Myc deregulation critically contributes to many cancer etiologies. Recent work suggests that Myc and its direct interactors can confer a distinct epigenetic state. Our goal is to better understand the Myc-conferred epigenetic status of cells. We have previously identified the long non-coding RNA (lncRNA), H19, as a target of Myc regulation and shown it to be important for transformation in lung and breast cells. These results prompted further analysis to identify similarly important Myc-regulated lncRNAs. Myc-regulated lncRNAs associated with the cell cycle and transformation have been identified by microarray analysis. A small number of candidate lncRNAs that were differentially expressed in both the cell cycle and transformation have been validated. Given the increasing importance of lncRNAs and epigenetics to cancer biology, the discovery of Myc-induced, growth associated lncRNAs could provide insight into the mechanisms behind Myc-related epigenetic signatures in both normal and disease states.

We investigated licking and grooming (LG) levels received by each pup from their dams and the locomotor activity, anxiety-like behaviors, and stress reactivity in adult female offspring. We also investigated glucocorticoid receptor (GR) gene expression and its DNA methylation status in the hippocampus, comparing pups between and with-in litters. Rats that receive more LG than their siblings showed less anxiety-like behaviors and increased locomotor activity, regardless of their litter type. Higher licked pups also showed increased expression of the GR gene. Gene expression levels of the GR 17 splice variant were not significantly different as a function of dam LG or LG received, whereas DNA methylation levels at two CpG sites within GR17 promoter were significantly higher in high LG pups than low LG pups. Our results indicate that naturally occurring intra- and inter-litter differences in maternal LG have a lasting effect on the phenotypic outcomes of adult female offspring.

A typical gene expression data set consists of measurements of a large number of gene expressions, on a relatively small number of subjects, classified according to two or more outcomes, for example cancer or non-cancer. The identification of associations between gene expressions and outcome is a huge multiple testing problem. Early approaches to this problem involved the application of thousands of univariate tests with corrections for multiplicity. Over the past decade, numerous studies have demonstrated that analyzing gene expression data structured into predefined gene sets can produce benefits in terms of statistical power and robustness when compared to alternative approaches. This thesis presents the results of research on gene set analysis. In particular, it examines the properties of some existing methods for the analysis of gene sets. It introduces novel Bayesian methods for gene set analysis. A distinguishing feature of these methods is that the model is specified conditionally on the expression data, whereas other methods of gene set analysis and IGA generally make inferences conditionally on the outcome. Computer simulation is used to compare three common established methods for gene set analysis. In this simulation study a new procedure for the simulation of gene expression data is introduced. The simulation studies are used to identify situations in which the established methods perform poorly. The Bayesian approaches developed in this thesis apply reversible jump Markov chain Monte Carlo (RJMCMC) techniques to model gene expression effects on phenotype. The reversible jump step in the modelling procedure allows for posterior probabilities for activeness of gene set to be produced. These mixture models reverse the generally accepted conditionality and model outcome given gene expression, which is a more intuitive assumption when modelling the pathway to phenotype. It is demonstrated that the two models proposed may be superior to the established methods studied. There is considerable scope for further development of this line of research, which is appealing in terms of the use of mixture model priors that reflect the belief that a relatively small number of genes, restricted to a small number of gene sets, are associated with the outcome.

