Methylation of cytosines within CpG dinucleotides is a feature of vertebrate DNA. The precise role of DNA methylation is unknown to date, although it has been implicated in several processes relating to transcriptional regulation. One approach to study DNA methylation is the characterization of proteins that bind specifically to methylated DNA. One such family of proteins is the methyl-CpG binding domain (MBD) containing family and MBD1 is a member of this family. MBD1 is implicated in transcriptional repression and various mechanisms by which it might bring about gene silencing have been proposed. These are mainly based on studies reporting interactions between MBD1 and various proteins that regulate chromatin structure. Also MBD1 function can be modified by PIAS proteins, which stimulate its conjugation to SUMO (small ubiquitinlike modifier).The original aim of this work was to address two questions about MBD1: (1) Does MBD1 form part of a stable complex with other factors, and if so, what are the identities of the other components? Purification of MBD1 revealed the presence of no stably bound interacting proteins. However, some evidence indicates MBD1 may interact with itself and form dimers, a finding which impacts on many aspects of the function of MBD1. Also a proteomics screen for transient interaction partners identified candidate binding partners for MBD1 and the related protein MeCP2, which may throw light on the function of these proteins. (2) Are there any activities which regulate MBD1 function by the removal of SUMO from this protein? No activities capable of removing SUMO from native MBD1 were found but it was demonstrated that this modification leads to the destabilization of MBD1 in vitro. The relevance of this finding in vivo is yet to be determined.

Genomic imprinting is the epigenetic modification of loci, primarily by DNA methylation, which results in parent-of-origin-specific monoallelic expression of a small subset of genes. In plants, imprinting occurs during endosperm development and a balance of maternally- and paternally-expressed imprinted genes is essential for normal seed development. Dependence on DNA methylation for imprinting highlights the potential to manipulate seed development, and consequently seed size, by altering DNA methyltransferase activity. DNA METHYLTRANSFERASE 1 (MET1) is the primary plant maintenance DNA methyltransferase and plays a significant role in imprinting. However, no evaluation of the potential role for other MET1 family members in genomic imprinting has been reported. The current model for the control of imprinting in plants suggests that maintenance DNA methyltransferases are required throughout development, yet the tissue-specific requirement of these enzymes is unconfirmed as analysis has relied solely on constitutive DNA methyltransferase mutants. To address these problems and to evaluate the potential to alter seed size, the work reported in this thesis investigated the potential involvement of putative maintenance DNA methyltransferases MET2a, MET2b and MET3 and the tissue-specific role of MET1 in imprinting. Imprinting was not significantly altered in met2a-1, met2b-1 and met3-1 mutants, indicating that MET1 is the sole DNA methyltransferase required for imprinting. Transcriptional analysis suggested MET1 is expressed throughout floral organ development and in the male and female gametophyte generation indicating that MET1 is potentially available to maintain imprinting-dependent methylation in these tissues. Tools to suppress MET1 tissuespecifically were developed to investigate the tissue-specific requirement of MET1 for imprinting. Analysis indicates that such tools could also be used to alter seed size by manipulating imprinting in commercially important species. Further work is needed to validate this approach.

