When the human genome project was completed, it revealed a surprising result. 98% of the genome did not code for protein of which more than 50% are repeats— later known as ”Junk DNA”. However, comparative genomics unveiled that many noncoding elements are evolutionarily constrained; thus luckily to have a role in genome stability and regulation. Though, their exact functions remained largely unknown. Several large international consortia such as the Functional Annotation of Mammalian Genomes (FANTOM) and the Encyclopedia of DNA Elements (ENCODE) were set to understand the structure and the regulation of the genome. Specifically, these endeavors aim to measure and reveal the transcribed components and functional elements of the genome. One of the most the striking findings of these efforts is that most of the genome is transcribed, including non-conserved noncoding elements and repeat elements. Specifically, we investigated the evolution and epigenetic properties of noncoding elements. 1. We compared genomes of evolutionarily distant species and showed the ubiquity of constrained noncoding elements in metazoa. 2. By integrating multi-omic data (such as transcriptome, nucleosome profiling, histone modifications), I conducted a comprehensive analysis of epigenetic properties (chromatin states) of conserved noncoding elements in insects. We showed that those elements have distinct and protective sequence features, undergo dynamic epigenetic regulation, and appear to be associated with the structural components of the chromatin, replication origins, and nuclear matrix. 3. I focused on the relationship between enhancers and repetitive elements. Using Cap Analysis of Gene Expression (CAGE) and RNASeq, I compiled a full catalog of active enhancers (a class of noncoding elements) during myogenesis of human primary cells of healthy donors and donors affected by Duchenne muscular dystrophy (DMD). Comparing the two time-courses, a significant change in the epigenetic landscape in DMD was observed that lead to global dysregulation of enhancers and associated repetitive elements.

Les cellules souches embryonnaires de souris (ESCs) et les cellules souches de l'épiblaste (EpiSCs) représentent, respectivement, les états naïf et amorcé de la pluripotence et sont maintenues in vitro par des voies de signalisation spécifiques. De plus les ESCs cultivées dans un milieu sans sérum avec deux inhibiteurs (2i) sont décrites comme étant les plus naïves. Plusieurs études ont suggéré que chaque type de cellules pluripotentes est caractérisé par une organisation différente de l'épigénome. Nous présentons ici une étude comparative de l'état épigénétique et transcriptionnel des séquences satellites répétées péricentromériques (PCH) entre les ESCs (2i et sérum) et EpiSCs. Nous montrons que H3K27me3 au PCH est très dynamique et peut discriminer les ESCs en 2i des autres cellules souches pluripotentes. Alors que la transcription des séquences satellites est élevée dans les ESCs en sérum, elle est plus faible dans ESCs en 2i et encore plus réprimée dans les EpiSCs. La suppression de la méthylation de l'ADN ou d'H3K9me3 dans les ESCs conduit à un dépôt important de H3K27me3 au PCH, mais peu de changements transcriptionnels de ces séquences. En revanche, l'absence d'H3K9me3 dans les EpiSCs n'empêche pas la méthylation de l'ADN au PCH, mais induit la transcription de ces séquences. La conversion in vitro des ESCs en EpiSCs est plus longue que le passage des cellules de l'ICM en épiblaste in vivo. Cette inefficacité ne peut pas être expliquée par une mise en place retardée du nouveau réseau transcriptionnel. Pour conclure notre étude a révélé que les EpiSCs ont perdu de la plasticité par rapport au ESCs sur l'hétérochromatine ainsi que l’euchromatine, comme le montre la réduction des niveaux d'H3K9ac et des domaines bivalents, étant ainsi plus proche épigénétiquement de cellules somatiques que de la pluripotence naïve. / Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent naïve and primed pluripotency states, respectively and are maintained in vitro using specific signaling pathways. Furthermore, ESCs cultured in serum-free medium with two inhibitors (2i) are described as being the most naïve. Several studies have suggested that each pluripotent cell type is characterized by a different epigenome organization. Here we present a comparative study of the epigenetic and transcriptional state of centromeric and pericentromeric (CH/PCH) satellite repeats in ESCs (2i and serum ones) and EpiSCs. We show that the pattern of H3K27me3 at PCH is highly dynamic and discriminate 2i-ESCs from the other pluripotent stem cells. Whereas satellites transcription is high in serum-ESCs, it is lower in 2i-ESCs and even more repressed in EpiSCs. Removal of either DNA methylation or H3K9me3 in ESCs leads to enhanced deposition of H3K27me3 but few changes in satellite transcription. By contrast, in EpiSCs removal of H3K9me3 does not prevent DNA methylation at PCH but de-represses the satellite transcription. In vitro conversion from naive to primed pluripotency showed an important delay compared to the in vivo development of ICM cells into post-implantation epiblast. Such inefficiency cannot be explained by a delayed switch to the new transcriptional network. Altogether our study reveals that EpiSCs have lost the chromatin plasticity of ESCs on heterochromatin as well as euchromatin, as shown by the reduction of H3K9ac levels and bivalent domains, thus being closer to somatic cells in terms of epigenetics than naive pluripotency.

