| Epigenetic modifications, such as methylation are essential for normal mammalian development. DNMT1 is the predominant and most active methylase, which is responsible for copying methylation patterns after replication. During early development, DNMT1 is highly regulated to support passive demethylation and normal reprogramming. In somatic cells, DNMT1 is localized in the nucleus and its introduction into cloned embryos is believed to be the main limiting factor for proper reprogramming of the donor genome. The aim of this study was to transiently suppress DNMT1 by RNA interference (RNAi) to investigate the role of DNMT1 during early development of embryos produced by somatic cell nuclear transfer (SCNT). RNA and protein extracts were collected from fibroblasts transfected with control or DNMT1 RNAi. Real-time PCR revealed a 93.4% reduction of DNMT1 transcript levels. Consistently, Western blot analysis showed a protein decrease of up to 98.8%. Subtle evidence of DNA demethylation after DNMT1 inhibition was consistently observed by MSP bisulfite sequencing and immunodectection. Real-time PCR and microarray analysis indicated that the expression of interferon response genes, IFITM3 and EIF2AK2 (PKR), and housekeeping genes, GAPDH, ACTB and lamin A/C, were not affected in fibroblasts after RNAi treatment however, expression of ACP5, MMP1, NDN and CCL2 (SCYA2) was increased. DNMT1 knockdown fibroblasts were used as donor cells for the production of SCNT embryos and developmental rates were compared with that of control SCNT embryos, no significant differences were observed between the developmental rates of both groups. Cell number of SCNT-derived blastocysts was significantly lower after bNMT1 inhibition. SCNT embryos overexpressed UBE3A, SLC16A1 and ACP5 compared to IVF embryos, however DNMT1 inhibition in SCNT embryos restored gene expression to normal levels observed in IVF embryos. This report demonstrates a specific and highly effective knockdown of bovine DNMT1 by RNAi at both mRNA and protein levels in fibroblasts, in which the majority of global DNA methylation is maintained and a small group of genes were consistently differentially expressed. Gene expression analysis on embryos indicated that DNMT1 RNAi is an useful tool to regulate gene expression in SCNT embryos and to better understand epigenetic reprogramming events. |
