Michael Kobor

Parkinson’s disease (PD) is a common neurodegenerative disorder with increasing worldwide prevalence. Although a number of genes and environmental factors influencing PD susceptibility have been identified, the mechanisms underpinning their joint contributions to disease pathogenesis are not fully understood. DNA methylation (DNAm) is an epigenetic mark that is sensitive to genetics and environment and can influence gene transcription. Due to these properties, DNAm has been proposed as a contributor to PD etiology and also as a potential early-stage biomarker. However, the complex interindividual etiology of PD and the inability to its study progression in human brain tissue present challenges in researching the role of DNAm in PD pathogenesis. In this dissertation, I investigated the associations of DNAm with genetic and environmental factors influencing PD susceptibility and with early-stage PD in human populations. Using a human dopaminergic neuron cell line, I found that overexpressing the wild type or A30P mutant form of the SNCA gene induced thousands of site-specific DNAm changes. These DNAm changes were associated with altered DNA hydroxymethylation (DNAhm) and transcription of glutamate signaling genes. Additionally, I investigated the impacts of overexpressing wild type SNCA in mice housed in a standard or enriched environment paradigm by profiling hippocampal DNAm and DNAhm, and integrating these results with previously generated RNA-seq and ChIP-seq (H3K4me1, H3K4me3, H3K27ac). SNCA overexpression was associated with similar DNAhm and H3K27ac alterations when mice were housed in a standard or enriched environment, and with environment-dependent changes to H3K4 and DNA methylation. In particular, environment prevented some SNCA-induced changes to H3K4me1, a select few of which correlated with gene expression. Finally, I investigated the contributions of sex, genetic background, and pesticide exposure to blood DNAm signatures of early-stage PD in a sample of 218 agricultural workers and their spouses. Differentially methylated regions associated with early-stage PD differed by sex and were influenced by genetic background to a greater degree than pesticide exposure. Altogether, I have enhanced our understanding of the impacts of PD-associated genotypes and environments on the brain and blood epigenome, and laid a foundation for further studies of the role of the epigenome in PD etiology.

View record

Interpersonal and societal adversity experienced throughout life, such as abuse or low socio-economic status (SES), have been associated with negative health outcomes. Many of these diseases share the common thread of inflammation, inspiring the hypothesis that chronic stress resulting from adverse experiences over activates the hypothalamic-pituitary- adrenal (HPA) axis resulting in dysregulation of the immune system. DNA methylation (DNAm), a methyl group covalently bound to cytosine bases for the purposes of this thesis, is one of many epigenetic mechanisms involved in responding to environmental signals in the genomic context. As such, this modification may be particularly pertinent to understanding how adverse experiences can become embedded in a way that results in lifelong health disparities.The overarching aim of this dissertation is to understand how various measures of adversity throughout the life-course could associate with DNAm differences between individuals. Initially, I compared whole blood DNAm patterns amongst elderly individuals with different years of education, household assets and self-reported measures of economic standing in childhood and older adulthood, in addition to a composite socioeconomic (SES) measure. I found there were significantly more DNAm associations with older adulthood relative to retrospective childhood SES measures, and the subjective SES measures displayed a dampened signal relative to objective ones. Next, I investigated the relationship between the inflammatory biomarker serum IL-6, lifetime SES measures and purified monocyte DNAm patterns amongst adults. Here, I found the relationship between some CpGs and IL-6 was partially explained by SES. Additionally, differences in SES-associated DNAm sites were seen predominantly amongst individuals who experienced low early life and high adulthood SES. Finally, I investigated how childhood abuse associated with DNAm in spermatozoa of adult men. Though this was a small pilot study, I found a subset of differentially methylated regions associated with childhood abuse. These associations were stable over a two-month period and survived adjusting for current life adversity measures. Overall, these findings provide evidence that the tissue, timing, and measure of adversity experienced are important considerations for social epigenetic studies and can yield unique DNAm patterns in a context-specific way.