Pendant la méiose, la division cellulaire qui forme les cellules haploïdes, les chromosomes homologues hérités de chacun des deux parents sont appariés et échangent des segments réciproques appelés crossing-overs (CO). Les CO ne sont pas distribués au hasard dans le génome et leur taux varie le long des chromosomes. Certains des mécanismes responsable ont été décrits chez les mammifères et la levure mais ne sont pas conservés chez les plantes. Les CO sont fortement inhibés dans l'hétérochromatine qui est riche en éléments répétés. Le degré élevé de méthylation d l'ADN qui caractérise les séquences répétées pourrait être un inhibiteur des CO. Cela a été clairement démontré chez le champignon Ascobolus immersus et des études récentes ont montré que la perte de méthylation modifiait la distribution des CO chez Arabidopsis thaliana. Le but de ma thèse a été de décrire plus précisément le rôle de la méthylation de l'ADN dans le contrôle des CO en l'absence de polymorphisme de séquence qui affecte aussi la recombinaison.Pour cela, j'ai mesuré la recombinaison dans différentes plantes dans lesquelles la méthylation de l'ADN a été partiellement ou totalement enlevée grâce à la mutation du gène ddm1. Pour tester l'effet opposé d'un gain de méthylation, j'ai aussi essayé de cibler la methylation de l'ADN à un point chaud de recombinaison connu. Mes résultats montrent que la parte de la méthylation de l'ADN entraîne une augmentation globale de la recombinaison. Paradoxalement, l'heterochromatine qui est normalement très méthylée est moins affectée par la perte de méthylation que le reste du chromosome, probablement car la méthylation de l'ADN a des effets à distance. L'augmentation de CO est accentuée dans les générations successives du mutant ddm1. Cependant, l'effet le plus important est observé dans les hétérozygotes où la moitié du génome seulement est hypométhylée, ce qui suggère un rôle complexe de la méthylation. Finalement, j'ai pu montrer que le polymorphisme affecte la recombinaison surtout dans l'hétérochromatine mais pas dans le sens attendu puisque les plantes homozygotes recombinent moins que les plantes hétérozygotes. / During meiosis, the cellular division that gives rise to haploid cells, homologous chromosomes inherited from each parent are paired and are subjected to reciprocal exchanges of chromosome segments called crossing-overs (COs). COs are not randomly distributed in the genome. Some of the involved mechanisms have recently been described in mammals and yeast bu they are not conserved in plants. Repeat-rich heterochromatin is suppressed for COs. The high level of DNA methylation associated with repeats could be an inhibitor of COs. This was clearly demonstrated in the fungus Ascobolus immersus and recent studies have shown that the loss of DNA methylation also affects COs in Arabidopsis thaliana. The aim of my thesis was to describe more precisely the role of DNA methylation in the control of CO distribution in the absence of any DNA sequence polymorphism which are known to affect recombination. For this purpose, I measured recombination in different plants where DNA methylation has been partially or completely removed thanks to the mutation of the DDM1 gene. To test the opposed effect of a gain of DNA methylation,.I also tried to target DNA methylation at a known recombination hotspot. My results show that the loss of DNA methylation induces a global increase of recombination. Paradoxically, the normally highly methylated heterochromatin is less affected by this loss than the rest of the chromosome, probably because DNA methylation has distal effects. The increased recombination is exacerbated in successive generations of the hypomethylated ddm1 mutants. However, the strongest effect is seen in the heterozygotes where only half of the genome is hypomethylated, suggesting a complex role in the control of CO distribution. Finally, I show that DNA sequence polymorphism affects mainly recombination in the heterochromatin but not in the expected sense, since homozygous plants recombine less than heterozygous.

Background: Over 200 million individuals worldwide are chronically exposed to arsenic (As) in drinking water at concentrations above the World Health Organization (WHO) guideline of 10 µg/L. Arsenic exposure is of particular concern in Bangladesh, where it is estimated that 35-77 million people are exposed to As in well water at concentrations above the WHO guideline. Chronic As exposure is associated with neurological impairments, respiratory disease, cardiovascular disease, skin lesions, and cancers of the skin, liver, lung and bladder. The mechanisms of As toxicity in humans are not well-characterized: there are considerable interspecies differences in As toxicokinetics, and until recently, there were no animal models to study As