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Half of the mammalian genome is made up of transposable elements (TEs), the vast majority of which are modified by DNA methylation and repressive histone marks. Intracisternal A-particles (IAPs) are evolutionarily young murine-specific endogenous retroviruses (ERVs). Some are capable of retrotransposition and many harbour regulatory sequences with the potential to influence host gene expression. Previous work has shown that, at the Agouti Viable Yellow (Avy) locus, an IAP insertion provides an alternative promoter for the Agouti coat colour gene. Variable methylation of this IAP is correlated with coat colour expressivity. In addition, the Avy allele exhibits transgenerational epigenetic inheritance, susceptibility to environmental exposures, and strain-specific modulation. A recent screen conducted in the C57BL/6J mouse strain identified a subset of IAPs that show variable methylation levels across genetically identical individuals. This thesis characterises this novel repertoire of variably methylated IAPs (VM-IAPs) and in doing so assesses the extent to which endogenous retroviruses in the mammalian genome display Avy-like properties. Findings indicate that VM-IAPs become hypermethylated in the male germline by the DNA methyltransferase DNMT3C and are reconstructed as variable loci in the next generation. An exploration of inter-individual VM-IAP methylation levels in the C57BL/6J population demonstrates that their frequency distributions form skewed bell curves that are locus-specific. Only one out of six VM-IAPs analysed via linear mixed effects models shows memory of maternal methylation level, and the effect size is small. This challenges the generalisability of epigenetic inheritance at these regions. In studying environmental influences on VM-IAPs, results suggest that these epigenetically dynamic loci are selectively susceptible to altered environmental conditions: abnormal folate metabolism appears to shift VM-IAP methylation levels and influence VM-IAP-associated gene expression, while no significant effects are observed following exposure to the endocrine-disruptor Bisphenol-A (BPA) or to an obesogenic diet. A longitudinal ageing analysis indicates that VM-IAPs are stable across the murine lifespan, with only modest increases in methylation detected for a subset of loci. Reciprocal hybrid breeding experiments reveal that VM-IAP methylation levels are subject to maternal and genetic background effects that can be harnessed to map strain-specific VM IAP modifiers. Finally, a naturally occurring genetically-conferred epiallele that influences neighbouring gene expression is identified and characterised. This body of work highlights the value of using VM-IAPs as a model to study TE regulation and mammalian epigenetic stochasticity, and serves as a foundation to elucidate the consequences of partially methylated repeat elements on genome structure and function.

Exposure to environmental stressors can impact our health and that of future generations even when they are not similarly exposed. How disease risk is inherited is unclear. My thesis focuses on a mouse model of transgenerational inheritance in which folate metabolism is disrupted by a mutation in Methionine synthase reductase (Mtrr^gt). Remarkably, Mtrr⁺/gt heterozygosity leads to an increased likelihood of a wide spectrum of congenital malformations in their wildtype offspring for at least four generations. Folate metabolism is required for DNA synthesis and cellular methylation. Folate and its metabolism have also been linked to spermatogenesis and male fertility. My thesis aims to explore three possible mechanisms for the transgenerational inheritance of congenital malformations in the Mtrr^gt model: germ cell morphology and function abnormalities, genetic instability and altered germ cell epigenetic patterns. We first considered if the Mtrr^gt mutation affected testes morphology, spermatogenesis or sperm parameters. These were largely normal in Mtrr^gt males. Next, we performed whole genome DNA sequencing of Mtrr embryos to determine whether abnormal folate metabolism affects genetic stability. Importantly, the frequency of structural variants and single nucleotide polymorphisms (SNPs) were similar in C57Bl/6 control and Mtrr^gt/gt embryos indicating that the Mtrr^gt mutant genome is relatively stability. We did however identify an increase in SNP and SV frequency at the Mtrr locus, linked to the generation of the Mtrr^gt mice in a 129/P2 genetic background prior to backcrossing into the C57Bl/6 background. Subsequently, we identified a large number of differentially methylated regions (DMRs) in sperm DNA of Mtrr⁺/+, Mtrr⁺/gt, and Mtrr^gt/gt males compared to C57Bl/6 control sperm. Few DMRs were associated with underlying SNPs or SVs. However, no sperm DMRs identified in Mtrr⁺/gt males persisted in embryonic or adult tissues of the wildtype F1 or F2 generations. Despite this, we identified two genes, Hira and Rn45s, that were adjacent to DMRs and were misexpressed in the F2 and F3 generation embryos. This suggested that abnormal DNA methylation in sperm may influence gene expression two generations later despite reprogramming events. Additionally, we identified a number of differentially expressed small non-coding RNAs in Mtrr⁺/gt and Mtrr^gt/gt sperm compared to C57Bl/6 control sperm. Overall, a complete understanding of the mechanisms behind transgenerational inheritance of phenotypes in the Mtrr^gt model remains elusive. Unravelling the mechanisms of transgenerational epigenetic inheritance could have important implications for the future prediction and prevention of human diseases.