Methyl reader proteins recognize and bind to post-translationally methylated residues and have functional roles in diverse cellular processes including gene regulation, development and oncogenesis. The CBX polycomb paralog family of methyllysine readers recognize trimethyllysine lysine residues on histone tail 3 and repress transcription by compacting chromatin. The polycomb paralogs form multi-protein complexes that silence the expression of tumour suppressor genes, and play important roles in regulating cell cycle and differentiation. Each paralog is structurally similar, yet has distinct functions, of which many are unknown. My work has focused on the design and synthesis of CBX inhibitors and on the development of new methodologies for the discovery of inhibitors targeting methyllysine readers. In this work, I report on a series of potent peptidic inhibitors that selectively target the CBX polycombs, as well as the first selective inhibitor for the family member CBX6, and dual-active inhibitors that target CBX6/CBX8. The results demonstrate the potential to achieve selectivity through interactions outside of the methyllysine binding domain. Structural determinants in the binding pocket of each protein that differ within the family and give rise to selectivity were discovered. I will also report on a series of peptidomimetic CBX inhibitors that are active in cells. Cellular active inhibitors are critical for understanding the biological role of each CBX protein and their potential as therapeutic targets. New high-throughput approaches are needed to efficiently target methyllysine readers by chemical inhibition. I describe in this work a strategy for creating massive libraries of phage-displayed peptidic inhibitors containing methyllysine mimics. Synthetic optimization on cysteine-containing peptide phage constructs allowed for the successful installation of Kme3 mimics. This is the first report of a post-translational methylated peptide phage library. The methodology I developed can be used in a synthetic chemistry-driven adaptation of traditional phage display for the screening of millions of peptide-based compounds. Strategies that allow for diversity and high throughput screening will aid in future efforts in targeting the highly similar CBX proteins. / Graduate / 2021-06-01

The goal of the ENCODE project has been to characterize regulatory elements in the human genome, such as regions bound by transcription factors (TFs), regions of open chromatin and regions with altered histone modifications. The ENCODE consortium has performed a large number of whole-genome experiments to measure TF binding, chromatin accessibility, gene expression and histone modifications, on a multitude of cell types and conditions in both human and mouse. In this dissertation I describe the analysis of numerous datasets comprising 66 epigenomes, chromatin accessibility and expression data across twelve tissues and seven time points, during mouse embryonic development. We defined chromatin states using histone modification data and performed integrative analysis on the states. We observed coordinated changes of histone mark signals at enhancers and promoters with gene expression. We detected evolutionary conserved bivalent promoters, selectively silencing ~3,400 genes, including hundreds of TFs regulating embryonic development. Second, I present a supervised method to predict TF binding across cell types, with features based on DNA sequence and patterns in DNase I cleavage data. We found that sequence and DNase read counts can outperform other features as well as state-of-the-art methods. I also describe our contribution to the ENCODE TF Binding DREAM challenge where we developed a method, using multiscale features and Extreme Boosting. Third, I describe methods, tools, and computational infrastructure that we have developed to handle large amounts of experimental data and metadata. These tools are fundamental to the selection and integration of large experimental datasets and are at the core of our pipelines, which are described in this dissertation. Finally, I present the protein docking server I developed, as well as algorithms and routines for post-processing predictions and protein structures. Collectively, this body of work encompasses computational approaches to the analyses of chromatin states, gene regulation, and the integration of large experimental datasets. / 2021-08-31T00:00:00Z

This thesis describes my work to develop methods and assays to study transgenerational inheritance in the widely used genetic model organism Caenorhabditis elegans (C. elegans). In the first chapter, I describe a novel method that uses an exogenous histamine-selective chloride channel (HisCl1) for negative selection in transgenesis. C. elegans transgenesis is a core technique used by most laboratories and often requires distinguishing between rare animals with a single-copy transgene inserted into the genome from more frequent animals that carry multiple copies of the transgene in extra-chromosomal arrays. I demonstrate that histamine-selection induces rapid and irreversible paralysis in only array animals thus allowing quick identification of the desired transgenic animals. In the second chapter, I develop a high-throughput assay for quantifying transgenerational epigenetic inheritance of endogenous gene silencing. Small RNA -mediated gene silencing leads to an increased incidence of males in the population which can be inherited for four to six generations. I identify a fluorescent marker that specifically fluoresces in males and show that I can use a large-particle particle sorter to quantify the frequency of males in a population. This automated system will allow me to follow inheritance patterns over at least ten generations in various mutant backgrounds in parallel to determine the genetic basis and the rules of epigenetic inheritance.