carcinogenesis. However, two of several proposed pathways of As toxicity in humans involve DNA methylation and oxidative stress. Arsenic metabolism, DNA methylation, and glutathione (GSH) are metabolically connected through the one-carbon metabolism and transsulfuration pathways, and their interactions are remarkably complex. The epidemiologic studies in this dissertation are designed to address the overarching hypothesis that one-carbon metabolism and the transsulfuration pathway interact to influence susceptibility to As toxicity. Introduction: Arsenic is methylated in the liver to monomethyl (MMA) and dimethyl (DMA) arsenical species by arsenic(III)-methyltransferase (AS3MT), which requires a methyl group from S-adenosylmethionine (SAM) and the presence of a reductant, such as glutathione (GSH). SAM is the universal methyl donor for transmethylation reactions, including DNA methylation, and is a product of folate-dependent one-carbon metabolism. GSH is the primary endogenous antioxidant and determinant of the intracellular redox state, and the rate-limiting precursor for GSH synthesis, cysteine (Cys), is a product of the transsulfuration pathway. One-carbon metabolism and the transsulfuration pathway are connected through homocysteine (Hcys). In humans, aberrant DNA methylation, oxidative stress, hyperhomocysteinemia (HHcys), and impaired As methylation capacity have been identified as risk factors for As-related conditions, including As-induced skin lesions. However, there are knowledge gaps regarding the relationships among these risk factors in humans, namely (1) the dose-response relationship between chronic As exposure and global DNA methylation over a wide range of As concentrations, as well as the influence of As exposure on the newly-discovered epigenetic modification, 5-hydroxymethylcytosine (5hmC); (2) whether an oxidized GSH redox state impairs the capacity to methylate As and DNA; and (3) whether variants in one-carbon metabolism genes are associated with HHcys and susceptibility to As-induced skin lesions. Methods: We addressed these questions in five self-contained epidemiological studies of As-exposed Bangladeshi adults, which employed cross-sectional (Chapters 3-6) and nested case-control (Chapter 7) designs. First, we examined the dose-response relationship between As exposure and global methylation of peripheral blood mononuclear cell (PBMC) DNA (Chapter 3). Second, we optimized a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure global 5-methylcytosine (5mC) and 5hmC content in human DNA samples, and we examined the associations of As exposure with global %mC and %hmC in two independent samples of As-exposed adults (Chapter 4). Third, we measured GSH and its "oxidized" form, glutathione disulfide (GSSG) in plasma, and we examined the interaction of plasma GSH redox state and folate nutritional status on As methylation capacity (Chapter 5). Fourth, we examined the relationships between blood GSH redox, blood SAM, and global methylation of PBMC DNA (Chapter 6). Fifth, we conducted a nested case-control study (Chapter 7) to determine whether nonsynonymous single nucleotide polymorphisms (SNPs) in methylene-tetrahydrofolate reductase (MTHFR) and other one-carbon metabolism genes were associated with HHcys and risk for As-induced precancerous skin lesions, and we conducted an exploratory genome-wide association study (GWAS) of Hcys in a subset of participants. Results: Chronic As exposure was associated with increased global DNA methylation over a wide range of well water As concentrations (Chapter 3), but the relationship between As exposure and global %hmC was gender-specific, with a positive association in males and negative association in females (Chapter 4). We found that an oxidized GSH redox state was associated with both decreased As methylation capacity (Chapter 5) and global DNA hypomethylation (Chapter 6). Finally, in the nested-case control study, we confirmed previous findings that serum HHcys was a risk factor for As-induced skin lesions, and gene variants in MTHFR were found to explain a substantial proportion of the variance in serum Hcys concentrations (Chapter 7). However, we did not find that one-carbon metabolism gene variants were risk factors for As-induced skin lesions. The GWAS of serum Hcys identified one genome-wide significant SNP in the pregnane X receptor (PXR) gene, along with other SNPs in genes involved in cell signaling and the establishment of epithelial cell polarity. Taken together, our findings suggest that indices of one-carbon metabolism and the transsulfuration pathway--DNA methylation, GSH redox, and As methylation--interact with one another to influence susceptibility to As toxicity in humans. In addition, to our knowledge, this is the first report of an association between As exposure and global 5hmC.

