AbstractHistones are important regulators of both chromatin structure and gene regulation. There are many subtypes of linker histones and, in contrast to core histones, they are very variable within and between species. Due to this, many linker histones have not been analysed thoroughly in all species. H1X, also known as H1.10, is the most recently identified linker histone variant.
The research presented in this thesis was focused on analysing the expression and function of H1X during embryonic development, using the vertebrate model organisms Xenopus laevis and Xenopus tropicalis.
The phylogenetic relationship of H1X between species was established by sequence analysis, revealing that h1fx genes were orthologs in many vertebrate species. A second h1fx gene, provisionally denoted h1fx.2, was found tandemly located only in anuran amphibians and coelacanths.
Temporal investigation of the expression of H1X.1 protein was performed by extraction of proteins from embryos at stages across early embryonic development and western blot using anti-H1X antibody. It showed that in X. laevis, H1X was expressed in all stages including the midblastula transition, indicating maternal stores of H1X in the oocyte. Analysis of h1fx.1 and h1fx.2 messenger RNA showed differences in expression in and between Xenopus species; most pronounced in X. laevis, where h1fx.2 mRNA was detected in low levels in only certain stages, compared to uniform h1fx.1 mRNA detection.
An RNA probe recognising X. tropicalis h1fx.1 was used for wholemount in situ hybridisation to determine the spatial expression of h1fx.1. The mRNA was detected in all stages probed, but restricted to the anterior tissues of the embryo, such as the head, during tailbud stages of embryonic development.
Gene editing by CRISPR-Cas9 to knockout the h1fx.1 gene successfully generated mosaic X. tropicalis embryos as shown by sequencing, with survival to maturity briefly shown.
Lastly, crosslinked chromatin immunoprecipitation (ChIP) followed by sequencing of precipitated DNA was carried out using early and late Xenopus chromatin and anti-H1X affinity antibody, in the first ChIP-Seq investigating H1X in non-mammalian cells. Peaks of H1X enrichment were seen in many genomic regions, and genes identified in both early and late X. laevis samples included genes encoding histones such as H2A.X and H2B, as well as multiple tRNA genes.
This research contributes to the understanding of the expression and function of linker histone subtypes. A previously undiscovered isoform of H1X has been characterised, the importance of H1X for survival and possibly growth has been shown and initial data about the location of H1X on the genome have been collected.
|Date of Award||Sep 2019|
|Supervisor||Fiona Myers (Supervisor), Matt Guille (Supervisor) & Frank Schubert (Supervisor)|