Comparative DNA Methylation Profiles of Somatic Cell Nuclear Transfer Embryos and In Vitro Fertilized Embryos

In the first days of embryo development, the paternal and maternal genomes are incorporated and reprocessed into the newly functional genome of the embryo itself. Epigenetic reprogramming of this new genome is integral to the proper development of the embryo. One epigenetic mechanism involved in reprogramming is DNA methylation in which a methyl group is added or removed from the DNA base cytosine. The functional form of DNA methylation occurs on a cytosine base when followed by a guanine base held together with a phosphate linkage (CpG). A high cluster of CpG sites is galled a CpG island and are found near the transcription start sites of genes. The presence of CpG methylation can inhibit gene transcription by adding to a crowded repressive chromatin state. The importance of classifying DNA methylation patterns in somatic cell nuclear transfer (SCNT) embryos can provide insight into the mechanisms in which a donor fibroblast is reconstructed and the ability to differentiate leading to further embryo development. Largely, SCNT embryos have poor success rates of live offspring compared to in vitro fertilization (IVF) embryos. Our data show differential success of pregnancies between SCNT embryos generated from different donor fibroblast lines. Our major goal of this study is to determine potential epigenetic memory or retained DNA methylation of donor fibroblasts in their respective SCNT embryos. We compared the DNA methylation profiles of developmentally important genes: NANOG, SOX2, LIN28, cMYC, KLF4, and OCT4 (POU5F1) in bovine SCNT and in vitro fertilized (IVF) embryos as well as 3 fibroblast cell lines used for SCNT. Fibroblast cell lines were selected based on pregnancy success with 2 lines showing high potential (WG and CT) and 1 showing low potential (KF). We used a high throughput bisulfate sequencing workflow to classify DNA methylation profiles of each gene region in 2-cell, 8-cell, morulae and blastocyst stage embryos as well as fibroblasts and oocyte samples. Our results showed differences in DNA methylation patterns on a per base resolution between SCNT embryos and IVF embryos across the 6 genes of interest. We also found differences between the 3 fibroblast cell lines in the NANOG and SOX2 gene regions. Specifically, our research showed 1) aberrant SOX2 promoter hypomethylation in KF SCNT embryos from the 2-cell stage up to blastocysts generated from low potential KF fibroblasts suggesting retained epigenetic memory or failure of de novo methylation and 2) Hypermethylation in 2-cell CT SCNT and significant hypomethylation in KF SCNT morulae in the NANOG gene region. Our data suggest potentially faulty DNA methylation of genes crucial to the development in pre-implantation embryos that could contribute to the poor success seen in select donor fibroblast cell lines used in SCNT.