Obesity and type 2 diabetes (T2D) are major risk factors for cardiovascular and other diseases and are currently undergoing an increase in global prevalence. The work presented in my thesis addresses the role epigenetics, specifically DNA methylation, plays in the susceptibility to obesity and T2D and deals with methodological issues in the analysis of DNA methylation data. I first combined epigenome-wide DNA methylation data across 38 adipose tissue samples with corresponding SNP and mRNA data for the same subjects. At 5% false discovery rate (FDR), methylation of 149 regions associated with at least one cis-SNP. When 19 of the 149 regions were tested for association in an additional 181 independent samples, five regions replicated. These results indicate a genetic influence on DNA methylation in adipose tissue. I then analysed 90 epigenome-wide methylation samples taken from 15 South Asian controls and 30 T2D cases participating in the LOLIPOP study at two time points ∼7 years apart. I found global differences at both follow-up and baseline between the normal glucose tolerant and T2D groups, as well as strong differences with aging. I further used the main EpiMigrant data from 2,687 individuals, with 36 samples measured in duplicate to assess approaches to quality control, data normalisation and batch correction through control probe adjustment. A null hypothesis for epigenome-wide association studies (EWAS) by permutation testing and I investigated the effects of correlation between individual methylation markers. Using the developed methods, I carried out an EWAS of body mass index (BMI) with subsequent meta-analysis amongst 10,261 individuals of European and South Asian ancestry. DNA methylation markers at 187 genetic loci were associated with BMI. Mendelian randomisation experiments suggested that association of DNA methylation with BMI is the consequence of BMI. Lastly, I tested haplotypes of 85 SNPs currently known to be associated with T2D and 118 SNPs associated with obesity traits for an enrichment of CpG creating or abrogating SNPs and found that 9 T2D and 23 obesity SNPs showed a significant difference in CpG count between the SNP alleles as established by permutation testing. Amongst these is FTO, a locus which has been previously been shown to have a haplotype-specific methylation effect. My work provides novel insights into the role of DNA methylation in metabolic diseases. The methods that I developed to robustly detect association are flexible and scalable and will further be useful for larger, future EWAS.

Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2-6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research. (C) 2017 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

Les éléments transposables (ET) sont une source majeure de variation génétique, ce qui leur confère un rôle essentiel dans l’évolution des génomes. Certes présents dans tous les génomes analysés à ce jour, leurs proportions sont fortement variables entre espèces et aussi entre populations, suggérant une relation unique entre génome hôte et ET. Grâce à un système modèle composé de populations naturelles de deux espèces proches (Drosophila melanogaster et D. simulans) avec des quantités différentes en ET, nous avons pu comparer les relations génome hôte/ET. Nous nous sommes particulièrement interessés à l’élément helena qui, chez D. simulans, montre une activité faible, malgré un nombre de copies élevé.Cette activité moindre est associée à de nombreuses délétions internes des copies, suggérant un mécanisme de régulation d’ET par des délétions de l’ADN. Un autre système de régulation de l’activité des ET utilise le contrôle épigénétique, ce qui permet le maintien des copies d’ET dans le génome mais un blocage de leur activité. Le remodelage de la chromatine est un système épigénétique bien décrit chez la drosophile. Les régions chromatiniennes des génomes sont associées à différents types de modifications d’histone. Nous avons mis en évidence, dans des populations de D. melanogaster et D. simulans, une variation conséquente de modifications d’histones de type hétérochromatique, H3K27me3 et H3K9me2, associées àdes copies de différents ET. De plus, nous avons décrit des populations chez D. simulans dites déréprimées, chez lesquelles certains éléments sont surexprimés et présentent des localisations probablement hétéchromatiques. Les ET sont donc contrôlés par le génome hôte par des délétions internes et probablement par un système épigénétique variable. De plus, dans certaines populations, des copies peuvent échapper à ce contrôle et envahir le génome. Les ET sont donc des grands créateurs de variabilité génétique mais permettent aussi une territorialisation chromatinienne du génome car ils portent des modifications épigénétiques précises et sont capables de les étendre à leurs environnements génomiques. Ceci leur confére la fonction "d'épigénétique mobile". / Transposable elements (TEs) are one major force of genome evolution thanks to theirability to create genetic variation. TEs are ubiquitous and their proportion is variable between species and also populations, suggesting that a tight relationship exists between genomes and TEs. The model system composed of the natural populations of the twin sisters Drosophila melanogaster and D. simulans is interesting to compare host/TE relationship, since both species harbour different amounts of TE copies. The helena element is nearly silenced in D.simulans natural populations despite a very high copy number. Such repression is associated to abundant internally deleted copies suggesting a regulatory mechanism of TEs based on DNA deletion. Another pathway of TE regulation is through epigenetics where the host genome is able to keep intact the DNA sequences of TEs and still silence their activities.Chromatin remodelling is well known in drosophila and specific histone modifications can be associated to specific chromatin domains. We observed an important variation on H3K27me3and H3K9me2, two heterochromatic marks, on TE copies in D. melanogaster and D. simulans natural populations. Also, we show that derepressed lines of D. simulans exist for specific elements, have high TE transcription rates and are highly associated to non constitutive heterochromatic marks. TEs are therefore controlled by the host genome through DNA deletion and a possible chromatin remodelling mechanism. Not only genetic variability is enhanced by TEs but also epigenetic variability, allowing the host genome to be partitioned into chromatin domains. TEs are therefore mandatory to gene network regulation through their ability of “jumping epigenetics”.

