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Depression is one of the most widespread and disabling mental health disorders affecting adults worldwide, and Black women bear a disproportionate burden of this disorder. With its varied symptom presentation, depression can be difficult to diagnose. In addition, Black women may be less likely to report symptoms due to cultural stigma. The purpose of this dissertation is to examine the associations between social determinants of health and depressive symptoms using DNA methylation data and machine learning to predict depressive symptoms in Black women. Chapter 2 contains two comprehensive literature reviews: a scoping review of machine learning methods used to analyze omics data to classify depressed cases and healthy controls and a concept analysis of depression in Black mothers. Chapter 3 examines associations between social determinants of health, depressive symptoms, and DNA methylation. Chapter 3A focuses on socioeconomic deprivation; Chapter 3B focuses on perceived income inadequacy; and Chapter 3C identifies differential methylation associated with depressive symptoms. Chapter 4 utilizes supervised machine learning algorithms to predict depressive symptoms and perform feature selection. These chapters show the harmful effects that perceived discrimination can have on the mental health of Black women. Additionally, the results indicate that DNA methylation is associated with depressive symptoms, an area which requires further research.

Epigenetic modifications, such as DNA methylation and histone modifications, play important roles in gene expression and regulation, and are highly involved in cellular processes such as stem cell pluripotency/differentiation and tumorigenesis. Chromatin immunoprecipitation (ChIP) is the technique of choice for examining in vivo DNA-protein interactions and has been a great tool for studying epigenetic mechanisms. However, conventional ChIP assays require millions of cells for tests and are not practical for examination of samples from lab animals and patients. Automated microfluidic chips offer the advantage to handle small sample sizes and facilitate rapid reaction. They also eliminate cumbersome manual handling. In this report, I will talk about three different projects that utilized microfluidic immunoprecipitation followed by next genereation sequencing technologies to enable low input and high through epigenomics profiling. First, I examined RNA polymerase II transcriptional regulation with microfluidic chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) assays. Second, I probed the temporal dynamics in the DNA methylome during cancer development using a transgenic mouse model with microfluidic methylated DNA immunoprecipitation followed by next generation sequencing (MeDIP-seq) assays. Third, I explored negative enrichment of circulating tumor cells (CTCs) followed by microfluidic ChIP-seq technology for studying temporal dynamic histone modification (H3K4me3) of patient-derived tumor xenograft on an immunodeficient mouse model during the course of cancer metastasis. In the first study, I adapted microfluidic ChIP-seq devices to achieve ultrahigh sensitivity to study Pol2 transcriptional regulation from scarce cell samples. I dramatically increased the assay sensitivity to an unprecedented level (~50 K cells for pol2 ChIP-seq). Importantly, this is three orders of magnitude more sensitive than the prevailing pol2 ChIP-seq assays. I showed that MNase digestion provided better ChIP-seq signal than sonication, and two-steps fixation with MNase digestion provided the best ChIP-seq quality followed by one-step fixation with MNase digestion, and lastly, no fixation with MNase digestion. In the second study, I probed dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, I developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ~100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled me to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using this data, I identified differentially methylated regions and their associated genes in different periods of cancer development. Interestingly, the results showed that methylomic features are dynamic and change with tumor developmental stage. In the last study, I developed a negative enrichment of CTCs followed