The histone code hypothesis: Studies of histone variants in mouse preimplantation embryos and extraembryonic tissues

The histone code forms part of a chromatin based epigenetic regulatory system. This system is important for setting up permissive and repressive chromatin environments which are critical for pluripotency and also for enabling the early differentiation events involved in embryonic lineage commitment. At the outset of this thesis, a complete pattern of expression for components of the histone code during critical stages of mouse preimplantation development was not known. In addition to this, the susceptibility of this system to in vitro handling procedures, which are becoming more widely used in both human and animal medicine, had not been well researched. This thesis has assessed the expression of genes coding for canonical histones H2A and H2B as well as all known histone variants, including H2A.Bbd, H2A.J, H2A.X, H2A.Y1, H2A.Y2, H2A.Z, Cenpa, H3.1, H3.2 and H3.3, specific chromatin remodelers including Swi/snf, Caf-1 and Hira. The expression of many factors known to help regulate chromatin based epigenetics including Hp1, Hdac1, Dmnt3b and Dnmt3l were also been assayed. These assays were conducted in in vivo, in vitro and parthenogenetic preimplantation embryos and were made possible following the optimization of an endogenous based reference gene approach for embryo qRT-PCR using Rplp0 as an endogenous housekeeping gene. In addition to these global assays of histone code expression patterns in the embryo, this thesis has put considerable research efforts into understanding the role of one histone variant, the H2A variant H2A.Z during preimplantation development. Assays of embryos generated in vivo showed that both the expression and protein levels of H2A.Z peaked at the blastocyst stage. Assays of in vitro embryos showed that this pattern could be disrupted by in vitro embryo manipulation. To further elucidate the role of H2A.Z in preimplantation development, H2A.Z’s expression and protein levels were assessed in a trophoblastic stem cell line. Results from the trophoblast cell studies, which included preliminary experiments into the effect that shRNA mediated knockdown of H2A.Z has on the differentiative potential of H2A.Z indicate that expression defects for this variant could contribute to trophoblast outgrowth failures commonly seen in mammalian embryos generated from in vitro based technologies. The results of this thesis provide a platform for future studies into how chromatin based epigenetic systems like the histone code are important in early mammalian development. This thesis also provides the basis for new hypotheses regarding the role of the H2A variant, H2A.Z in early lineage commitment of the extraembryonic tissues