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published_or_final_version / abstract / Medicine / Master / Master of Philosophy

La tuberculose demeure un problème majeur de santé publique. Mycobacterium tuberculosis infecte un tiers de la population mondiale et environ 3 millions de décès, des suites de l’infection, sont recensés chaque année dans le monde. La mise au point d’une stratégie vaccinale efficace constitue dès lors la solution idéale pour tenter d’éradiquer la bactérie. Le système immunitaire humain répond à l’infection par l’induction d’une réponse cellulaire, caractérisée essentiellement par la sécrétion de médiateurs pro-inflammatoires, comme l’interféron-gamma (IFN-?). A la fois les lymphocytes T CD4+ et T CD8+ produisent cette cytokine, mais les seconds sont également doués de propriétés cytotoxiques, entraînant la mort de la cellule infectée et du bacille. L’évaluation du potentiel de nouveaux candidats vaccins implique dès lors la caractérisation exhaustive de la réponse immunitaire induite. La « heparin-binding hemagglutinin (HBHA) » est une protéine de 28-kDa, sécrétée et exprimée à la surface de M. tuberculosis et de M. bovis BCG. Montrant une affinité importante pour les gycoconjugués sulfatés, elle favorise la dissémination hématogène du bacille de Koch. Nos résultats démontrent que la HBHA stimule l’immunité à médiation cellulaire humaine avec, toutefois, des différences selon que le sujet infecté souffre ou non de tuberculose active. En effet, les cellules mononuclées circulantes, de la majorité des individus infectés mais non-malades, secrètent de l’IFN-? en réponse à la HBHA, alors qu’une minorité de sujets malades produit de faibles quantités d’IFN-?, après stimulation in vitro avec l’antigène. Lors de l’infection naturelle par le bacille de Koch, la HBHA devient dès lors une cible pour le système immunitaire, et plus particulièrement au sein des sujets, généralement considérés, comme protégés. L’analyse de la réponse cellulaire, spécifique à l’adhésine, démontre que les lymphocytes T CD4+, mais également T CD8+, des sujets infectés mais non-malades, produisent de l’IFN-?. L’antigène est effectivement présenté aux lymphocytes T grâce aux glycoprotéines du complexe majeur d’histocompatibilité de classe I et de classe II. Le phénotypage des cellules productrices d’IFN-? témoigne également la participation des cellules « natural killer (NK) » dans la réponse immunitaire contre la HBHA. En l’absence des lymphocytes T restreints à l’antigène, les cellules NK se montrent toutefois incapables de secréter de l’IFN-?, au contact de la HBHA. Les interactions entre les lymphocytes T, spécifiques à l’antigène, déterminent également la production de cytokines. Alors que la déplétion des cellules T CD8+ diminue légèrement la production d’IFN-?, l’absence des lymphocytes T CD4+ abolit toute sécrétion résiduelle d’IFN-?, lors de la stimulation avec la HBHA. Par contre, les lymphocytes T CD8+, pré-stimulés avec l’antigène en présence de cellules T CD4+, répondent secondairement à la présentation de la HBHA par des macrophages. Ce résultat suggère une coopération entre ces deux sous-populations cellulaires, afin de produire de l’IFN-? à l’encontre de la HBHA. Grâce à un contact cellulaire, les lymphocytes T CD4+ spécifiques à la HBHA soutiennent effectivement l’activation des cellules T CD8+. Outre la production de cytokines, la participation des lymphocytes T CD8+ à la lutte contre M. tuberculosis, se traduit également par leurs fonctions cytotoxique et bactéricide. La caractérisation des cellules T CD8+, spécifiques à la HBHA, s’est dès lors poursuivie par l’évaluation de leur potentiel cytolytique. Après expansion clonale, les lymphocytes T CD8+ induisent la mort des macrophages présentant la HBHA. Le mécanisme cytotoxique engage la libération du contenu des granules cytoplasmiques, comme le montre l’augmentation de la synthèse de perforine et de granzyme A, lorsque les cellules T CD8+ sont stimulées avec la HBHA. Privés de ces médiateurs solubles, les lymphocytes T CD8+, spécifiques à la HBHA sont alors incapables de lyser les cellules cibles. En définitive, l’activité microbicide constitue actuellement le meilleur corrélat de protection. La culture de macrophages infectés par M. bovis BCG, en présence de cellules T CD8+ spécifiques à la HBHA, limite partiellement la croissance de la bactérie phagocytée, soulignant le pouvoir anti-mycobactérien de l’immunité cellulaire induite par la HBHA, chez le sujet infecté mais non-malade. D’autre part, l’analyse biochimique, menée à l’Institut Pasteur de Lille, démontre que la HBHA subit une modification post-traductionnelle, lors de sa synthèse. Il s’agit d’une méthylation des multiples résidus lysine, qui composent son extrémité C-terminale. La comparaison des formes native méthylée et recombinante non-méthylée de la HBHA démontre que la méthylation détermine l’immunogénicité et le pouvoir protecteur de la HBHA. En effet, contrairement à la HBHA native, la forme recombinante stimule faiblement la production d’IFN-?, chez les individus infectés mais non-malades, et ne protège pas la souris contre l’infection par le bacille de Koch. La sécrétion d’IFN-? est, par ailleurs, partiellement restaurée lorsque la HBHA est artificiellement méthylée in vitro. Les splénocytes murins se comportent également différemment, selon qu’ils ont été immunisés avec la forme méthylée ou non. Alors que la HBHA recombinante est immunogène chez la souris et chez l’homme, l’immunité cellulaire murine induite demeure impassible face à l’infection des phagocytes par les mycobactéries, ce qui se traduit par l’absence de protection. En conclusion, la HBHA se compose d’épitopes protecteurs, qui dépendent de la présence des groupements méthyls, associés à son domaine C-terminal. Il s’agit, à notre connaissance, de la première mise en évidence de l’implication de la méthylation dans la réponse d’immunité cellulaire à l’encontre d’une protéine. De plus, l’immunité adaptative spécifique à la HBHA, chez le sujet infecté mais non-malade, se caractérise par les trois principaux corrélats de protection, actuellement décrits chez l’homme. Le potentiel vaccinal de cette adhésine mycobactérienne est donc bien réel.