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

<p>Aging is associated with a decline in visual function and increased prevalence of ocular disease, correlating with changes in the transcriptome and epigenome of cells in the eye. The extended photoreceptor cell lifespan, in addition to its high metabolic needs due to phototransduction, makes it critical for these neurons to continually respond to the stresses associated with aging by mounting an appropriate gene expression response. My work, in collaboration with fellow lab members and colleagues, has focused on better understanding the regulatory mechanisms that result in age-dependent transcriptional changes in photoreceptors, and if these changes not only correlate with but cause the decrease in function with age. In order to better characterize photoreceptor specific changes my initial work first focused on expanding the gene expression toolkit for eye specific expression. In chapter 1 we describe a previously unnoticed sevenless mutation present in the majority of the TRiP RNAi collection. In chapter 2 we characterized the currently available eye- and photoreceptor-specific binary expression system drivers in <i>Drosophila</i>. Using a luciferase and fluorescent reporter, we characterized the relative expression and cell type-specificity of each driver in the 10-day old adult eye. Also, wecharacterized the expression pattern of these drivers in various developmental stages. We then compared several Gal4 drivers from the Bloomington Drosophila Stock Center (BDSC) including GMR-Gal4, longGMR-Gal4 and Rh1-Gal4 with newly developed Gal4 and QF2 drivers that are specific to different cell types in the adult eye. In addition, we generated drug-inducible Rh1-GSGal4 lines and compared their induced expression with an available GMR-GSGal4 line. Although both lines had significant induction of gene expression measured by luciferase activity, Rh1-GSGal4 was expressed at levels below the detection of the fluorescent reporter by confocal microscopy, while GMR-GSGal4 showed substantial reporter expression in the absence of drug by microscopy. This study systematically characterized and compared a large toolkit of eye- and photoreceptor-specific drivers, while also uncovering some of the limitations of currently available expression systems in the adult eye.</p><p>In chapter 3, we sought to untangle the more general neuronal age-dependent transcriptional signature of photoreceptors with that induced by light stress. To do this, we aged flies or exposed them to various durations of blue light, followed by photoreceptor nuclei-specific transcriptome profiling. Using this approach, we identified genes that are both common and uniquely regulated by aging and light induced stress. Whereas both age and blue light induce expression of DNA repair genes and a neuronal-specific signature of death, both conditions result in downregulation of neurotransmitters important for synaptic transmission. Interestingly, blue light uniquely induced genes that directly counteract the overactivation of the phototransduction signaling cascade. Lastly, unique gene expression changes in aging photoreceptors included the downregulation of genes involved in membrane potential homeostasis and mitochondrial function, as well as the upregulation of immune response genes. We proposed that light stress contributes to the aging transcriptome of photoreceptors, but that there are also other environmental or intrinsic factors involved in age-associated photoreceptor gene expression signatures.</p><p>In chapter 4, we sought to test if age-associated changes in gene expression patterns in the eye directly contribute to the increased risk of retinal degeneration. To do this, we performed a targeted photoreceptor specific RNAi screen in <i>Drosophila </i>to identify gene regulatory factors that result in premature, age-dependent retinal degeneration. From an initial set of 155 RNAi lines each targeting a unique gene and spanning a diverse set of gene regulatory factors, we identified 18 high confidence target genes whose decreased expression in adult photoreceptors leads to premature and progressive retinal degeneration. The 18 target genes were enriched for factors involved in the regulation of transcription initiation, pausing, and elongation, suggesting that these processes are essential for maintaining the health of aging photoreceptors. To identify the genes regulated by these factors, we profiled the photoreceptor transcriptome in a subset of lines. Strikingly, two of the 18 target genes, <i>Spt5</i> and <i>domino</i>, show substantially similar changes in gene expression to those observed with advanced age.</p><p>Together, our data suggests that dysregulation of the mechanisms involved in transcription initiation and elongation plays a key role in shaping the transcriptome of aging photoreceptors. Further, our findings indicate that the age-dependent changes in gene expression not only correlate, but might also contribute to increased risk of retinal degeneration.</p>

Physical exercise offers a highly effective health-endowering activity as has been evidence using rodent models of Parkinson’s disease (PD). It is a particularly useful intervention in individuals employed in sedentary occupations or afflicted by a neurodegenerative disorder, such as PD. The several links between exercise and quality-of-life, disorder progression and staging, risk factors and symptoms-biomarkers in PD all endower a promise for improved prognosis. Nutrition provides a strong determinant for disorder vulnerability and prognosis with fish oils and vegetables with a mediterranean diet offering both protection and resistance. Three factors determining the effects of exercise on disorder severity of patients may be presented: (i) Exercise effects upon motor impairment, gait, posture and balance, (ii) Exercise reduction of oxidative stress, stimulation of mitochondrial biogenesis and up-regulation of autophagy, and (iii) Exercise stimulation of dopamine (DA) neurochemistry and trophic factors. Running-wheel performance, as measured by distance run by individual mice from different treatment groups, was related to DA-integrity, indexed by striatal DA levels. Finally, both nutrition and exercise may facilitate positive epigenetic outcomes, such as lowering the dosage of L-Dopa required for a therapeutic effect.