The first half of this dissertation demonstrates development of a novel method for DNA methylation profiling based on site specific conversion of cytosine in CpG sites catalyzed by DNA methyltransferases. DNA methylation, a chemical process by which DNA bases are modified by methyl groups, is one of the key epigenetic mechanisms used by cells to regulate gene expression. It predominantly occurs at the 5-position of cytosines in CpG sites and is essential in normal development. Aberrant methylation is associated with many diseases including cancer. Bisulfite Genomic Sequencing (BGS), the gold standard in DNA methylation profiling, works on the principle of converting unmethylated cytosines to uracils using sodium bisulfite under strong basic conditions that cause extensive DNA damage limiting its applications. This dissertation focuses on the research and development of a new method for single cell whole-genome DNA methylation profiling that will convert the unmethylated cytosines in CpG sites to thymine analogs with the aid of DNA methyltransferase and photo-irradiation. Previously we synthesized a model deoxycytidine containing an optimized allyl chemical group at the 5-position and demonstrated that this molecule undergoes photo-conversion to its deoxythymidine analog (C to T conversion) with irradiation at 300 nm. The C to T conversion also proved feasible using synthetic DNA molecules. In this thesis, we demonstrate the conversion of a novel modified deoxycytidine molecule (PhAll-dC) using 350 nm photo-irradiation and a triplet photosensitizer (thioxanthone, TX) to avoid potential DNA damage. The new photoproduct was identified as the deoxythymidine analog of the starting molecule as assessed by IR, MS and NMR. An AdoMet analog containing the optimized chemical group was also synthesized and tested for enzymatic transfer to the C5-position of CpG cytosines using DNA methyltransferases. DNA methyltansferase M.SssI was engineered for more efficient enzymatic transfer. In the future, we will incorporate a triplet photosensitizer into the photoreactive moiety on AdoMet to increase energy transfer efficiency for photo-conversion of C to the T analog. Incorporating this into an overall method followed by amplification and sequencing should allow us to assess the methylation status of all CpGs in the genome in an efficient manner. The second half of this dissertation demonstrates development of a DNA sequencing by synthesis (SBS) method, The Sequence Walking Approach, using novel nucleotide reversible terminators (NRTs) together with natural nucleotides. Following the completion of The Human Genome Project, next generation DNA sequencing technologies emerged to overcome the limitations of Sanger Sequencing, the prominent DNA sequencing technology of the time. These technologies led to significant improvements in throughput, accuracy and economics of DNA sequencing. Today, fluorescence-based sequencing by synthesis methods dominate the high-throughput sequencing market. One of the major challenges facing fluorescence-based SBS methods is their read length limitation which constitutes a big barrier for applications such as de novo genome assembly and resolving structurally complex regions of the genome. In this regard, we have developed a novel SBS method called ‘The Sequence Walking Approach’ to overcome current challenges in increasing the single pass read length of DNA sequencing. Our method utilizes three dNTPs together with one nucleotide reversible terminator in reactions called ‘walks’ that terminate at predetermined bases instead of after each incorporation. In this method, the primer extended via 4-color SBS is stripped off and replaced by the original primer for walking reactions. By reducing the accumulation of cleavage artifacts of incorporated NRTs in a single run, our method aims to reach longer read lengths. In this thesis, we have demonstrated a variation of The Sequence Walking Approach in which 4-color sequencing steps are interspersed with walking steps over a continuous length of DNA without stripping off extended primers and reannealing the original primer. The improvements introduced with this method will enable the use of fluorescence-based SBS in many applications such as detection of genomic variants and de novo genome assemblies while preserving low costs and high accuracy.

