The developing mammalian embryo is subjected to low O2 tensions, which owing to the short diffusion distance of O2 may form a gradient within the early embryo, and thus may function as a developmental morphogen. It was observed that culture of mouse embryonic stem cells (mESCs) in 1% O2, compared to atmospheric [O2], acted to skew differentiation. The cause of this effect was studied with respect to alterations in chromatin structure, via regulation of epigenetic modifications. The ten-eleven translocation (Tet) demethylase enzymes, Tet1/2/3, oxidise methylated DNA to form 5-hydroxymethylcytosine (5-hmC); a stable epigenetic mark that is implicated in developmental events. The mechanism(s) which underlie the regulation of their function(s) are unknown. However, intriguingly the catalytic activity of these enzymes is mediated via a conserved O2-dependent hydroxylase domain. Therefore, it was hypothesised that the Tet enzymes may be differentially regulated by [O2] to influence cellular specification and thus contribute to asymmetry within the developing embryo. Here it was identified that Tet1 is the most likely isoform to be inhibited by O2 tensions deemed physiologically relevant during embryogenesis. Further, differentiating mESCs displayed a transient, Tet1-mediated, O2-dependent burst of hydroxylation. This hydroxylation was predominantly targeted to a CG rich region of a Tet3 promoter, driving expression of a truncated protein isoform that lacks a CXXC DNA binding domain. This promoter region was shown to associate with Tet1, and also both the repressive (H3K27me3) and activating (H3K4me3) histone marks, characteristic of a bivalent promoter. Here it was also confirmed that 2 distinct Tet3 protein isoforms are expressed in differentiated mESCs, which were found to have differential transcriptional regulation and are thus likely to serve distinct cellular functions. It is suggested that Tet1 activity, in part determined by [O2] within the early embryo, may regulate Tet3 expression spatially to control cell fate decisions.
The Role of Molecular Oxygen in the Epigenetic Regulation of Cellular Differentiation
Burr, S. J. (Author). 2018
Student thesis: Doctoral Thesis › Doctor of Philosophy