It is well established that epigenetic modifications to the genome are crucial for the exquisite control of gene expression required for an organism to develop and differentiate. These modifications are maintained through mitotic rounds of cell division, but must be cleared and reset through meiosis in order for the cells of the early embryo to achieve totipotency. Although we know these mechanisms exist, the rules determining which modifications are established where on the genome and the genes involved in these processes remain poorly characterised. Much of what is known about epigenetic processes has come from studies in non-mammalian organisms, such as Drosophila. However, in our laboratory we have developed a mammalian system for identifying modifiers of epigenetic gene silencing. An ENU mutagenesis screen is being carried out using an inbred mouse line carrying a GFP transgene, with an erythroid-specific promoter, that is particularly sensitive to changes in epigenetic modifications. Currently, 14 mutant lines that display a heritable shift in GFP expression have been recovered. These have been termed Modifiers of Murine Metastable Epialleles (Mommes). When I began my PhD in 2005, we had not identified any of the mutations underlying the phenotypes observed. To confirm the efficacy of the screen, I have tested the effect of heterozygosity for null alleles of two known epigenetic modifiers, Dnmt3a and Dnmt3b, on expression of the GFP transgene. Heterozygosity for the Dnmt3b knockout allele does shift expression while heterozygosity for the Dnmt3a knockout allele does not. This highlights the limitations of the screen. With this particular screen we will only detect modifiers that are expressed during haematopoiesis in the bone marrow. I have also worked on MommeD5. MommeD5 is a semi-dominant, homozygous embryonic lethal mutation that acts as an enhancer of variegation. I have found that the MommeD5 allele carries a 7 bp deletion in the major histone deacetylase, Histone deacetylase 1 (Hdac1), and this significantly alters the C-terminus of the mutant protein. The finding of Hdac1 attests to the screen design. The MommeD5 homozygous mutants die at approximately the same time as the published knockout of Hdac1 and the heterozygous mutants show increased levels of Hdac2 and acetylated histone H3, as reported in Hdac1-deficient embryonic stem cells. In addition, I have studied the effect of heterozygosity for each of the mutations on the phenotype of the mouse. In general, heterozygous Momme mutants are viable and fertile, but show subtle abnormal phenotypes. However, in the case of MommeD5 none were observed and this may relate to the compensatory upregulation of other histone deacetylases. In the case of Dnmt3a and Dnmt3b a sex ratio distortion is seen in the colonies, with less males seen than expected. Also, Dnmt3a heterozygous mutant males that inherited the mutant allele from the dam are smaller and show an increased range of body weights compared to their wild-type male littermates. This may be an example of intangible variation, i.e. phenotypic variation observed in isogenic individuals raised in standardised environments. These results suggest that epigenetic mechanisms have a role in intangible variation, also known as developmental noise. Despite the fact that it is now acknowledged by many that stochastic events occur at the level of the cell, the idea that it can happen at the level of the whole organism is rarely considered.

Selfish genetic elements can distort the sex ratios of their hosts by increasing their own transmission to the next generation in a non-mendelian fashion. These elements can be either nuclear genes on a sex chromosome or cytoplasmically inherited microbes, and achieve an increased transmission by manipulating gametogenesis or host reproduction. Often these selfish elements benefit from a female biased population (for example heritable microbes are passed on maternally in the egg cytoplasm), while non-selfish, autosomal genes are selected to produce a balanced sex ratio. These differing reproductive strategies cause a genetic conflict that results in an “evolutionary arms race” that can promote the evolutionary change of sex determination systems. In this thesis, I investigate an extreme sex ratio distortion in a species of booklouse, Liposcelis sp. This species contains two distinct female types, one of which carries a maternally transmitted selfish genetic element that results in exclusively female offspring being produced. Recently, a candidate for the sex ratio distortion was identified as a horizontally transferred bacterial gene, that we have called Odile, and that is present in the genome of the (distorter) female carrying the distorting element. The gene originates from the endosymbiotic bacterium Wolbachia that is well known for its ability to distort the sex ratio of its hosts. I investigated this horizontal gene transfer event and attempt to characterize Odile. I provide evidence that this Wolbachia gene has been integrated into the genome of the distorter females and is not a bacterial contaminant. I found that the Odile gene has been duplicated and may have been horizontally transferred from Wolbachia independently to at least three other insect genomes. Additionally, I found that Odile is transcribed at low levels in a life-stage specific manner that is suggestive of a role in development. Additionally, I looked into male mate choice in this species as one aspect of the persistence of the distorting element. I found that male Liposcelis sp. do not discriminate between the two female types and do not spend more time mating with one female type over the other. These results contribute to ongoing research into the extreme sex ratio distortion found in this species and the candidate gene that may be the cause. Selfish genetic elements are an important driver of sex determination evolution, and Liposcelis sp. provides a unique and exciting system to investigate the implications of selfish elements in a genome further. / Graduate / 2019-12-17