title: Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development.
abstract: 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.
authors.affiliation: Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, USA. mgolding@cvm.tamu.edu
authors.forename: Michael C
authors.initials: MC
authors.lastname: Golding
authors.forename: Gayle L
authors.initials: GL
authors.lastname: Williamson
authors.forename: Todd K
authors.initials: TK
authors.lastname: Stroud
authors.forename: Mark E
authors.initials: ME
authors.lastname: Westhusin
authors.forename: Charles R
authors.initials: CR
authors.lastname: Long
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology; D002417:Cattle; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000378:metabolism*; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D053652:Nuclear Transfer Techniques; D010312:Parthenogenesis; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D034741:RNA, Small Interfering; D004248:DNA (Cytosine-5-)-Methyltransferases