Autoimmune diseases are clinical manifestations of a loss-of-tolerance of the immune system against the body’s own substances and healthy tissues. Primary Sjögren’s syndrome (pSS) and systemic lupus erythematosus (SLE) are two chronic inflammatory autoimmune diseases characterized by autoantibody production and an activated type I interferon system. Although the precise mechanisms leading to autoimmune processes are not well defined, recent studies suggest that aberrant DNA methylation and gene expression patterns may play a central role in the pathogenesis of these disorders. The aim of this thesis was to investigate DNA methylation and gene expression in pSS and SLE on a genome-wide scale to advance our understanding of how these factors contribute to the diseases and to identify potential biomarkers and novel treatment targets. In study I, differential DNA methylation was analyzed in multiple tissues from pSS patients and healthy controls. We identified thousands of CpG sites with perturbed methylation; the most prominent finding was a profound hypomethylation at regulatory regions of type I interferon induced genes in pSS. In study II, a cases-case study comparing DNA methylation in pSS patients with high fatigue to patients with low fatigue, we found methylation patterns associated to the degree of fatigue. In study III, RNA-sequencing was applied to investigate the transcriptome of B cells in pSS in comparison to controls. Increased expression of type I and type II interferon regulated genes in pSS was observed, indicating ongoing immune activation in B cells. In study IV, the impact of DNA methylation on disease susceptibility and phenotypic variability in SLE was investigated. We identified DNA methylation patterns associated to disease susceptibility, SLE manifestations and different treatments. In addition, we mapped methylation quantitative trait loci and observed evidence for genetic regulation of DNA methylation in SLE.   In conclusion, the results presented in this thesis provide new insights into the molecular mechanisms underlying autoimmunity in pSS and SLE. The studies confirm the central role of the interferon system in pSS and SLE and further suggest novel genes and mechanisms to be involved in the pathogenesis these autoimmune diseases.

<p>How organisms adapt and survive in continuously fluctuating environments is a central question of evolutionary biology. Additionally, organisms have to deal with the inherent stochasticity in all cellular processes. The purpose of this thesis is to gain insights into how organisms can use epigenetics and the stochasticity of gene expression to deal with a fluctuating environment. To accomplish this, two cases at different temporal and structural scales were explored: (1) the early transcriptional response to an environmental stimulus in single cells, and (2) the evolutionary dynamics of a population adapting to a recurring fluctuating environment. Mathematical models of stochastic gene expression, population dynamics, and evolution were developed to explore these systems.</p><p>First, the information available in sparse single cell measurements was analyzed to better characterize the intrinsic stochasticity of gene expression regulation. A mathematical and statistical model was developed to characterize the kinetics of a single cell, single gene behavior in response to a single environmental stimulus. Bayesian inference approach was used to deduce the contribution of multiple gene promoter states on the experimentally measured cell-to-cell variability. The developed algorithm robustly estimated the kinetic parameters describing the early gene expression dynamics in response a stimulus in single neurons, even when the experimental samples were small and sparse. Additionally, this algorithm allowed testing and comparing different biological hypotheses, and can potentially be applied to a variety of systems.</p><p>Second, the evolutionary adaptation dynamics of epigenetic switches in a recurrent fluctuating environment were studied by observing the evolution of gene regulatory circuit in a population under multiple environmental cycles. The evolutionary advantage of using epigenetics to exploit the natural noise in gene expression was tested by competing this strategy against the classical genetic adaptation through mutations in a variety of evolutionary conditions. A trade-off between minimizing the adaptation time after each environmental transition and increasing the robustness of the phenotype during the constant environment between transitions was observed. Surviving lineages evolved bistable, epigenetic switching to adapt quickly in fast fluctuating environments, whereas genetic adaptation with high robustness was favored in slowly fluctuating environments.</p> / Dissertation