Haematopoiesis requires strict regulation in order to maintain a balanced production of the various blood cell components. Escape from this regulation contributes to the development of cancers such as leukemia. SOCS2 is a member of the Suppressor of cytokine signalling (SOCS) family, and normally functions as a negative regulator of the JAK/STAT pathway. I examined gene expression and promoter methylation in acute myeloid leukemia (AML) cell lines and patient samples. SOCS2 expression was quite variable in AML patients, and very low in acute promyelocytic leukemia (APL) patients. Promoter hyper-methylation was found in these patients, particularly those with high white blood cell count and a FLT3-ITD. I speculate that SOCS2 interacts with an aspect of the signalling complex to inhibit cell growth in these patients, and silencing SOCS2 is necessary for leukemia progression. Treating these patients with a de-methylating agent, such as decitabine, may show promise in the clinic.

I have investigated epigenetic mechanisms of acquired endocrine-resistance in breast cancer using an in vitro model system based on estrogen-dependent MCF7 cells and their derivatives, LCC1 and LCC9. LCC1 cells, derived from MCF7 after passage in ovariectomised mice and routinely cultured in vitro in the absence of estrogen, exhibit estrogen-independent growth. They retain sensitivity to tamoxifen and fulvestrant. LCC9 cells, derived from LCC1 cells by growing them in increasing concentrations of fulvestrant, are completely estrogen-independent and are resistant to fulvestrant and cross-resistant to tamoxifen. When compared to MCF7 cells, LCC1 cells have marked up-regulation of the estrogen receptor α (ERα) protein that is not concomitant with increased estrogen receptor 1 (ESR1) transcription, suggesting a role for estrogen in controlling the proteasomal degradation of ERα. However, despite being grown in the same estrogen-deprived conditions, LCC9 cells do not have up-regulated ERα levels. As LCC1 cells retain sensitivity to tamoxifen and fulvestrant, these data suggest that LCC1 have developed estrogen-independence through ERα uncoupled from its ligand. However, LCC9 cells appear to have developed an alternative mechanism which is not dependent on ERα, presumably explaining their resistance to fulvestrant. I have studied global gene expression changes in the presence and absence of estrogen in these cell lines, using oligonucleotide microarrays, and correlated these data with global DNA methylation data derived from methylation arrays, which interrogate the methylation status of approximately 27,000 CpG dinucleotides in the genome. The analysis led to the discovery of more than 5,000 genes that were potentially either up-regulated or down-regulated by estrogen in MCF7 cells, either directly or indirectly. The transcriptional response to estrogen was generally muted in LCC1 and LCC9 compared with MCF7, but was not completely absent. I used various methods based on differential gene expression to parse the data, including gene ontology analysis, aiming to select genes for further mechanistic study. However, none of these methods led to the conclusive identification of a specific gene (or set of genes) that might have accounted for the physiological differences between the cell lines. In one strategy, I reasoned that, as the endocrine-resistant cells had lost their estrogen-dependence, genes involved might be regulated in an estrogen-dependent manner in MCF7 cells, without exhibiting misregulation in LCC9. This led to the identification of DUSP1 as a candidate gene, which was taken forward for mechanistic study because of its potential role in regulating ERα expression. However, when over-expressing DUSP1 in LCC9 cells, I could not demonstrate any effect on ERα levels. The final approach taken was to identify genes that might have been epigenetically deregulated, being both estrogen-regulated and deregulated in association with aberrant DNA methylation in the estrogen-independent cell lines. Surprisingly, given the phenotypic differences between the cell lines, only a very few genes were significantly methylated between cell lines. Of those that were differentially methylated between MCF7 cells and LCC1/9, only three exhibited the expected inverse correlation between methylation and expression. Of these, the gene CYBA was selected for further investigation. CYBA is a critical component of the NAPDH oxidase complex which is involved in generating oxygen free-radicals. My work suggests CYBA expression is estrogen-dependent, and that chronic estrogen deprivation leads to the epigenetic inactivation of CYBA in breast cancer cells. I speculate that the epigenetic suppression of CYBA may protect cells from the oxidant damage that results from estrogen deprivation and may be part of the mechanism that leads to acquired endocrine-resistance in previously sensitive cells.