View record

Cells are routinely confronted with DNA damage and depend on genome maintenance factors to prevent cell death, as well as mutations and genomic rearrangements. The DNA damage response includes DNA repair and cell cycle checkpoints, which arrest sensitive processes such as DNA replication while damage is repaired. Rtt107 is an evolutionarily conserved Saccharomyces cerevisiae protein that is required for resistance to agents that cause replicative stress and DNA damage. Rtt107 has been best characterized as a scaffold that acts in response to DNA damage, localizing to phosphorylated H2A and binding to diverse protein partners, including Slx4. However, other functions of Rtt107 remain unclear. Mutants lacking Rtt107 exhibit prolonged checkpoint activation after replicative stress, and the “checkpoint dampening” model proposes that Rtt107-Slx4 reduces checkpoint activity by limiting formation of a Rad9-Dpb11 complex that promotes checkpoint activity. In this dissertation, I identified higher levels of DNA damage in cells lacking Rtt107 during replicative stress. This presented an alternative cause of checkpoint activity, and I showed that the Rad9-Dpb11 complex was not primarily responsible for prolonged checkpoint activity in this context. Based on these findings, I proposed a revised model of Rtt107 function during replicative stress, in which Rtt107 primarily reduced checkpoint activity by limiting DNA damage levels. I next explored Rtt107’s less well-understood function in limiting spontaneous genome instability, and found that this was partially distinct from its function in the context of treatment with replicative stress. Specifically, Rtt107 bound to the DNA repair protein Rad55 to limit spontaneous loss of heterozygosity, and spontaneous crossover events. I next focused on the regulation of Rtt107, specifically the phosphorylation of Rtt107’s SQ/TQ cluster domain (SCD) by the Mec1 kinase after treatment with DNA damaging agents. To address inconsistencies between prior reports on the importance of Rtt107 phosphorylation, I reviewed prior work on this subject and demonstrated that phosphorylation of Rtt107’s SCD was dispensable for growth during chronic treatment with DNA damaging agents. Collectively, my dissertation reveals how Rtt107 acts with various partners and in different contexts to confer resistance to agents that cause replicative stress and DNA damage, and to prevent spontaneous genome instability.

View record

DNA Methylation (DNAm) is an epigenetic modification that is present across the human genome, primarily in the context of CpG di-nucleotides. In human population studies, high throughput bead chip microarray assays are the prevalent way to simultaneously measure the methylation state of many thousands of genomic CpG sites. Proximal genomic CpGs have correlated methylation state within a single cell and often function as a single biological unit. The prevailing common methylation state of such multiple CpGs within a common biological unit has been the subject of intense study, due to its immediate relevance for gene expression regulation and ultimately for health and disease. I designed and implemented a method for a biologically motivated DNAm array data reduction, which constructs co-methylated regions (CMRs), while incorporating information about the genomic CpG background from the reference human genome annotation. The method relies on the correlations of CpG methylation across individuals for proximal CpG probes. The method aims for enhanced statistical performance in terms of statistical power and specificity, including for downstream applications. For example, Epigenome Wide Association Studies (EWAS), an important such application, often places the focus on group “hits” with multiple adjacent CpGs that are significant, because their gnomic proximity makes it more likely that the detected correlations are not spurious. The CMRs capture such groups and I showed that the CMRs constructed in whole blood public data have high statistical specificity in the context of EWAS for chronological age and biological sex. When the composite CMR methylation measures were used to perform EWAS for age and sex, they had high sensitivity and specificity, including uncovering additional associated CpGs not detected by conventional EWAS. The utility of the data reduction method was further discussed within the broader context of applying machine learning algorithms for high dimensional DNAm array data analysis.

View record

Many early experiences and exposures are known to cause health disparities later in life, suggesting that they are somehow ‘biologically embedded’. The mechanisms underlying ‘biological embedding’ are currently not well understood. However, emerging evidence has implicated a potential contribution from epigenetic modifications, such as DNA methylation (DNAm), which has been shown to associate with early life experiences of low socioeconomic status. Additional related experiences have also been connected with DNAm, including parental stress, childhood maltreatment or deprivation, and maternal mental health problems during the perinatal period. The relationship between early life experience and epigenetics is complicated by internal psychological and physiological factors, as well as genetic variation, which can account for 20% to 80% of inter-individual epigenetic differences. As well, stress reactivity and temperament are predictive of how a child may interact with his or her environment and can affect how such exposures are internalized. Thus, the main objectives of my dissertation were to elucidate the relationships between childhood environment, DNAm, genetic variation, and behaviour, to understand how these systems influence one another. Using matched DNAm profiles from blood and buccal tissue from a cross-sectional cohort of Canadian children, I uncovered tissue-specific and -shared DNAm signatures in order to glean the utility of accessible tissues in epigenetic association studies. In a longitudinal cohort, I tested the hypothesis that indicators of children’s early internal, biological and behavioural responses to stressful challenges are linked to stable patterns of DNAm later in life; I found relationships between biobehavioural response propensities in early life and patterns of DNAm in DLX5 and IGF2 genes at ages 15 and 18. Finally, I examined the epigenetic correlates of familial socioeconomic status in matched childhood peripheral blood and dried neonatal blood spot samples, allowing me to assess the DNAm pattern over time. Together these findings build upon our current understanding of the role of DNAm in biological embedding and more broadly, the field of social epigenetics.

