Examination of DNA Methyltransferase Expression in Cloned Embryos Reveals an Essential Role for Dnmt1 in Bovine Development

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, USA.
Molecular Reproduction and Development (Impact Factor: 2.53). 05/2011; 78(5):306-17. DOI: 10.1002/mrd.21306
Source: PubMed


In studies of somatic cell nuclear transfer (SCNT), the ability of factors within the oocyte to epigenetically reprogram transferred nuclei is essential for embryonic development of the clone to proceed. However, irregular patterns of X-chromosome inactivation, abnormal expression of imprinted genes, and genomic DNA hypermethylation are frequently observed in reconstructed embryos, suggesting abnormalities in this process. To better understand the epigenetic events underlying SCNT reprogramming, we sought to determine if the abnormal DNA methylation levels observed in cloned embryos result from a failure of the oocyte to properly reprogram transcription versus differential biochemical regulation of the DNA methyltransferase family of enzymes (DNMTs) between embryonic and somatic nuclei. To address this question, we conducted real-time quantitation of Dnmt transcripts in bovine preimplantation embryos generated though in vitro fertilization (IVF), parthenogenic activation, and SCNT. By the 8-cell stage, transcripts encoding Dnmt1 become significantly down-regulated in cloned embryos, likely in response to the state of genomic hypermethylation, while the de novo methyltransferases maintain an expression pattern indistinguishable from their IVF and parthenote counterparts. Depletion of embryonic/maternal Dnmt1 transcripts within IVF embryos using short-interfering RNAs, while able to lower genomic DNA methylation levels, resulted in developmental arrest at the 8/16-cell stage. In contrast, SCNT embryos derived from a stable, Dnmt1-depleted donor cell line develop to blastocyst stage, but failed to carry to term. Our results indicate an essential role for Dnmt1 during bovine preimplantation development, and suggest proper transcriptional reprogramming of this gene family in SCNT embryos.

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Available from: Charles R Long, Apr 23, 2015
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    • "The epigenetic abnormality of cloned blastocysts relative to those produced by IVF was further indicated by a higher transcript level of DNMT1, which maintains established patterns of methylation during DNA replication, although that of DNMT3a, which participates in establishing the methylation pattern de novo during preimplantation, was not different among the three groups. Drastically reduced transcript levels of Dnmt1 have been reported in cloned than in IVF or parthenote bovine embryos by Golding et al. (2011), who suggested that the hypermethylated status of the genome in cloned embryos could be because of attenuation of transcription of Dnmt1 in clones as a result of high levels of CpG methylation within the donor genome. "
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    • "In early embryonic development, subsequent to fertilization, the embryo's DNA undergoes demethylation followed by processes of active and passive remethylation (Reik et al., 2001). Therefore, the zygote is in state of totipotency (Golding et al., 2011; Mason et al., 2012). Then, the process of de novo remethylation that begins at the 8-to 16-cell stage in the bovine embryo will act to drive the differentiation and development of the embryo (Reik et al., 2001). "
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