15 Identification of developmental genes regulated by H3K9me2 and H3K27me3 histone marks in bovine somatic cells and their somatic cell nuclear transfer embryos

Histone H3 lysine 9 dimethylation (H3K9me2) and Histone H3 lysine 27 trimethylation (H3K27me3) are common markers associated with repression of gene expression. Our aim was to evaluate the presence of H3K9me2 and H3K27me3 histone markers in the context of their association with five genes known to contribute to maintaining totipotency: POU5F1, NANOG, SOX2, KLF4, and cMYC, and a control gene (GAPDH) in 8-cell and blastocyst-stage embryos generated by IVF or somatic cell nuclear transfer (SCNT). Fertilisation was performed using our standard IVF protocol. The SCNT embryos were produced using three fibroblast cell lines, Whirligig (WG), Crushiertime (CT), and Kung Fu heifer (KF). These cell lines have been used extensively by our laboratory, and development to term based on total number of transfers averages ∼10% (WG), 2% (CT), and 0% (KF). All SCNT embryos were produced by enucleation of IVM oocytes, which were fused with a nuclear donor cell and subsequently activated in ionomycin followed by 6-(dimethylamino)purine (6-DMAP) and cycloheximide. The IVF and SCNT embryos were cultured in microdrops of synthetic oviductal fuid (SOF) medium at 38.5°C in 5% CO₂. After culture to the appropriate stage of development, chromatin was isolated from all samples using a rapid micro chromatin immuno-precipitation (μChIP) protocol developed by Dahl and Collas (2008 Nat. Protoc. 3, 1032-1045). A µChIP assay using antibodies specific for each histone modification was utilised, followed by real-time quantitative PCR (qPCR) analysis using Fluidigm (BioMark). The cycle threshold (ct) value was used to calculate the DNA amount in nanograms per microliter, which was compared with input chromatin DNA to estimate the percentage of each gene regulated by each histone marker. Our H3K9me2 results indicate the majority of the totipotent genes were roughly associated with the H3K9me2 mark in 100% of both 8-cell and blastocyst stages of IVF embryos, although NANOG amplification was absent in the blastocyst stage, and therefore not regulated by this histone mark. Interestingly, KF 8-cell embryos did not present H3K9me2 mark associated with the NANOG and POU5F1 genes and exhibited ∼60% of H3K9me2 mark in cMYC compared with the IVF control. The other two cell lines presented similar patterns compared with the IVF embryos. For H3K27me3, the mark was absent in NANOG and cMYC genes in IVF 8-cell embryos and in the KLF4 gene in IVF blastocysts. A notable difference from IVF embryo samples was the presence of H3K27me3 associated with NANOG in the 8-cell stage of WG, and the absence of this histone mark in POU5F at the 8-cell stage of KF SCNT embryos. Also, the H3K27me3 mark was not detected for SOX2 in CT 8-cell embryos, and all SCNT blastocysts presented gene amplification for KLF4. These differences are likely to affect embryo viability associated with proper reprogramming. We continue to collect additional data to confirm these results through robust statistical analysis.