All mC5-MTases are monomeric enzymes, except M. EcoHK31I and M. AquI which are MTases composed of two poly peptides. M.EcoHK31I is a mC5-MTase which recognizes the sequence 5-YGGCCR-3' and consists of polypeptide alpha and beta, with the latter gene encoded in an alternative reading frame of the former. All of the conserved motifs in mC5-MTases can be found in polypeptide alpha, except motif IX, which is located in polypeptide beta. Both polypeptides are required for in vitro methylation. / Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. In higher eukaryotic organisms cytosine-C5 methyltransferase (mC5-MTase) is the only type of DNA MTase and it plays an important role in controlling a number of cellular processes including transcription genomic imprinting and DNA repair. In bacteria, there are three types of MTases, mC4-, mC5- and mAb-, classified according to the methylation site of the DNA. MTase and its cognate restriction endonuclease (ENase) form restriction-modification system. The role of MTase is to protect the host from its own ENase digestion while the ENase acts to degrade the invasion of foreign DNA. Sequence comparison of nearly 50 bacterial mC5-MTases has shown that these enzymes share an overall common protein architecture. Ten conserved motifs (I to X), each 10 to 20 amino acids in length, have been identified, five of which are highly conserved (I, IV, VI, VIII and X). In addition, all of these enzymes have a hypervariable region lying between motifs VIII and IX. It is called the target recognition domain (TRD), and is responsible for the specificity of DNA recognition and the choice of base to be methylated. / Since both of the polypeptides alpha and beta of M.EcoHK31I are sequenced and cloned into the expression vector separately, the role of DNA recognition and subunits interaction of individual polypeptides can be studied. By electromobility shift assay, we found that polypeptides alpha and beta complex recognize specific double strand oligos substrate. Polypeptide alpha-DNA formed aggregates and polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists the proper binding of polypeptide alpha to DNA substrate. Complex of polypeptide alpha and a polypeptide beta variant with N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive MTase. By surface plasmon resonance assay, the dissociation equilibrium constant (KD) of polypeptides alpha and beta complex was found to be 56.2nM and the KD for polypeptide alpha and DeltaN46-polypeptide beta complex was increased by about 95 folds, contributing by a drastic decrease in dissociate rate constant (kd) and an increase in association rate constant (ka). This indicated that the N-terminal region of polypeptide beta takes part in subunit interaction. / To pinpoint which amino acid residues located at the variable region of polypeptide alpha are important for DNA binding and subunits interaction, "charge-to-alanine scanning mutagenesis" were performed on 16 charge residues between Asp213 and Glu271 in the small domain. It was found that the five charge residues upstream of motif X are not required for activity. For other residues except K225, E240 and D245, the protein is active when the same charge is maintained. / Fung Wai To. / "March 2006." / Adviser: P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6376. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 180-201). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The phenomenon of cell type-specific DNA methylation has received much attention in recent years and a number of DNA methylation differences have been described between cells of the immune system. Of particular interest when studying DNA methylation are CpG islands (CGIs) which are distinct from the rest of the genome due to their elevated CpG content, generally unmethylated state and promoter association. In the instances when they become methylated this is associated with gene repression although it is unclear the extent to which differential methylation corresponds to differential gene expression. I have used an immune system model to assess the role of CGI methylation and the role of the methylation reader MBD2 in regulation of gene expression. A relatively small number of DNA methylation differences were seen between immune cell types with the most developmentally related cells showing the fewest methylation differences. Interestingly, the vast majority of CGI-associated cellspecific methylation occurred at intragenic CGIs located, not at transcription start sites, but in the gene body. Increased intragenic CGI methylation tended to associate with gene repression, although the precise reason for this remains unclear. Most differentially methylated CGIs were depleted for the active chromatin mark H3K4me3 regardless of their methylation state but some of these were associated with the silencing mark H3K27me3 when unmethylated. These findings suggest that intragenic CGIs are a distinct class of genomic element particularly susceptible to cell type-specific methylation. I also looked at the effect of removing the methyl- CpG binding domain protein MBD2 from immune system cells. Immune cells from Mbd2-/- mice showed a number of previously uncharacterised phenotypes as well as a number of differences in gene expression compared to wild-type animals. Most of these genes increased their expression in the absence of MBD2 consistent with MBD2’s role as a transcriptional repressor and Mbd2-/- Th1 cells showed increases in histone H3 acetylation compared to wild-type Th1 cells. This work provides an insight into the role played by cell-specific CGI methylation and MBD2 in regulating gene expression.

