Williamson, Gayle Linger (2011-08). Characterization and Analysis of the Bovine Epigenome during Preimplantation Embryo Development In Vitro. Master's Thesis. Thesis uri icon

abstract

  • During early mammalian embryogenesis, the embryonic genome undergoes critical reprogramming events that include changes in both DNA methylation and histone modifications necessary to control chromatin structure and thus, gene expression. Improper reprogramming of the epigenome during this window of development can lead to a vast number of imprinting anomalies, which are increased in children and livestock conceived in vitro. In the bovine, which closely resembles human preimplantation development, epigenetic changes occur from fertilization through the blastocyst stages. In particular, and concurrent with embryonic genome activation (EGA), de novo DNA methylation begins at the 8-cell stage. In order to explore the roles of histone-modifying enzymes during this crucial period of development, we characterized the transcript expression of several enzymes key enzymes across in vitro bovine preimplantation development using quantitative real-time PCR. Two of the 7 genes analyzed (Suz12 and Lsh) exhibited notable increases at the 8-16 cell stages, with basal levels observed both before and after this. These increases coincided with both EGA and de novo DNA methylation. We further explored their roles in bovine preimplantation embryos by knocking down expression via the use of gene-specific targeting siRNAs. Independent suppression of either Suz12 or Lsh via cytoplasmic microinjection of targeting siRNAs resulted in lower development rates (p < 0.0001), and poorer embryo quality of the morulas and blastocysts that survived. In addition, Suz12 suppression led to reductions in both H3K27 (p < 0.0001) and H3K9 (p = 0.07) trimethylation, and an increase in DNA methylation levels (p < 0.0001), as compared to the null-injected controls. Lsh suppression did not change H3K27, but led to a reduction in H3K9 trimethylation (p = 0.006) and an increase in DNA methylation (p < 0.0001). Clearly our data demonstrate that these epigenetic modifiers play a critical role in formation of the embryonic epigenome, but further research would be necessary in order to fully characterize gene activities during this developmental window.

publication date

  • August 2011