Abstract
BackgroundCloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI).ResultsMost of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1) showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns.ConclusionThese results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a good predictor of whether the foetus would develop to term or not.
Highlights
Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development
The phenotypes commonly observed in SCNT foetuses bear many similarities to some of those seen in experimentally-created imprinting disruptions in mice, or to naturally-occurring human syndromes, such as Beckwith-Wiedemann syndrome (BWS) [9,10,11,12,13]
We have used the MassARRAY technology to look at multiple regions in the genome and found that for SCNT foetuses that survived to mid-gestation, albeit with phenotypic abnormalities in some cases, the methylation patterns were very similar to those of naturally conceived foetuses, at least for the three organs examined
Summary
Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. It is thought that a substantial part of this gene regulation is mediated through epigenetic modifications such as DNA methylation, histone tail modifications and the binding of non-histone proteins to chromatin [17,18,19] so that each somatic cell in the organism has its own epigenetic signature (epigenome) which reflects its genotype, developmental history and environmental influences, which determines the phenotype of the cell and the organism This is clearly illustrated in the events following fertilization, where the majority of the genome undergoes active paternal demethylation, passive maternal demethylation. How the developmental programs are coordinated and orchestrated from the genomic blueprint is still poorly understood, even in normal development
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