In higher eukaryotes, the DNA base Cytosine can exist in a variety of modified forms when in the dinucleotide CpG. Although a methylated form tends to dominate within the genome, approximately 1% of all CpG dinucleotides are found unmodified at high densities spanning around 1Kb and tend to co-localise to the 5’ ends of around 60% of annotated gene promoters. These unique DNA sequences are known as CpG islands (CGIs) and their role within the genome to date is largely unknown. Methylation of CGIs in cancers however has been linked to silencing of associated genes implying a role in gene regulation. Furthermore these sites are also interesting as they remain specifically nonmodified within a genome rich in methylated CpG. We set out to better understand the roles for CGIs through the characterisation of any specific CGI binding proteins. Digestion of nuclei with methyl sensitive restriction enzymes facilitates the purification of CGI fragments. Subsequent immunohistochemistry on the CGI chromatin fragments along with ChIP-PCR over several CGIs revealed an enrichment of the “active” histone modifications including H3K4me3, a depletion of the “silencing” marks such as H3K27me3, as well as a group of CGI specific binding factors. These latter proteins contained a domain previously shown to bind to non-methylated CpG dinucleotides (the CXXC domain) and as such were ideal candidates for CGI specific factors, in particular a protein called Cfp1. Genome wide sequencing revealed a striking correlation between Cfp1 and H3K4me3 which were both seen at around 80% of islands. Furthermore, the presence of Cfp1/H3K4me3 at islands tended to have a negative correlation with the presence of chromatin rich in the silencing histone modification H3K27me3. Closer investigation of the Cfp1 protein reveals it to be a true non-methyl CGI binding factor in vivo and shRNA reduction of Cfp1 levels to around 10% of wild type resulted in a precipitous drop in H3K4me3 levels over CGIs without a dramatic reduction in global H3K4me3 levels. As Cfp1 has been shown to be part of the Set1 histone H3K4 methyltransferase complex responsible for this modification, this CXXC protein may be attracting this histone modifying complex and as such represents a method whereby the underlying DNA sequence (CpG) can drive the overlying epigenetic state. This study may go some way to understanding the functional significance of CGIs within the genome.

Thousands of chemical pollutants enter the environment continuously, each with the potential to cause adverse effects in both terrestrial and aquatic organisms. As a result, organisms are often exposed to a mixture of stressors within their habitat. Populations of fish inhabiting most aquatic environments are exposed to time-varying or repeated pulses of exposure, driven by run-off events or spills, or due to their mobility between polluted and clean waters. Therefore, the sustainability of fish populations is critically dependent on their ability to adapt to frequent changes in their local environment. Despite this, legislation to protect the environment from chemical contamination are generally based on toxicological measurements following exposures to single stressors, conducted under optimal laboratory conditions, and that do not take into account the variation in susceptibility of wild populations, or the potential consequences of exposure for the susceptibility of the population during future exposures, including across generations. Increasing evidence is suggesting that a number of chemicals may interact with the epigenome, and that differential responses to pollutants may be modulated, at least in part, via epigenetic mechanisms. However, our understanding of the role of epigenetic mechanisms in normal development in fish models or its susceptibility to exposure to environmental stressors is currently very limited. This thesis aimed to document the mechanisms of genetic and epigenetic responses to industrial pollutants in fish, and to explore the extent to which differential responses can be induced in the lab following exposure during the critical window of embryonic development or in adults. To address these objectives, I performed a series of experiments using both the zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus) as fish models. I first used the zebrafish (Danio rerio) model to investigate the sex-specific transcription and DNA methylation profiles for genes involved in the regulation of reproduction and in epigenetic signalling in the livers and gonads. I provide evidence of the sex-specific transcription of genes involved in reproduction and their regulation by epigenetic signalling in this commonly used vertebrate model and highlight important considerations regarding the use of whole tissues comprised of multiple cell types in epigenetic and transcriptomic studies. I then investigated the potential for exposure to Bisphenol A (BPA) to cause adverse effects on reproduction and to disrupt the expression profiles and promotor DNA methylation