by ultrasensitive microfluidic ChIP-seq technology for profiling histone modification (H3K4Me3) of CTCs to resolve the technical challenges associated with CTC isolation and difficulties related with tools for profiling whole genome histone modification on tiny cell samples. / Ph. D. / The human genome has been sequenced and completed over a decade ago. The information provided by the genomic map inspired numerous studies on genetic variations and their roles in diseases. However, genomic information alone is not always sufficient to explain important biological processes. Gene activation and expression are not only associated with alteration in the DNA sequence, but also affected by other changes to DNA and histones. Epigenetics refers to the molecular mechanisms that affect gene expression and phenotypes without involving changes in the DNA sequence. For example, the DNA can get methylated, the histone protein that is wrapped around by DNA can also get methylated or acetylatied, and transcription factors can bind to different part of DNA. All of these can affect gene expression without alter the DNA sequences. Epigenetic changes occur throughout all stages of cell development or in response to environmental cues. They change transcription patterns in a tissue/cell-specific fashion. For example, transcriptional silencing of tumor-suppressor genes by DNA methylation plays an important role in cancer development. Therefore, understanding of epigenetic regulations will help to improve various aspects of biomedicine. For instance, personalized medicine can be vi tailored based on epigenetic profile of certain patient to specifically control gene expression in the disease treatment. However, the technology for profiling epigenetic modifications, i.e. Chromatin Immunoprecipitation (ChIP), suffers from serious limitations. The key limitation is the sensitivity of the assay. Conventional assay requires a large number of cells (>10⁶ cells per ChIP). This is feasible when using cell lines. However, such requirement has become a major challenge when primary cells are used because very limited amounts of samples can be generated from lab animals or patients. Population heterogeneity information may also be lost when a large cell number is used. In this project, we developed an automated ultrasensitive microfluidic chromatin/DNA immunoprecipitation followed by next-generation sequencing (ChIP/MeDIP-Seq) technology for profiling epigenetic modifications (e.g., histone modifications, transcriptional regulations, and DNA methylation). We extensively optimized design parameters for each and every step of ChIP/MeDIP (e.g. sonication/crosslinking time, antibody concentration, washing conditions) in order to reach highest sensitivity of 0.1 ng DNA (or ~50-100 cells) as starting material for IP, which is roughly 4-5 orders of magnitude higher than the prevailing protocol and 2-3 orders of magnitude higher than the-state-of-the-art(~50 ng). With such sensitivity, we were able to study temporal dynamics in the DNA methylomes during the various stages of mammary cancer development from a transgenic mouse mode. We were able to investigate transcriptional regulation of RNA polymerase II from scarce cell samples. We were also able to study histone modification (H3K4Me3) of circulating tumor cells during cancer metastasis.

Die Geschlechtsbestimmung bei Säugetieren erfolgt über gegensätzliche Netzwerke von ovariellen und testikulären Genen, die recht gut charakterisiert sind. Die epigenetischen Mechanismen, insbesondere diejenigen, die die 3D-Chromatinorganisation beeinflussen, sind jedoch größtenteils unbekannt. Ich habe die 3D-Chromatinlandschaft der Geschlechtsbestimmung in vivo untersucht, indem ich FACS-sortierte embryonale Mausgonadenpopulationen vor und nach der Geschlechtsbestimmung in beiden Geschlechtern analysierte. Dabei wurde eine begrenzte Variation in der dreidimensionalen Chromatindynamik beobachtet, insbesondere bei den Topologically Associating Domains (TADs). Konventionelle Hi-C-Analysemethoden sind hauptsächlich auf vordefinierte 3D-Strukturen ausgerichtet und könnten potenziell andere Veränderungen in der Chromatinorganisation übersehen, die für die Genregulation relevant sein könnten. Um diese Einschränkungen zu überwinden, wurde METALoci eingesetzt - ein