View record

Aging is a multifaceted process occurring in all living organisms, and it involves the breakdown of biological robustness. Although much research has revealed fascinating features of cellular mechanisms related to aging and lifespan, we have yet to understand the underpinnings driving this inevitable progression. Epigenetics is one area of aging research that has developed significant interest as certain modifications, such as DNA methylation, have been proposed to mediate the relationship between the environment and gene expression as well as have age-associated patterns. Interestingly, predictors of age based on DNA methylation of
View record

Epigenomic variation represents an emerging focus in human health research, particularly in regards to neurobiological disease susceptibility and pathogenesis. DNA methylation (DNAm), which involves the covalent attachment of a methyl group to a cytosine primarily at CpG dinucleotides, has been widely assessed in the context of epigenome-wide association studies (EWASs), with DNAm associations identified across a broad range of disease states, environmental exposures and genetic backgrounds. However, DNAm profiling in neurobiological diseases is challenged by the fact that DNAm variation is highly tissue-specific and target brain tissues may be difficult or impossible to collect from postmortem samples, in living individuals undergoing treatment interventions or in pediatric populations. As such, the use of cell-culture models or accessible peripheral tissues such as blood or buccal swabs represent alternative approaches used in human neurobiological DNAm studies to identify potential biomarkers of disease or treatment response. The overarching aim of my dissertation was to apply and evaluate various tissue-specific approaches to investigate DNAm variation across different neurobiological diseases. To this end, I performed four separate studies to assess disease-associated DNAm from a) post-mortem brain samples, b) primary brain-derived cell culture models and c) accessible peripheral tissues. Specifically, I examined DNAm patterns related to Huntington’s disease pathogenesis and tissue-specific Huntingtin gene expression in postmortem human cortex samples. I subsequently compared DNAm profiles from glioblastoma multiforme tumours and matched primary cell cultures enriched for brain-tumour initiating cell populations, identifying a homeobox-enriched signature of differential DNAm between the paired samples. Beyond brain-specific DNAm patterns, I also explored the use of a disease-relevant blood cell type, CD³⁺ T-lymphocytes, to detect DNAm alterations associated with alcohol dependence in patients undergoing a clinical intervention. Finally, I assessed DNAm variability and the influence of genetic variation on DNAm in peripheral blood and buccal epithelial cells from two pediatric cohorts, highlighting a number of potential considerations and practical implications for the appropriate design and interpretation of early-life EWAS analyses in these tissues. Overall, these findings provide evidence to implicate DNAm variation in neurological function and pathology as well as present potential opportunities for the identification of novel biomarkers in accessible tissues.

View record

Prenatal alcohol exposure (PAE) can alter the development, function, and regulation of neurobiological and physiological systems, causing lasting cognitive alterations, behavioral deficits, immune dysfunction, and increased vulnerability to mental health problems. In humans, the spectrum of these deficits is known as fetal alcohol spectrum disorder (FASD). Although the molecular underpinnings are not fully elucidated, epigenetic mechanisms are a prime candidate for the programming of physiological systems by PAE, as they may bridge environmental stimuli and neurodevelopmental outcomes. DNA methylation is also emerging as a potential biomarker of early-life events, which may aid in earlier FASD diagnoses. Thus, my overarching aim was to identify epigenetic mechanisms that may contribute to the deficits associated with FASD and act as biosignatures of PAE. Specifically, I used genome-wide approaches to assess underlying gene expression programs and epigenomic profiles in a rat model of PAE and clinical cohorts of individuals with FASD. In the rat model, I identified alterations to gene expression programs in the brain of adult PAE females under steady-state and immune challenge conditions. Building on these long-term alterations to transcriptomic programs, I identified altered DNA methylation patterns persisting from birth to weaning in the hypothalamus PAE animals, suggestive of early reprogramming of neurobiological systems. In parallel, I found concordant alterations to DNA methylation profiles in the hypothalamus and white blood cells of PAE animals, which may reflect systemic effects and potential biomarkers of PAE. To complement the animal model, I also investigated DNA methylation patterns in two clinical cohorts of FASD, where I identified an epigenetic signature of FASD in buccal epithelial cells. As these results raised the possibility of an epigenetic biomarker, I investigated the relevance of DNA methylation as a diagnostic method for PAE, and successfully generated a predictive algorithm that could classify individuals with FASD versus controls. Overall, these findings provide evidence for the biological embedding of PAE’s effects through changes in gene expression and DNA methylation, while setting the stage for the development of novel biomarkers. Ultimately, these may aid in the development of targeted interventions and early screening tools to mitigate the deficits associated with FASD.

View record

No abstract available.

No abstract available.

No abstract available.

No abstract available.

No abstract available.

No abstract available.