Asthma is a heterogeneous group of conditions that typically begin in early life and result in recurrent, reversible bronchial obstruction. The role played by epigenetic mechanisms in the pathogenesis of childhood asthma is understood only in part. Here we discuss asthma epigenetics within a developmental perspective based on our recent demonstration that the epigenetic trajectory to childhood asthma begins at birth. We next discuss how this trajectory may be affected by prenatal environmental exposures. Finally, we examine in vitro studies that model the impact of asthma-associated exposures on the epigenome. All of these studies specifically surveyed human DNA methylation and involved a genome-wide component. In combination, their results broaden our understanding of asthma pathogenesis and the role the methylome plays in this process.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine / Fetal Alcohol Syndrome (F AS), the most severe form of Fetal Alcohol Spectrum Disorder (F ASD), has traditionally been associated with maternal alcohol consumption during pregnancy. However, a number of animal studies have shown an association between paternal preconception alcohol consumption and developmental abnormalities in the offspring that resemble the features of F AS. Dysregulation of epigenetic factors (such as DNA methylation) in the presence of alcohol may provide a plausible mechanism by which paternal alcohol consumption could result in offspring affected with features of F AS. Imprinted genes are expressed in a parentof- origin manner due to DNA methylation at distinct differentially methylated regions (DMRs) and are essential for normal embryonic development. There are only two known paternally methylated DMRs in humans, with an additional one described in mice - associated with Rasgrfl. The first aim of this study was to determine whether the human RASGRFl gene contains a DMR and whether this DMR is paternally methylated. In order to assess the imprint status of RASGRF 1, a number of computational assessments were done to identify key features of imprinted loci. Pyrosequencing analysis was used to assess the methylation status of various CpO islands surrounding RASGRFi in peripheral blood and sperm DNA samples. The RASGRF i-associated CpO regions were not found to exhibit differential methylation in a parent-of-origin manner. The second aIm of the study was to examine the effect of paternal alcohol consumption on the methylation status of the IG-DMR locus in male gametes and to detennine whether alcohol is correlated with methylation in a dose-dependant manner. Methylation assessment was done using the quantitative pyrosequencing technology. While an overall reduction in methylation was noted in males who consumed alcohol after adjusting for confounding variables, the amount of alcohol consumed did not correlate with overall methylation. When analyzed by individual CpG sites, alcohol consumption was found to correlate preferentially with demethylation at CpG 3 while alcohol-dosage preferentially correlated with demethylation at CpG 7. Age was significantly correlated with an increase in the overall methylation at JG-DMR and at individual sites within JG-DMR. In conclusion, these findings support the hypothesis that paternal preconception alcohol consumption can lead to hypomethylation of nonnally hypennethylated DMRs of specific imprinted genes in human spenn. This in tum could have significant implications with regard to the regulation of developmentally significant genes in the zygote and fetus, resulting in developmental, behavioral and neurocognitive disorders.