innovatives Werkzeug, das Hi-C- und ChIP-seq-Daten integriert und räumliche Autokorrelationsanalyse nutzt, um dreidimensionale Enhancer-Hubs im gesamten Genom zu identifizieren. METALoci zeigte eine deutliche Umverdrahtung von Chromatininteraktionen während der Geschlechtsbestimmung, die die regulatorischen Landschaften von Hunderten von Genen beeinflusste. Darüber hinaus führte die Vorhersagekraft von METALoci in Kombination mit funktionalen Validierungen an transgenen Mäusen zur Identifizierung eines neuen Fgf9-regulatorischen Hubs. Die Deletion dieses Hubs führte zu teilweisem Geschlechtsumkehr von männlich zu weiblich, mit einer Hochregulierung ovarieller spezifischer Marker und der Einleitung der Meiose. So erweist sich die räumliche Autokorrelationsanalyse als eine effektive Strategie zur Identifizierung von regulatorischen Netzwerken, die mit biologischen Prozessen verbunden sind, und zur anschließenden Charakterisierung der funktionalen Rolle des dreidimensionalen Genoms. / Mammalian sex is determined by opposing networks of ovarian and testicular genes that are relatively well characterized. Yet, the epigenetic mechanisms governing sex determi- nation, in particular those involving 3D chromatin organization, remain largely unknown. This gap of knowledge constrains our understanding of a fundamental process for species reproduction and perpetuation. Here, I explored the 3D chromatin landscape of sex deter- mination in vivo, by profiling FACS-sorted embryonic mouse gonadal populations, prior and after sex determination, in both sexes. Using conventional Hi-C analysis tools, limited variation in the 3D chromatin dynamics was observed, especially at the level of Topolog- ically Associating Domains (TADs). This contrasts with the broad transcriptional differ- ences occurring during sex determination. Yet, conventional Hi-C analysis methodologies are largely focused on predefined 3D structures, potentially overlooking other types of changes in chromatin organization that might be relevant for gene regulation. To ad- dress these limitations, METALoci was applied- an innovative tool that integrates Hi-C and ChIP-seq data and relies on spatial auto-correlation analysis to identify 3D enhancer hubs distributed throughout the genome. METALoci uncovered a prominent rewiring of chromatin interactions during sex determination, affecting the regulatory landscapes of hundreds of genes. Furthermore, METALoci ’s predictive capacity, in combination with functional validations in transgenic mice led to the identification of a novel Fgf9 regulatory hub, which deletion resulted in partial male-to-female sex reversal with the upregulation of ovarian-specific markers and the initiation of meiosis. Thus, spatial auto-correlation anal- ysis proves to be an effective strategy to identify regulatory networks linked to biological processes and to subsequently characterize the functional role of the 3D genome.

Direkte zelluläre Reprogrammierung wird durch den Einsatz von Transkriptionsfaktoren (TFs) erreicht, die das Zellschicksal induzieren und die Umwandlung in einen gewünschten Zelltyp direkt einleiten. Die Fähigkeit der TFs, die Identität von Zelltypen umzuprogrammieren, wird jedoch durch den zellulären Kontext bestimmt und ist durch hemmende Mechanismen eingeschränkt. Diese hemmenden Mechanismen schützen und erhalten das Zellschicksal und wirken daher als Barrieren für die Reprogrammierung. Ein Faktor, der als Barriere der Reprogrammierung fungiert, ist das Histon-Chaperon FACT. Es ist jedoch nicht bekannt, wie FACT das Zellschicksal sichert. Dieses Projekt entschlüsselt die zugrundeliegenden Reprogrammierungsmechanismen bei der Deletion von FACT in C. elegans. Das Aurora-Kinase B kodierende Gen air-2 wurde als Promotor der Reprogrammierung identifiziert. Aurora-Kinase B fördert die Umwandlungdes Zellschicksals, indem sie das Chromatin durch Phosphorylierung von H3S10-Resten umgestaltet. Darüber hinaus identifiziere ich die Histon-Acetyltransferase CBP-1 als Promotor der Reprogrammierung durch die Acetylierung von H3K18 und H3K27. Die Deletion des Cytochrom c-Oxidase - 1 kodierenden Gens cco-1, einer Untereinheit des mitochondrialen Atmungskettenkomplexes, ermöglicht eine von CBP-1 abhängige Reprogrammierung von Darmzellen zu Neuronen. Diese Beobachtung wirft ein neues Licht