No description available.

No description available.

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.

No description available.

No description available.

Multiple myeloma is a bone marrow malignancy characterized by the presence of monoclonal plasma cells. In 50-75% of myeloma patients, chromosome translocations at the IgH locus are observed, which result in overexpression of oncogenes from the translocated chromosome due to linkage with the IgH enhancers. IgH enhancer activity is mediated by the B cell-specific transcription factors Bob1 and Oct2. We hypothesized that inhibiting the IgH enhancer, through inhibition of Bob1 and Oct2, is a potential therapeutic strategy for translocation-positive myeloma. The expression and prognostic value of Bob1 and Oct2 in myeloma patient samples were assayed. High Bob1 expression was associated with increased survival, whereas high Oct2 expression was associated with reduced survival. In a t(4;14) myeloma cell line, Bob1 inhibition led to decreased expression of the translocated oncogene, FGFR3; however, this did not lead to decreased proliferation or increased apoptosis. To fully understand the roles of Bob1 and Oct2 in myeloma, further research is required. / Experimental Oncology

Cellular development and function necessitate precise patterns of gene expression. Control of gene expression is in part orchestrated by a class of remote regulatory elements, termed enhancers, which are brought into contact with promoters via DNA looping. Enhancers typically contain clusters of transcription factor binding sites, and TF recruitment to them is thought to play a key role in transcriptional control. In this thesis I have addressed two issues regarding gene regulation by enhancers. First, with recent genome-wide enhancer mapping, it is becoming increasingly apparent that genes are commonly regulated by multiple enhancers in the same cell type. How a gene’s regulatory information is encoded across multiple enhancers, however, is still not fully understood. Second, numerous recent studies have found that enhancers are enriched for expression-modulating and disease-associated genetic variants. However, understanding and predicting the effects of enhancer variants remains a major challenge. I focussed on a human lymphoblastoid cell line (LCL), GM12878, for which ChIP-Seq data are available for 52 different TFs from the ENCODE project. Significantly, Promoter Capture Hi-C data for the same LCL are available, making it possible to link enhancers to target genes globally. In the first part of the thesis, I investigated how gene regulatory information is encoded across enhancers. Specifically, I asked whether a gene tends to use multiple enhancers to bring the same or distinct regulatory information. I found that there was a general trend towards a “shadow” enhancer architecture, whereby similar combinations of TFs were recruited to multiple enhancers. However, numerous examples of “integrating” enhancers were observed, where the same gene showed large variation in TF binding across enhancers. Distinct groups of TFs were associated with these contrasting models of TF enhancer binding. To investigate the functional effects of variation at enhancers, I additionally took advantage of a panel of LCLs derived from 359 individuals, which have been genotyped by the 1000 Genomes Project, and for which RNA-Seq data are publically available. I used TF binding models to computationally predict variants impacting TF binding, and tested the association of these variants with the expression of the target genes they contact based on Promoter Capture Hi-C. Compared to the standard eQTL calling approach, this offers increased sensitivity as only variants physically contacting the promoter and predicted to impact TF binding are tested. Using this approach, I discovered a set of predicted TF-binding affinity variants at distal regions that associate with gene expression. Interestingly, a large proportion of these binding variants fall at the promoters of other genes. This finding suggests that some promoters may be able to act in an enhancer-like manner via long-range interactions, consistent with very recent findings from alternative approaches.