of target genes important for reproductive function and epigenetic signalling in the zebrafish. To do this, I exposed breeding zebrafish to a range of BPA concentrations over 15 days and found that BPA disrupted reproductive processes in zebrafish, likely via estrogenic mechanisms, but only at high concentrations. Importantly, exposure to environmentally relevant concentrations of BPA resulted in altered transcription of key enzymes involved in DNA methylation maintenance, and caused changes in promoter DNA methylation. I also conducted a series of repeated exposures to copper in the three-spined stickleback to investigate the extent to which differential susceptibility can be induced in the lab. This work provides evidence that pre-exposure to copper results in differential responses in future exposure scenarios both when the initial exposure occurred in adults and during embryogenesis. For adults, fish appeared to recover completely from the initial exposure following a period of depuration of 30 days, but displayed decreased susceptibility upon re-exposure. In contrast, for fish exposed during the critical windows of embryonic development when epigenetic reprogramming are hypothesised to occur, differential copper accumulation was maintained throughout life. Importantly, the initial exposure caused increased tolerance in the offspring, which was inherited up to the F2 generation. This work provides valuable information regarding potential critical windows of development which may be more susceptible to effects associated with pre-exposure, highlighting that early life exposure to a low concentration of copper can induce differential responses to copper across generations. These data highlight the extent of differential responses to chemical stressors likely to be present in wild populations, and point towards the possibility that effective population management will likely require an in-depth understanding of the exposure history of a given population in order to manage restocking initiatives, and to inform conclusions drawn from toxicity testing studies conducted using individuals originating from wild populations. In addition, these data suggest that it is likely that both epigenetic and genetic changes can contribute to the adaptation of individual populations to their local environment. Finally, other vertebrates including humans have been shown to be exposed to the chemicals tested in this thesis. Therefore, this highlights the potential for these chemicals to also cause toxic effects in humans, potentially via (epi) genetic mechanisms, and advocate the testing of the potential for inheritable phenotypes, such as those described in this thesis, to occur in mammalian models.

Epigenetic mechanisms are of fundamental importance for resolving and maintaining cellular identity. The mechanisms regulating muscle stem and progenitor cell identity have ramifications for understanding all aspects of myogenesis. The epigenetic mechanisms regulating muscle stem cells are therefore important aspects for understanding the regulation of muscle regeneration and maintenance. Important roles for the trithorax H3K4 histone methyltransferase (HMT) MLL1 have been established for early embryogenesis, and for hematopoietic and neural identity. Here, using a conditional Mll1 knockout (KO), we find that in vivo, MLL1 is necessary for efficient muscle regeneration, and for maintenance and proliferation of muscle stem and progenitor cells. Loss of Mll1 in cultured myoblasts reveals an essential role for expression of the myogenic specification gene Pax7. Mll1 KO results in a minor decrease in Pax7 mRNA and a strong decrease of Pax7 protein. While MLL1 was found to bind the Pax7 promoter, Mll1 KO results in a minor decrease of H3K4me3 at Pax7, supporting a recognized non-HMT role for Mll1 at Pax7. Microarray analysis of mRNA expression in Mll1 KO myoblasts finds that Myf5 is the most strongly downregulated of all genes, unexpectedly, mRNA expression of previously identified MLL1 targets are unaffected by loss of MLL1 in myoblasts. Pax7 activates Myf5 expression through recruitment of a H3K4 HMT, and in Mll1 KO myoblasts expression of, and H3K4me3 at Myf5 is lost. Exogenous Pax7 rescues Myf5 expression and H3K4me3 at Myf5 in the absence of MLL1, indicating that Myf5 expression is conditional on Pax7, but not MLL1. We also show that Myf5 DNA is methylated in non-myogenic cells, and in satellite stem cells that have never expressed Myf5, but is not methylated in satellite cells that are committed to the myogenic lineage, indicating that demethylation of Myf5 may be a fundamental step in myogenic commitment. Intriguingly, Myf5 promoter DNA becomes remethylated in Mll1 KO myoblasts. This work finds that Pax7 expression and myogenic identity is partly dependent on MLL1 expression. Further, evidence is uncovered that myogenic commitment is initiated by demethylation of Myf5. These findings add to the understanding of the epigenetic mechanisms that regulate and define muscle stem cells.