auf die Art und Weise, wie zelluläre Störungen, die in verschiedenen Kompartimenten durch die Deletion zellulärer Schutzmechanismen entstehen, zu ähnlichen Effekten bei der Reorganisation des Chromatins führen können, welche die Reprogrammierung vorantreiben. Darüber hinaus beschreibe ich eine mögliche Rolle der mitochondrialen Funktion bei der durch FACT-Deletion vermittelten Reprogrammierung durch die Induktion des mitochondrialen Chaperons HSP60. Schließlich kläre ich auf, wie FACT zelluläre Schicksale schützt, indem es die Integrität des Chromatins während der Transkription bewahrt. / Direct cellular reprogramming is achieved by using cell fate-inducing transcription factors (TFs) that directly induce conversion to a desired cell type. However, the ability of TFs to reprogram cells is defined by cellular context and is usually restricted by inhibitory mechanisms. Studying barriers of cellular reprogramming in vivo is a crucial step to attaining its therapeutic potential and provides important insights into the basic biology of cell fate regulation. One factor that acts as a barrier of reprogramming is the histone chaperone FACT. However, how FACT safeguards cellular fate is not yet known. Here, we unravel the underlying reprogramming mechanisms upon FACT depletion in C. elegans. To this end, an enhancer/suppressor screen with epigenetic regulators was performed. This screen identified the kinase Aurora B encoding gene air-2 as a promotor of reprogramming, promoting cell fate conversion by remodelling chromatin through the phosphorylation of H3S10. Additionally, I identify the histone acetyltransferase CBP-1 as a promotor of cell fate conversion through the acetylation of H3K18 and H3K27. Moreover, I characterize another reprogramming event where CBP-1 promotes reprogramming. Depleting the cytochrome c oxidase – 1 encoding gene cco-1, a subunit of the mitochondrial respiratory chain complex, allows for gut-to neuron reprogramming that is dependent on CBP-1. FACT and cco-1-depletion-mediated reprogramming show an overlap in reprogramming pathways. This observation sheds new light on how cellular perturbations originating in different compartments through depletion of cellular safeguards can produce similar effects on chromatin reorganization that drive reprogramming. I describe a potential role for mitochondrial function in FACT-depletion-mediated reprogramming through the induction of the mitochondrial chaperone HSP60. Lastly, I elucidate how FACT protects cellular fates through its role as a safeguard of chromatin integrity during transcription.

Die zentromerischen Regionen eukaryotischer Chromosomen sind notwendig für die Segregation der Schwesternchromatiden während der Zellteilung. In den Nukleosomen in diesen Regionen ist das kanonische Histon H3 durch die zentromerische Histon H3 Variante CENP-A ersetzt. Diese wird in Saccharomyces cerevisiae Cse4 genannt. Indem CENP-A die geordnete Assemblierung einzelner Untereinheiten des Kinetochors vermittelt, spezifiziert es die zentromerische Chromosomenregion und ist damit essentiell für die korrekte Segregation der Chromosomen während der Zellteilung. Kürzlich wurde gezeigt, dass Cse4 posttranslational modifiziert wird. Von Bedeutung für diese Arbeit sind die in der essentiellen Domäne des N-terminus liegenden Methylierung von Arginin 37 und die Acetylierung von Lysin 49, die durch phänotypische Suppression eine genetische Interaktion und eine antagonistische Funktion bei der epigenetischen Regulation in der korrekten Assemblierung der Kinetochoruntereinheiten zeigen. In dieser Arbeit wurde gezeigt, dass die Acetylierung an Cse4K49 von der Histonacetyltransferase Gcn5 abhängt, und dass dieses Enzym Komponenten des SAGA-Komplexes, aber nicht des ADA-Komplexes benötigt. Außerdem konnte in dieser Arbeit gezeigt werden, dass die Acetylierung von K49 in der frühen S-Phase ansteigt und zum Ende der S-Phase abnimmt. Es konnte weiterhin eine biochemische Interaktion der N-terminalen Domäne von Cse4 mit der COMA-Untereinheit Ctf19, der zentralen Region des Kinetochors, nachgewiesen werden, möglicherweise mit einer Präferenz zu einer nicht acetylierten Form von Cse4K49. In dieser Arbeit konnte gezeigt werden, dass der Transkriptions-Co-Aktivator Komplex SAGA eine Funktion an der zentromerischen Region in S. cerevisiae