Investigating the coordinated transcriptional networks regulating Xenopus myogenesis

Myogenesis is the process by which mesodermal progenitor cells are committed to a myogenic fate and differentiate to form myofibers via a complex transcriptional network largely controlled by Myogenic Regulatory Factors (MRFs). One such MRF, MyoD, has been extensively investigated and its regulatory network shown to include not only protein-coding mRNAs, but non-coding RNAs and epigenetic modifications too, leading to highly coordinated transcriptional activation of target genes. The advances in high-throughput and small-RNA sequencing have revealed that transcriptional output by RNA Polymerase III, like Polymerase II, is differentially regulated between tissues and during development. This indicates that developmental transcriptional networks are potentially more complex than previously thought, involving cross-talk between multiple polymerases. Polr3G is a subunit unique to RNA Polymerase III, is associated with maintaining pluripotency and proliferation in hESCs and exists in 2 distinct forms in mammals and in Xenopus. This thesis presents the distinct expression profiles of Polr3G and its paralog Polr3gL during Xenopus tropicalis development, the specific expression of Polr3G in the skeletal muscle lineage and investigation into its regulation by myogenic factors. Custom tRNA Xenopus microarrays show the regulation of tRNAs by Polr3G at the earliest point of embryonic transcriptional activation, and the differential regulation of tRNAs in the muscle lineage in response to changes in expression of Polr3G and Polr3gL and expression changes to RNA Polymerase III targets and transcriptional machinery in animal caps undergoing myogenic differentiation. This thesis also presents finding from an RNA-seq study analysing founder (F0) embryos where MyoD was disrupted using CRISPR/Cas9 methods to determine early gene targets of MyoD. Together, this thesis proposes that a conserved downregulation of RNA Polymerase III activity during differentiation is shared between this model and previous cell culture studies but that the novel factors, Polr3G and Polr3gL, may be implicated in its mediation.