Introduction: Gestational diabetes mellitus (GDM) is a significant public health concern, due to its association with short- and long-term complications in both mothers and offspring. DNA methylation and single nucleotide polymorphisms (SNPs) offer potential to serve as molecular biomarkers, which may lead to improved detection of GDM with positive effects on health outcomes. Aim: The aim of this study was to investigate whether DNA methylation and SNPs are associated with GDM and may offer potential as molecular biomarkers for GDM in South Africa (SA). Methods: This study followed a two-pronged approach. Firstly, literature searches were conducted to collate and synthesise all published articles reporting on the prevalence of GDM in SA, the screening and diagnostic strategies used, and the current status of DNA methylation and SNPs as biomarkers for GDM. Secondly, we conducted experiments to investigate global (n=201), genome-wide (n=24) and gene-specific DNA methylation (n=286) of the adiponectin gene (ADIPOQ) in whole blood of women with and without GDM, using an Enzyme-Linked Immunosorbent Assay, a methylationEPIC BeadChip Array and pyrosequencing, respectively. In addition, genotype and allele frequencies of ADIPOQ rs266729 and rs17300539, and methylenetetrahydrofolate reductase (MTHFR) rs1801133 were determined, using quantitative real-time PCR (n=449) and DNA sequencing for validation. Results: The literature search showed that the prevalence of GDM in SA has increased over the years. Furthermore, it showed that the lack of uniformity in screening and diagnosis between and within countries hamper the accurate detection of GDM. Lastly, the literature search identified several studies that support the use of DNA methylation and SNPs as potential biomarkers for GDM. Experimentally, we showed no differences in global DNA methylation between GDM and non-GDM groups. Interestingly, global DNA methylation levels were 18% (p=0.012) higher in obese compared to non-obese pregnant women. Genome-wide methylation analysis identified 1046 differentially methylated CpG sites (associated with 939 genes) (Cut-off threshold: M>0.06 and p<0.01). Among the top five CpG sites identified, one CpG mapped to the calmodulin-binding transcription activator 1 (CAMTA1) gene, which has been shown to regulate insulin production and secretion. Two CpG sites (-3410: p=0.048 and -3400: p=0.004) in the ADIPOQ promoter were hypomethylated during GDM in HIV negative, but not in HIV positive women. Lastly, no association between the ADIPOQ and MTHFR polymorphisms and GDM was observed in our population. Conclusion: To our knowledge, this is the first study to investigate the association between DNA methylation or ADIPOQ (rs266729 and rs17300539) and MTHFR (rs1801133) polymorphisms and GDM in SA. Findings suggest that gene-specific, but not global methylation nor SNPs rs266729, rs17300539 and rs1801133, may offer potential as molecular biomarkers of GDM in this population. Future longitudinal studies in larger samples that include both HIV negative and positive pregnant women are warranted to explore the candidacy of DNA methylation as molecular biomarkers for GDM. / Thesis (PhD)--University of Pretoria, 2019. / National Research Foundation (NRF) of South Africa, Thuthuka Grant (unique grant no. 99391). / South African Medical Research Council (SAMRC) / Obstetrics and Gynaecology / PhD / Unrestricted

There have been leaps in both fields of epigenetics and reproductive technology. This has culminated in the production of bi-maternal mouse offspring through a few studies utilizing direct gene mutations as functional-imprints. While these genetic interventions result in positive results, it has yet to be described, in full, what mechanisms underlie the epigenetic barriers to human gynogenesis. Between maternal and paternal imprints, differences in methylation patterns of CpG island promoters, non-coding regions, microsatellites, transposons, and histones result in differences in higher order chromatin structure, which ultimately impacts expression of certain genes. While the necessary components of a minimal paternal epigenetic program are described, programming this imprint onto m2, a hypothetical, experimentally-produced maternal genome with a paternal epigenome is still not elucidated. Sequential timing of imprint acquisition and maintenance and the numerous regulatory factors associated with them illuminate how difficult potential assisted reproductive epigenetic interventions will be. Other processes like histone-protamine exchange which also plays a crucial factor in structural regulation of imprints, as well as signaling through and after fertilization, pose logistical problems to creating a gynogenote through epigenetic means. Lastly, ethics surrounding the introduction of dangerous mutations and epialleles into the human population add another wall of caution and hesitance in exploring human gynogenesis as a reproductive technology.