aufweist. Des Weiteren ist die Acetylierung an Cse4K49 in die Rekrutierung der Kinetochoruntereinheit Ctf19 involviert, und gibt damit einen Hinweis auf einen epigenetischen Regulationsmechanismus während der Chromosomensegregation. / The centromeric regions of eukaryotic chromosomes are essential for proper chromosome segregation. The nucleosomal composition in these regions differs from that of canonical nucleosomes in that histone H3 is replaced by the H3 variant CENP-A, which is termed Cse4 in Saccharomyces cerevisiae. CENP-A is the most important epigenetic mark on centromeres and essential for accurate kinetochore establishment at centromeric sites, which in turn are necessary to connect the centromeric sites to the microtubules. Whereas the histone fold domain of Cse4 shares a sequence homology with canonical histone H3, the N-terminal domain is rather long as compared to canonical histone tails and the centromeric histone variants of higher eukaryotes. One part of this flexible, positively charged histone tail has been shown to represent an essential N-terminal domain (END). Lysine 49 was defined as an acetylation site in this region, but the modification remains to be characterized in detail. In this study, we found that the Cse4K49 acetylation is dependent on the histone acetyltransferase Gcn5, and that the enzyme in this context acts within the SAGA/SLIK complex, whereas an involvement of the smaller ADA complex was not observed. Furthermore, we addressed the cell-cycle dependence of the K49 acetylation and found an increase in early S-phase and a decrease in late S-phase, whereas the R37 methylation persisted throughout S-phase. Ctf19 was found to bind acetylated and unacetylated Cse4K49 with a preference for unacetylated Cse4. The findings presented here show that the transcriptional co-activator complex SAGA functions at centromeric regions in S. cerevisiae, and that the Cse4K49 acetylation has an influence on the recruitment of the kinetochore subunit Ctf19. This suggests an epigenetic regulatory mechanism involving a specific acetylation on the N-terminus of Cse4 in chromosome segregation.

Im Fokus der Dissertation stand die Evaluierung molekularer Möglichkeiten zur Findung geeigneter Biomarker für das Prostatakarzinom (PCa). Zunächst wurde die epigenetische Promotormethylierung der Gene GSTP1, RARbeta2, APC und PITX2 im Hinblick eines möglichen Methylierungsfeldeffektes untersucht. Die RARβ2-Promotormethylierung konnte als potentieller Marker zur Tumorvorhersage im histologisch normal erscheinenden Gewebe bestimmt werden. GSTP1 zeigte eine äußerst spezifische Tumormethylierung, was dessen diagnostisches Potenzial unterstreicht. Die Methylierung korrelierte zudem mit pathologischen Parametern. Ein Screening im Array-Format identifizierte weitere Gene, deren Promotormethylierung diagnostisch interessant sein könnten. Des Weiteren wurden Genexpressionsanalysen angiogenetischer und Endothel-assoziierter Faktoren durchgeführt, deren diagnostische und prognostische Aussagekraft für das PCa ermittelt wurden. Dabei konnte für Caveolin-1 (CAV1) eine signifikante Herunterregulierung der mRNA-Menge im Tumor gezeigt werden. Die Bisulfit-Sequenzierung von sieben CpG-Dinukleotiden im CAV1-Promotor ergab zwischen Tumor- und tumorangrenzendem Gewebe differenzielle Methylierungsmuster, die zu einer verminderten CAV1-Transkription führen könnten. Zudem führte eine 5-Aza-2-desoxycytidin-Behandlung der CAV1-defizienten LNCaP-Zelllinie zur Reexpression. Weiterhin assoziierte CAV1 invers mit pathologischen Parametern und zeigte prognostische und diagnostische Relevanz. Immunhistologisch wurde CAV1 z.T. deutlich verringert in Endothelzellen und Fibroblasten des Tumors nachgewiesen. Ko-Kultivierungsversuche von HUVEC mit konditionierten Zellmedien ergaben z.T. signifikant reduzierte CAV1-Mengen in HUVEC. Die Ergebnisse zeigen, dass epigenetische Veränderungen wertvolle Informationen zur Diagnostik, Prognostik und Progression des PCa liefern. Zudem konnte CAV1 als potentieller Marker des PCa identifiziert werden. / The focus of the thesis was to evaluate molecular possibilities to find suitable biomarkers for prostate cancer (PCa). First, the epigenetic promoter methylation patterns of several genes (GSTP1, RARbeta2, APC and PITX2, respectively) were investigated