In this dissertation, genomic tools were applied to investigate populations of two endangered native species of California for conservation purposes. The first species is the McCloud River Redband Trout (MRRT; Oncorhynchus mykiss stonei), a unique subspecies of rainbow trout that inhabits the isolated Upper McCloud River of Northern California. A major threat to MRRT is introgressive hybridization with non-native rainbow trout from historical stocking and contemporary unauthorized introductions. To help address this concern, I collected RAD-sequencing data on 308 total individuals from MRRT and other California O. mykiss populations and examined population structure using Principal Component and admixture analyses. I found that populations of MRRT in Sheepheaven, Swamp, Edson, and Moosehead creeks are nonintrogressed. Additionally, I saw no evidence of introgression in Dry Creek, and suggest further investigation to determine if it can be considered a core MRRT conservation population. Sheepheaven Creek was previously thought to be the sole historical lineage of MRRT, but our analysis identified three: Sheepheaven, Edson, and Dry creeks, all of which should be preserved. Finally, I discovered diagnostic and polymorphic SNP markers for monitoring introgression and genetic diversity in MRRT. The results provide a valuable resource for the conservation and management of MRRT.The second species is the endangered delta smelt (Hypomesus transpacificus). A refuge population of delta smelt has been maintained in a conservation hatchery system at the Fish Conservation and Culture Lab at UC Davis (FCCL). The FCCL uses a pedigree-based breeding system to keep the hatchery fish genetically close to the wild population. Despite intense genetic management at the FCCL, evidence of fitness differences between wild and cultured fish was reported. To investigate domestication selection, I used RAD-sequencing and Whole Genome Bisulfite Sequencing to collect genomic and epigenomic data on archived FCCL fin clip samples. For the genomic experiment, I grouped 240 individuals based on their level of hatchery ancestry (Domestication Index = DI) to low, medium, and high DI groups plus wild group. Selection signatures were examined using two FST comparisons: early (wild/low ID) vs. late (medium/high DI) groups, and wild vs. hatchery (low/medium/high DI) groups. I identified one candidate region on chromosome 22 as being putatively under domestication selection and discovered four genes located in this region through its annotation. Other candidate regions identified in this study need to be investigated further. My results also suggest domestication selection happens quickly (i.e., in early generations). For the epigenomic experiment, I investigated the methylation change between the wild and hatchery environments, and the inheritance patterns of the methylation changes after three generations in captivity. To do this, I collected 104 males and females from wild and hatchery delta smelt. I chose families with individuals grouped based on their generation number: G0, G1, G2, and G3. The most informative differentially methylated regions (DMRs) between the two environments were discovered by comparing methylation differences of individuals between G0 (n=12) and G3 (n=10) groups. A total of 201 significant DMRs (p-value < 0.05) were discovered across 26 chromosomes. The ratio of hypermethylated DMRs was 2.4 times more than hypomethylated DMRs in the hatchery group versus the wild group (71% to 29%). Genes associated with the DMRs in our study were predominantly involved in swimming and transmembrane activity. Methylation status of those DMRs in all the individuals at the group level, revealed possible evidence of methylation inheritance across generations but further examination at the level of individual family did not illustrate a clear inheritance pattern. Collectively, our findings may ultimately help the FCCL to adjust their protocol to minimize the rate of hatchery adaptation, and thus improve the likelihood of success for future supplementation.