La différentiation des lymphocytes Th17 dépend fortement du facteur de transcription RORγt. RORγt a été identifié pour la première fois chez la souris comme un facteur spécifique des thymocytes, jouant un rôle important dans le développement du thymus. En effet, RORγt est exprimé sélectivement dans le stade double positif (DP) et il est ensuite réprimé dans le stade simple positif (SP) ainsi que dans les cellules CD4+ naïves périphériques. Les mécanismes moléculaires qui régulent l’expression transitoire de RORγt dans le thymus et en périphérie sont peu connus. Le but de ce projet est de définir les paramètres transcriptionnels et épigénétiques en corrélation avec l'expression de RORγt chez l’homme. Afin de caractériser les mécanismes moléculaires qui contrôlent l'expression de RORγt dans les thymocytes humains, nous avons effectué une analyse des modifications épigénétiques sur l’ensemble du locus RORC. Nos résultats montrent que le locus RORC subit un remodelage étendu, qui lui confère une conformation "permissive" lors de la transition du stade Double Négatif (DN) au stade DP (augmentation de H4ac et diminution de H3K27me3). Alors que, H3K4me3, qui est un marqueur de promoteurs actifs, est présente spécifiquement au niveau du promoteur RORC2, et uniquement dans les cellules qui expriment RORC2. Une analyse similaire des marqueurs épigénétiques pour les enhancers (H3K27ac) nous a permis d'identifier plusieurs régions avec une fonction potentiellement régulatrices de l'expression de RORC2. Nous avons démontré que certaines de ces régions avaient une fonction d'enhancer in vitro. Dans les cellules T humaines CD4+ naïves périphériques, nous avons observé que la stimulation du TCR en présence de TGFβ est suffisante pour induire RORγt et un remodelage du locus RORC. Nous avons également constaté que la cyclosporine A, un inhibiteur de la voie calcineurine/NFAT, inhibe fortement l'expression de RORC2. Cette inhibition est associée ˆ une diminution des marqueurs positifs de la chromatine. De plus, nous avons démontré que l’activation des cellules induit une activation de NFAT et sa liaison au promoteur RORC2 et aux enhancers. Cette liaison coïncide avec le recrutement de p300/CBP au promoteur qui est dépendant de la calcineurine, ce qui peut permettre le recrutement d'autres facteurs (tel que NFkB) importants pour la différenciation des Th17. Nous avons Également détecté la liaison de NFAT au locus RORC dans les thymocytes. Ces données démontrent un rôle central de NFAT dans la régulation Epigénétique et transcriptionnelle du locus RORC / Generation of inflammatory CD4+ Th17 cells is strongly dependent on the transcription factor retinoid-related orphan receptor, RORγt. RORγt was first identified in the mouse as a thymocyte-specific factor and was shown to play a critical role in the regulation of thymopoiesis. In the thymus RORγt is selectively expressed at the double positive stage (DP: CD4+ CD8+), and is down regulated in later stages of thymocyte development, as well as in naïve peripheral CD4+ T cells. The molecular mechanisms by which RORγt is transiently expressed during thymopoiesis and re-expressed in selected peripheral lymphocytes are poorly understood. The goal of this project is to define the transcriptional and epigenetic settings that correlate with RORγt expression. To start addressing the molecular mechanisms that control expression of human RORγt in thymocytes, we performed an analysis of epigenetic modifications at the RORC locus. Our findings show that the whole RORC locus undergoes extensive remodeling, assuming a more "permissive" conformation at the transition between the Double Negative and the DP stage (increase of H4ac and decrease of H3K27me3). However, H3K4me3, a mark of active promoters, is present specifically at the RORC2 promoter only at stages where RORC2 is expressed. A similar analysis of epigenetic marks for enhancers (H3K27ac) has allowed us to identify several regions with potential regulatory function on RORC2 expression, which displayed enhancer function in vitro. In human naïve peripheral CD4+ T cells we observed that TCR stimulation in the presence of TGFβ is sufficient to induce low levels of RORγt expression, and further remodeling of the RORC locus. We also found that cyclosporine A, an inhibitor of the calcineurin/NFAT pathway, strongly inhibited RORC2 expression, and was associated with a decrease in the positive chromatin marks at the RORC locus. Consistently, we demonstrated that cell stimulation induced NFAT activation, and binding to the RORC2 promoter and enhancers. This binding coincided with calcineurin-dependent p300/CBP recruitment and remodeling of the locus, which may allow binding of the transcriptional machinery and other factors (such as NFkB) important for Th17 differentiation. NFAT binding to the RORC locus could be also detected in thymocytes. These data demonstrate a central role for the NFAT pathway in the epigenetic priming the RORC locus for transcriptional competence.

The advancement of medicine around the world is happening at a rapid speed. There is not a day that one does not hear about a disease being cured or a wonder-drug being developed. Medicine has always been a field that treated a sick patient to bring that person back to “normal” function, which could be said to be the state he or she was in before acquiring the disease in question. However, due to scientific breakthroughs humanity is in a position that historically it has never been before. A place where a person can increase their mental and physical performance using pharmaceuticals to a level where genetics and upbringing alone would not have allowed. Pharmaceuticals have been created which can do for the brain what has been done for the body. These drugs provide an avenue in which a person’s cognitive functions can be increased beyond what was otherwise possible. The ethical and legal dilemmas posed by prescribing or allowing people to use nootropics is hotly debated. Greely, a leading figure in the bioethical debate, in connection with nootropics, has stated that the ethical concerns comprise three main aspects: safety, fairness and coercion., It is clear that these three aspects cover a wide ambit. The arguments for and against nootropics will not merely be rehashed but rather placed into the South African context to ascertain how best to provide for the ethical concerns relevant to South Africa. The ethics of nootropic use will be examined through the lens of the Beuchamp Childress model which determines ethical problems using the principles of autonomy, justice, beneficence and non-maleficence. The aim and contribution of this thesis will be to build a legal framework for the use of nootropics in South Africa, to create this one must ascertain what the current legal position is. Law is built from precedent in context of case law and legislation. The current law will be examined to see what can be used, what may need to be discarded and what would need to be added. A layered approach is used to determine what the legal position in South Africa currently is. A layered approach looks at the constitution and proceeds to legislation, case law, and legal articles and books. A major aspect around the use of nootropics will be informed consent and how it relates to the use of nootropics especially with regards to adolescents i.e. under 18 years of age. This thesis follows an MPhil in Medical Law and Ethics which outlined the doctrine of informed consent in South Africa and thus allows for the application of this doctrine to a pressing issue in South African society. / Thesis (PhD)--University of Pretoria, 2019. / Public Law / PhD / Unrestricted

Yes / DNA breaks at protein-coding sequences are well-established threats to tissue homeostasis and maintenance. They arise from the exposure to intracellular and environmental genotoxins, causing damage in one or two strands of the DNA. DNA breaks have been also reported in non-coding regulatory regions such as enhancers and promoters. They arise from essential cellular processes required for gene transcription, cell identity and function. One such process that has attracted recent attention is the oxidative demethylation of DNA and histones, which generates abasic sites and DNA single-strand breaks. Here, we discuss how oxidative DNA breaks at non-coding regulatory regions are generated and the recently reported role of NuMA (nuclear mitotic apparatus) protein in promoting transcription and repair at these regions.