for a possible methylation field effect. The RARbeta2 promoter methylation could be determined as a possible marker for tumor prediction in histologically normal-appearing tissue. GSTP1 showed a highly specific tumor methylation, underlining its diagnostic potential. The methylation also correlated with pathological parameters. Screening in an array format identified other genes whose promoter hypermethylation could be diagnostically interesting. Furthermore, gene expression analysis of angiogenic and endothelial-associated factors was performed to determine their diagnostic and prognostic potentials for PCa. For CAV1, a significant down-regulation of its mRNA level could be determined in the tumor. Bisulfite sequencing of seven CpG dinucleotides in the CAV1 promoter showed different methylation patterns between tumor and tumor adjacent tissue that could cause a reduced transcription. Furthermore, treatment of the CAV1-deficient LNCaP cell line with 5-aza-2-deoxycytidine led to CAV1 reexpression. Additionally, CAV1 inversely associated with pathological parameters and showed prognostic and diagnostic relevance. Immunohistochemical analysis clearly demonstrated decreases in CAV1 protein expression in endothelial cells and fibroblasts of the tumor. Conditioned media of cultivated tumor cells partly induced downregulation of the CAV1 protein level in HUVEC. The results show that epigenetic and angiogenic processes play crucial roles and provide valuable information for diagnosis, prognosis and progression of PCa. Moreover, CAV1 could be identified as a potential marker of prostate cancer.

Significant alterations in the environmental conditions can have pronounced effects on plant genome stability. Recent evidence argues for a global involvement of the components of epigenetic modules in the regulation of genome homeostasis both immediately after stress exposure and long after environmental cues were acquired. The last observation is of particular interest as the memory of imposing stress can be maintained at the molecular level throughout plant ontogenesis and may be faithfully propagated into the following generation. Our study provides evidence that epigenetic repercussions exerted by stress exposure of parental plants manifest themselves in untreated progeny at all three levels of the epigenetic module: DNA methylation, histone posttranslational modifications and small RNA metabolism. Additionally, the results of our study shed new light on the engagement of the epigenetic machinery in the maintenance of plant genome integrity by counteracting the activity of invading nucleic acids. / xv, 280 leaves : ill. ; 29 cm

Phenotypic plasticity is the ability of an organism to express different phenotypes in response to biotic or abiotic environmental cues. The ability of an organism to make changes during development to adjust to changes in its environment is a key to survival. Sexually reproducing organisms that have short life cycles and that are easy to raise in the laboratory are more conducive for developmental phenotypic plasticity. Considerable research has already been carried out on the brine shrimp, Artemia franciscana, regarding its morphology due to changing salinities. There is, however, little research considering subsequent generations and how there morphology might be affected by parental experiences. This study has examined: 1) the morphological effects of different rearing regimes of different salinity levels, and 2) the epigenetic transgenerational transfer of these morphological traits in A. franciscana. Measurements included rate of growth (as measured by instar), body size, body length, and other morphological traits. A gradual increase to more hyperosmotic conditions during development produced brine shrimp that were larger in size and also more developmentally advanced. Salinity stress experienced by adults had increased the growth rate in the F1 offspring of A. franciscana. Collectively, these data indicate that Artemia franciscana is a tractable model for investigating phenotypic plasticity. These findings have added to the ever-growing field of developmental phenotypic plasticity while also providing more information on the natural history and adaptive abilities of A. franciscana.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.