significantly lower than that of control group. The result suggested that DNA methylation reprogramming of studied domain was abnormal in clones.