DNA methylation plays a crucial role in human health, especially cancer. Traditional DNA methylation analysis aims to identify CpGs/genes with differential methylation (DM) between experimental groups. Differential variability (DV) was recently observed that contributes to cancer heterogeneity and was also shown to be essential in detecting early DNA methylation alterations, notably epigenetic field defects. Moreover, studies have demonstrated that environmental factors may modify the effect of DNA methylation on health outcomes, or vice versa. Therefore, this dissertation seeks to develop new statistical methods for epigenetic data focusing on DV and interactions when efficient analytical tools are lacking. First, as neighboring CpG sites are usually highly correlated, we introduced a new method to detect differentially methylated regions (DMRs) that uses combined DM and DV signals between diseased and non-diseased groups. Next, using both DM and DV signals, we considered the problem of identifying epigenetic field defects, when CpG-site-level DM and DV signals are minimal and hard to be detected by existing methods. We proposed a weighted epigenetic distance-based method that accumulates CpG-site-level DM and DV signals in a gene. Here DV signals were captured by a pseudo-data matrix constructed using centered quadratic methylation measures. CpG-site-level association signal annotations were introduced as weights in distance calculations to up-weight signal CpGs and down-weight noise CpGs to further boost the study power. Lastly, we extended the weighted epigenetic distance-based method to incorporate DNA methylation by environment interactions in the detection of overall association between DNA methylation and health outcomes. A pseudo-data matrix was constructed with cross-product terms between DNA methylation and environmental factors that is able to capture their interactions. The superior performance of the proposed methods were shown through intensive simulation studies and real data applications to multiple DNA methylation data.

Aberrant methylation of CpG island promoters is a frequent observation in cancer and is known to affect many genes, including tumour suppressor genes. Genes with methylated promoters are usually repressed and inactive, and there is good evidence that most genes that become methylated in cancer are already repressed in the normal tissues from which tumours arise. However, the methylation of some genes appears to arise at previously active loci, suggesting either a stochastic epigenetic event or that these genes are somehow predisposed to becoming methylated. The DNA mismatch repair gene MLH1 is expressed in normal colonic epithelial cells but methylated and down-regulated in some sporadic mismatch repair-deficient colon tumours. These tumours are almost invariably associated with the simultaneous methylation of multiple cancer-specific loci, termed the CpG island methylator phenotype (CIMP) and an activating mutation of BRAF (V600E), raising the possibility that a hypermethylator phenotype may arise in cancer in direct association with a specific genetic alteration. The possibility that MLH1-deficiency caused BRAF mutation was discounted as genetic deficiency of MLH1 is not associated with BRAFV600E. I explored the possibility that BRAFV600E might induce MLH1 methylation but found no evidence in support of this. I then focused on factors that might mediate CIMP gene methylation, of which MLH1 methylation is known to be a part. Bioinformatic analysis of the genes methylated in BRAFV600E colon tumours indicated a significant enrichment in binding sites for the transcription factor MAZ (MYC-associated zinc finger protein). I hypothesised that loss of MAZ might lead to MLH1 down-regulation and its subsequent methylation. In this thesis I provide evidence that both MAZ and MLH1 expression are deregulated during normal colonic epithelial differentiation. The down-regulation of MAZ by RNA interference led to a reduction in MLH1 expression and methylation of its promoter. I speculate that MLH1 methylation may be associated with BRAF mutation because transformation by BRAFV600E allows progenitor cells to undergo a degree of differentiation whilst maintaining their malignant proliferation. I speculate that it is during this process of differentiation that MLH1 becomes susceptible to methylation.

Transcriptional repression is maintained by many molecular processes, including DNA methylation and polycomb repression. These two systems are both associated with chromatin modification at the promoters of silent genes, and are both essential for mammalian development. Previous work has shown that DNMT proteins are required for correct targeting of polycomb repressive complexes (PRCs). In this thesis, I investigate whether targeting of DNA modification has a reciprocal dependence on the polycomb machinery by mapping DNA modification in wild-type and PRC-mutant ES cells (Ring1B null, EED null, and Ring1B/EED duble null). I find that the loss of PRCs results in increased DNA modification at sites normally targeted by de novo DNA methyltransferase which lose H3K4 methylation upon PRC removal. This increased DNA modificaiton is associated with increased gene expression when found at CpG island shores of genes marked by the PRC-mediated histone modifications H3K27me3 and H2AK119ub, but not genes lacking these marks. Gene misregulation may be further linked to DNA modification changes by increased DNA modification at enhancers. While loss of either Ring1B or EED led primarily to increases in DNA modification at regions dependant on DNMT3A/DNMT3B, the combined loss of Ring1B and EED results in widespread loss of DNA modification at sites more dependent on DNMT1 activity. This thesis suggests an interplay between PRCs and DNA modification placement which is relevant to the cntrol of gene expression.