Histone-lysine N-methyltransferase SETDB1 is required for development of the bovine blastocyst.

Transcripts derived from select clades of transposable elements are among the first to appear in early mouse and human embryos, indicating transposable elements and the mechanisms that regulate their activity are fundamental to the establishment of the founding mammalian lineages. However, the mechanisms by which these parasitic sequences are involved in directing the developmental program are still poorly characterized. Transposable elements are regulated through epigenetic means, where combinatorial patterns of DNA methylation and histone 3 lysine 9 trimethylation (H3K9me3) suppress their transcription. From studies in rodents, SET domain bifurcated 1 (SETDB1) has emerged as the core methyltransferase responsible for marking transposable elements with H3K9me3 and temporally regulating their transcriptional activity. SETDB1 loss of function studies in mice reveal that although extraembryonic tissues do not require this methyltransferase, establishment of the embryo proper fails without it. As the bovine embryo initiates the processes of epigenetic programming earlier in the preimplantation phase, we sought to determine whether suppressing SETDB1 would block the formation of the inner cell mass. We report here that bovine SETDB1 transcripts are present throughout preimplantation development, and RNA interference-based depletion blocks embryo growth at the morula stage of development. Although we did not observe alterations in global histone methylation or transposable element transcription, we did observe increased global levels of H3K27 acetylation, an epigenetic mark associated with active enhancers. Our observations suggest that SETDB1 might interact with the epigenetic machinery controlling enhancer function and that suppression of this methyltransferase may disrupt the bovine developmental program.

Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development.

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.