Epigenetics is the study of heritable changes in gene expression that do not involve changes in the underlying DNA sequence. Epigenetic pathways appeared in eukaryotes where multicellular organisms differentiate into different cell types, associated with different phenotypes. The differentiation process is achieved by modifications of the chromatin structure which, by altering the access of trans-factors to the DNA, result in gene activation and repression. Epigenetic mechanisms are therefore viewed as an "extra" layer of information that modulate the genetic information in time and space, necessary for the development and the response to environment stimuli. Although the recent development of high-throughput sequencing technologies has provided an unprecedented insight into epigenetic pathways, the mechanisms controlling the chromatin dynamic as well as their downstream effects on cellular processes are far from being fully understood. In this thesis, our focus will be restricted to mechanisms acting on the nucleosome level. The first chapter will present the factors known to influence nucleosome positioning as well as the challenges related to the measurement of the nucleosome architecture. The second chapter will introduce a statistical approach, NucleoFinder, capable of identifying nucleosomes consistently positioned in a population of cells. In chapter three, we will make use of NucleoFinder to investigate the importance of cis and trans-factors on the nucleosome architecture in human and show that, despite variation across functional regions, cis-factors have a very modest in influence on nucleosome positioning. Finally, in chapter four, we will aim to identify clusters of histone modifications specific to functionally distinct regions, characterize their function and their association with gene expression level.

Improved survival for women receiving chemotherapy for breast cancer (BC) has been accompanied by the development/persistence of psychoneurological symptoms (PNS) that compromise their quality of life. The biological basis for these PNS is unknown, but could reflect the acquisition of soma-wide chromosomal/epigenetic alterations. An important first step in testing this hypothesis is to determine if somatic genetic/epigenetic changes arise and persist following treatment. To answer this question we longitudinally studied 71 women (ages 23-71) with early-stage BC and collected measures before chemotherapy (baseline), and 4 weeks (mid-chemo); six months (during radiation therapy for a subset of women); and one year following the initiation of chemotherapy. Acquired lymphocyte chromosomal instability (scored by micronuclei frequencies [MNF]) showed a significant increase in post-treatment compared to baseline time-points (p<0.0001), with these increases persisting for at least one year following chemotherapy. Significant predictive associations were observed between MNF and tumor characteristics [luminal B (lower MNF; p=0.0182); triple negative (higher MNF; p=0.0446)], radiotherapy (higher MNF; p=0.0004), the type of chemotherapy received (p=0.0463), race (Caucasians > African Americans; p=0.0037), perceived stress levels (positive-association; p=0.0123), and cognitive flexibility domain measures (positive-association; p=0.0238). Genome-wide acquired methylation changes were also measured in peripheral blood cells, with 1265 sites showing significant differential methylation following chemotherapy. These sites were localized to open sea, shores, shelves, and CpG island sequences and included sites within genes involved in cell cycle, DNA repair, transcription regulation, signal transduction pathways, neuronal regeneration, and immunity. To determine if the genetic/epigenetic alterations acquired in peripheral blood cells correlated with those in tumor cells, BC tumors from 10 participants were analyzed using a genome-wide copy number/targeted mutations (CN/M) microarray. While no clear blood-tumor cell correlations were detected, genome-wide CN/M evaluations showed promise for stratifying tumors. Lastly, in an unrelated project studying a rare case of fetuses in fetu, methylation changes acquired in embryogenesis were shown to be influenced by both environmental and genetic cues. In summary, acquired chromosomal/epigenetic alterations do arise following chemotherapy (and in embryogenesis). Further delineation of these acquired changes could increase our understanding of the biological basis for cancer-related side-effects and help to identify “at risk” individuals.