Molecular structure of bovine Gtl2 gene and DNA methylation status of Dlk1-Gtl2 imprinted domain in cloned bovines

Abstract

Somatic cell nuclear transfer (SCNT) is an inefficient process, which is due to incomplete reprogramming of the donor nucleus. DNA methylation of imprinted genes is essential to the reprogramming of the somatic cell nucleus in SCNT. Dlk1- Gtl2 imprinted domain has been widely studied in mouse and human. However, little is known in bovine, possibly because of limited appropriate sequences of bovine. In our study, we first isolated the cDNA sequence and found multiple transcript variants occurred in bovine Gtl2 gene, which was conserved among species. A probably 110-kb-long Dlk1- Gtl2 imprinted domain was detected on bovine chromosome 21. We identified the putative Gtl2 DMR and IG-DMR corresponding to the mouse and human DMRs and assessed the methylation status of the two DMRs and Dlk1 5′ promoter in lungs of deceased SCNT bovines that died within 48 h after birth and the normal controls. In cloned bovines, Gtl2 DMR exhibited hypermethylation, which was similar to controls. However, the methylation status of IG-DMR and Dlk1 5′ promoter in clones was significantly different from controls, with severe loss of methylation in IG-DMR and hypermethylation in the Dlk1 5′ promoter region. Our data suggested that abnormal methylation patterns of IG-DMR may lead to the abnormal expression of Gtl2 and Dlk1 5′ hypermethylated promoter is associated with the aberrant development of lungs of cloned bovines, which consequently may contribute to the low efficiency of SCNT.