The effect of somatic cell nuclear transfer on the stability and regulation of X chromosome inactivation and genomic imprinting

Somatic cell and early embryonic nuclei represent opposite ends of the differentiation spectrum. Somatic cells are severely limited in their potency while cells of the early embryo can give rise to all cell types: therefore it is surprising that the somatic nucleus can replace the egg nucleus in somatic cloning. Somehow the epigenetic modifications present on the somatic nucleus must be modified to an embryonic pattern during the cloning procedure. For this reason nuclear transfer embryos provide a tool to investigate the processes involved in epigenetic reprogramming during normal mammalian development. Recent studies have found that somatic cell nuclear transfer embryos exhibit extensive epigenetic abnormalities, including aberrant genomic methylation and abnormal gene expression. In this study, a thorough analysis of imprinted gene methylation and X chromosome inactivation was performed in nuclear transfer embryos. Cloned blastocysts exhibited a significant decrease in methylation at the imprinting control regions of two genes, H19 and Snurf/Snrpn. This result suggests that misregulation of imprinted genes through loss of methylation may contribute to the poor survival of cloned embryos. Reactivation of the somatic X chromosome was confirmed in these embryos; however, imprinted X-linked gene expression that is expected at this stage of development was not recapitulated. Even in later stage cloned placentae, which normally exhibit imprinted X inactivation, expression of X-linked genes was not monoallelic. To determine the extent of X chromosome inactivation disruption, Xist RNA FISH and Eed protein immunohistochemistry was performed on cloned blastocysts. Both analyses demonstrated that cells within cloned blastocysts have varying degrees of X inactivation. Additionally, a significant fraction of cells were found to have supernumerary X chromosomes. One process that was successful in the clones was recapitulation of the Xce effect in later stage embryos. This indicates that the Xce phenomenon is not affected by nuclear transfer and can be implemented by cloned embryos as development progresses. Overall, our results describe abnormalities in two critical epigenetic processes in cloned embryos. The dysregulation of these processes indicates the complexity of epigenetic changes that routinely occur during development and suggests that the low rate of clone survival may be due to epigenetic problems.