Intelligent robots have been widely studied and investigated to replace, fulfilling a complex mission in a hazardous environment. Lately, swarm robotics, a group of collaborative robots, has become popular because it offers benefits over a single intelligent system. Many strategies have been developed to achieve collective and decentralised control applying evolutionary algorithms. However, since the evolutionary algorithm relies principally on an individual fitness function to explore the solution space, achieving swarm robotics' collaborative behaviour in a dynamic environment becomes a problem. This is due to the lack of adaptation in most of the evolutionary methods. In order to thrive in such environment, external stimuli and rewards from the environment should be utilised as ``knowledge'' to achieve the intelligent behaviour currently lacking in evolutionary swarm robotics. The aims of this research are: (1) to develop novel reward-based evolutionary swarm learning using mechanisms of epigenetic inheritance; and (2) to identify an efficient learning method for the epigenetic layer achieving a decision-making strategy in a dynamic environment. This research's contributions are the development of reward-based co-learning algorithm and co-evolution using epigenetic-based knowledge backup. The reward-based co-learning algorithm enables the swarm to obtain knowledge of the dynamic environment and override the objective-based function to evaluate internal and external problems. An advantage of this is that the learning mechanism also enables the swarm to explore potentially better behaviour without the constraint of an ill-defined objective function. Simulated search-and-rescue missions using a swarm of UAVs shows that individual behaviour evolves differently although each member has the same physical characteristics and the same set of actions. As an addition to reward-based multi-agent learning mechanisms, epigenetics is introduced as a decision-making layer. The epigenetic layer has two functions: there are genetic regulators, as well as an epigenetic inheritance (the epigenetic mechanism). The first is the function of an epigenetic layer regulating how genetic information is expressed as agent’s behaviour (the ``phenotype''). Thus, utilising the regulatory function, the agent is able to switch genetic strategy or decision-making based on external stimulus from the aforementioned reward-based learning. The second function is that epigenetic inheritance enables sharing of genetic regulation and decision-making layer between agents. In summary, this research extends the current literature on evolutionary swarm robotics and decentralised multi-agent learning mechanisms. The combination of both advances the decentralised mechanism in obtaining information and improve collective behaviour.

In evolutionary terms, propagation of a species is the only biological imperative. In sexually reproducing species, species survival requires the formation of specialized gametes that exist in a perpetual cycle of fertilization and establishment of totipotency, differentiation into the next generation of gametes, and then fertilization and the re-establishment of totipotency. This makes the germline an immortal cell lineage. To successfully repeat this cycle at every generation, the germline must maintain its replicative immortality. Equally important is the accurate transmission of genetic and epigenetic information to the next generation to facilitate the correct execution of the developmental program. Thus, pathways that protect genome fidelity and ensure appropriate gene expression at every generation are essential for germline immortality. In animals, small non-coding RNAs that are expressed in the germline and transmitted to progeny control gene expression to promote fertility. Germline-expressed small RNAs, including endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), drive the repression of deleterious transcripts such as transposons, repetitive elements, and pseudogenes. Small RNA pathways are highly conserved in metazoans and have been best described in the model organism Caenorhabditis elegans. Many features of C. elegans make it ideal for the study of small RNA biology and germline maintenance, including genetic tractability, rapid development, an invariant cell lineage, and propagation via self-fertilization. In C. elegans, endo-siRNAs are deposited from the maternal germline into the embryo, where they trigger the amplification of another generation of endo-siRNAs as well as heritable heterochromatin formation at target genes. Transgenerational inheritance of endo-siRNAs is critical for germline maintenance, as mutants that are heritable RNAi defective (Hrde) are germline mortal (progressively sterile). How endo-siRNAs control chromatin state and germline maintenance is unknown. I have identified morc-1, the sole C. elegans homolog of the highly conserved Microrchidia family of chromatin-binding proteins, as a crucial link between endo-siRNAs and multigenerational chromatin organization. I also identify and describe the first suppressor of the germline mortality phenotype of hrde pathway mutants. This work establishes a critical role for endo-siRNAs and MORC-1 in the control of transgenerational chromatin architecture to promote fertility.

