Analysis of the Histone Methyltransferase ASH2L via RNA Interference and CRISPR-Cas9 during Bovine Early Embryonic Development
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Epigenetic patterns established during early bovine embryogenesis via DNA methylation and histone modification patterns are essential for proper gene expression and embryonic development. Epigenome patterns established during this period, if improperly maintained, can lead to developmental anomalies and may partially explain the lower pregnancy rates of in vitro-produced embryos. We hypothesized that the histone methyltransferase of ASH2L would alter preimplantation development, epigenetic reprogramming, and gene expression profiles in the early bovine embryo. We observed that the depleted and deleted ASH2L embryos developed to the blastocyst stage with suppressed ASH2L having comparable development rates with its respective control (31.3 ± 2.0%, n = 466 v. 34.8 ± 1.9%, n = 418). To see if errors were in the chromatin structure at the blastocyst stage, DNA methylation and histone modifications were examined to further explain the role of ASH2L during embryonic development. Blastocysts from each treatment (N = 601) were fixed and prepared for immunocytochemistry following standard laboratory protocol. Our findings show ASH2L may play a role in DNA methylation by decreasing 5mc and 5hmc conversion, which is a key event during early embryonic development. Suppression of ASH2L also alters global levels of H3H4me3 and H3K27me3 (p<0.001), which may lead to transcription aberrations. RNA-seq showed altered gene expression profiles in 407 genes in the morphologically comparable Day 17 conceptus (p<0.05). Closer examination showed that there is altered mesenchymal stem cell differentiation present in the Day 17 conceptuses. Analysis of ASH2L shows to not have a detrimental effect during preimplantation development but altered chromatin status and gene expression profiles suggest that ASH2L could play a vital role later in development.
Snyder, Matthew D (2016). Analysis of the Histone Methyltransferase ASH2L via RNA Interference and CRISPR-Cas9 during Bovine Early Embryonic Development. Master's thesis, Texas A & M University. Available electronically from