Sperm and oocyte deliver all the information, both genetic and epigenetic, that will specify development of the next generation. Histone modifications are one form in which the gametes deliver epigenetic information to the embryo. Our understanding of how histone modifications inherited on parental genomes influences offspring transcription and development is poorly understood. I investigated a paradigm in which worms inherit the sperm genome lacking H3K27me3, a widely conserved mark of repression. I used genetic, genomic and microscopy approaches to investigate how distributions of histone marking are established in parental germlines, transmitted to offspring and influence offspring transcription and development. I demonstrate that altered inheritance of H3K27me3 results in misregulation of many genes both in the soma and in the germline of offspring. I show that there are developmental consequences to germline health and function as a result of this altered inheritance. My analysis reveals that the derepression of numerous genes in offspring is the result of increased transcription from sperm alleles specifically, which demonstrates that sperm-inherited histone marks can directly influence offspring transcription. I found that in the germlines of fathers lacking H3K27me3, H3K36me3, a mark associated with gene transcription, invades domains normally occupied by H3K27me3 in wild-type male germlines. These changes in H3K36me3 distribution in the germlines of fathers correlate with changes in transcription that occur in offspring both in somatic cells and in the germline. This work demonstrates that gamete-inherited histone marks are one mechanism through which epigenetic information can be transmitted to the next generation to influence transcription and development in offspring. A deeper understanding of this process will inform our broader understanding of how changes to the parental epigenome may influence the development and health of future generations.

The inheritance of environmentally-induced traits is an established phenomenon, however the underlying molecular mechanism is yet to be elucidated. In some cases, environmental factors may act directly on the germline, however in other cases this appears impossible. While ‘soma-to-germline feedback’ conflicts with the long-held dogma that heritable genetic information flows solely from germline to soma, there is no reason to suppose that germline- associated somatic cells cannot communicate with developing germ cells. A prime candidate for the means of such communication is small RNAs carried within extracellular vesicles (EVs). Small RNAs have been associated with inheritance of acquired phenotypes across phyla, and EVs are known intercellular messengers produced by nearly all cells. In this thesis, I have taken the first steps towards testing this idea by investigating the hypotheses that EVs produced by germline-associated somatic cells transfer small RNA cargo to germ cells, and that the RNA cargo is susceptible to environmental influence. I isolated and characterised EVs from two types of germline-associated somatic cells, Sertoli cells (in vitro) and epididymal epithelial cells (in vitro and in vivo), and characterised their small RNA profiles using high-throughput sequencing. By co-incubating labelled Sertoli EVs with spermatogonial stem cells (SSCs) and labelled epididymal EVs with living sperm, I showed that these EVs interact with germ cells. Furthermore, I also demonstrated the transfer of many Sertoli EV small RNAs to SSCs. I also established that the small RNA profiles of EVs can be influenced by the environment. I used Bisphenol A and dimethyl sulfoxide as environmental stressors of Sertoli cells, varied the concentration of folate available for in vitro epididymal cells, and modelled the response of in vivo epididymal EVs to dietary methyl donor supplementation. The small RNAs affected by these factors included miRNAs associated with transcription and nucleic acid-binding (some of which were transferred from Sertoli EVs to SSCs), and tRNA-derived fragments that have previously been associated with the vertical transmission of diet-induced phenotypes. Taken together, my data show that small RNA cargo from somatic EVs is susceptible to environmental influence, and thus such somatic RNA is capable of being transferred to the germline.

Epigenetic variation between individuals can provide a mechanism for phenotypic variation and differences in disease risk. While a proportion of epigenetic variation can be ascribed to genetic factors, epigenetic variation can persist even in the absence of genetic differences and in some cases this variation can be inherited between generations. Only a handful of such loci have so far been identified in animals, but importantly no systematic assessment of the frequency of epigenetic inheritance has been performed. The underlying aim of this work was to determine the extent to which transgenerational epigenetic inheritance occurs in the mammalian genome, with a starting hypothesis that transgenerational epigenetic inheritance is widespread. Genome-wide DNA methylation analysis was performed on two related pedigrees of isogenic mice. Investigations of pedigree-specific epigenomic differences were hampered by batch effects in RRBS data, but a systematic approach to this confounder led to the identification of epigenomic variation across the genome that was specific to each pedigree of the isogenic animals. As this investigation progressed, two other areas came in to focus: gender-specific cytosine methylation differences across autosomal DNA of several tissue types, and the problem of low concordance of results from differential methylation analysis methods. Gender-specific methylation differences are widespread and tissue specific, and a fraction of them appear to be independent of sex-specific hormonal factors. Furthermore, a considerable degree of under-calling was found among differential DNA methylation analysis methods, suggesting that a majority of genuine differentially methylated loci may be going undetected in a range of experimental scenarios.

DNA methylation is a conserved epigenetic modification, usually of the 5th carbon of cytosine in eukaryotes, and in plants is known to regulate gene expression and silence transposable elements. Posttranslational histone tail modifications are also conserved epigenetic regulators, known in the plant kingdom to interact with DNA methylation and regulate chromatin structure. Both DNA methylation and histone modifications are reversible and, collectively, play an important role in the orchestration of dynamic transcriptional profiles during the entire life cycle of the plant. In plants, DNA methylation is found in the symmetric CG and CHG contexts, and the asymmetric CHH context (where H is A, T or C). Several studies have shown that CG methylation is catalyzed by the mammalian DNMT1 methyltransferase ortholog, MET1, and CHG methylation is maintained by the plant specific chromomethylase 3 (CMT3), through a self-reinforcing loop between CMT3 and the heterochromatic dimethylation of lysine 9 of the histone H3 subunit (H3K9me2). Due to its asymmetry, CHH methylation was thought to be maintained solely by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2, an ortholog of mammalian DNMT3-type methyltransferases) through the plant-specific RNA-directed DNA methylation (RdDM) pathway. However, DRM2-mediated CHH methylation only accounts for about 35% of total CHH methylation in Arabidopsis, suggesting that other proteins contribute to the maintenance of CHH methylation. To identify which proteins are involved in the maintenance of CHH methylation in addition to DRM2, I performed a reverse genetic screen on Arabidopsis mutants to identify genes that can potentially affect plant DNA methylation by performing whole-genome bisulfite sequencing of each individual homozygous mutant seedling. Eventually, I found that CMT2, a homolog of CMT3, is an active methyltransferase that maintains ~70% of total CHH methylation in Arabidopsis, independently of RdDM. Furthermore, I determined that the DRM2-mediated RdDM pathway mainly carries out CHH methylation in more euchromatic regions, including short TEs in chromosome arms and the edges of long TEs in pericentromeric regions, whereas CHH methylation at more heterochromatic sites, such as the bodies of long TEs, is mediated by CMT2. This maintenance activity is thought to allow DNA methylation to carry epigenetic information through cell division and reproduction, influencing gene expression and phenotype across generations. Trans-generational inheritance is mediated by a small group of cells that includes gametes and their progenitors in flowering plants. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of trans-generational inheritance are inferred from such studies. The male gametophyte pollen, consisting of two sperm cells and a vegetative cell, was reported to undergo DNA reprogramming during sexual reproduction: a gain of heterochromatic CHH methylation in vegetative cells and a loss of heterochromatic CHH methylation in sperm cells were attributed to the activation of DRM2 and the silencing of DRM2, respectively. To gain further insight into how DNA methylation is inherited and reprogrammed during sexual reproduction, I analyzed Arabidopsis thaliana sperm and vegetative cells purified from pollen with mutations in the DRM (both DRM1 and DRM2. DRM1 is specifically expressed during early seed development and functions redundantly with DRM2), CMT2, and CMT3 methyltransferases. I found that although the gain of heterochromatic CHH methylation in the vegetative cell is primarily mediated by CMT2 instead of DRM, the effect of the cmt2 mutation in the vegetative cell was weaker than expected, and a small but significant fraction of new CHH methylation targets in heterochromatic TEs was mediated by DRM through RdDM pathway, which supports the observation that many heterochromatic TEs are derepressed in the vegetative cell. I also showed that lack of histone H1, which elevates heterochromatic DNA methylation in somatic tissues, does not have this effect in pollen. Instead, levels of CG methylation in wild-type sperm and vegetative cells, as well as in wild-type microspores from which both pollen cell types originate, are substantially higher than in wild-type somatic tissues and are similar to those of H1-depleted roots. In summary, I discovered a novel DNA methyltransferase in plants, CMT2, which maintains CHH methylation independently from RdDM. I showed that the DNA methylation of germlines are maintained similarly to somatic tissues, which further explains why the reprogramming of CHH methylation in pollen is most likely dependent on CMT2. Although the mechanisms of methylation maintenance are similar between pollen and somatic cells, I demonstrated that the efficiency of CG methylation is higher in pollen, allowing methylation patterns to be accurately inherited across generations. The biological significance of the reduced CHH methylation found in sperm remains unknown. Overall, my findings expand the previous incomplete DNA methylation maintenance model with the discovery of CMT2-mediated CHH methylation. My work on the pollen methylome contributes to our further understanding of the regulation and reprogramming of DNA methylation during sexual reproduction, providing insights into transgenerational epigenetic inheritance in plants.