No description available.

<p>Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.</p> / Dissertation

<p>Low-dose ionizing radiation (LDIR) exposure (under 10.0 centigray (cGy)) from man-made sources, such as diagnostic imaging, predominates in the US population and comprises nearly 50% of an average individual's yearly radiation exposure (Ullrich, Brooks et al. 2009). The increase in such exposures has led to public and government alarm about the impact of LDIR on human health (Ullrich, Brooks et al. 2009). Besides the mutational effects of radiation exposure, there is concern it might also result in modifications of the epigenome. Such aberrations can disrupt normal development and are involved in the progression of numerous diseases, including cancer (Gasser and Li 2011). High doses of radiation (>100.0 cGy) have been shown to cause epigenetic disruption (Kaup, Grandjean et al. 2006; Tamminga, Koturbash et al. 2008; Ilnytskyy, Koturbash et al. 2009), which is necessary for the persistence of radiation-induced genomic instability (Rugo, Mutamba et al. 2011); however, it is presently unclear to what extent LDIR in vivo alters the epigenome. </p><p>The viable yellow agouti (Avy) mouse was used here to characterize the dose-dependent epigenetic response to LDIR. The Avy mouse is a unique biological model that functions as a biosensor for environmentally induced epigenetic changes and disease susceptibility due to the presence of a metastable epiallele that modulates coat color (Waterland and Jirtle 2003). Pregnant dams were whole-body exposed to one of five doses of X-ray radiation ranging from 0-10.0 cGy on gestational day 4.5. Using a phantom mouse model, the intrauterine doses were estimated to be 0.0 cGy, 0.4 cGy, 0.7 cGy, 1.4 cGy, 3.0 cGy, and 7.6 cGy, respectively. At weaning, offspring coat colors were assessed and tissues were collected for methylation analysis. First, methylation changes at CpG sites in the Avy and Cdk activator binding protein (CabpIAP) metastable epialleles and at intracisternal a particle (IAP) elements across the genome were quantified using Sequenom technology. Second, three imprinted genes, Peg3, Nnat, and H19, were assessed for methylation changes in differentially methylated regions (DMRs) that regulate their parent-of-origin monoallelic expression using Sequenom technology. Lastly, it was postulated that the epigenetic changes at the Avy locus could be counteracted with dietary alterations. To test this hypothesis, female mice were placed on an antioxidant-supplemented diet prior to pregnancy and throughout gestation and lactation. Pregnant dams were irradiated with 3.0 cGy of whole-body X-rays. Offspring coat colors were assessed and methylation changes at the Avy allele were measured with the Sequenom platform. </p><p>Herein, I demonstrate that in utero LDIR exposure induced epigenetic changes in the Avy mouse in a dose-dependent and sex-specific manner. Acute, whole-body exposure to 0.7 cGy, 1.4 cGy, 3.0 cGy or 7.6 cGy X-rays significantly shifted offspring coat color distribution toward pseudoagouti. Acute exposure to 1.4 cGy, 3.0 cGy, and 7.6 cGy significantly increased methylation at multiple CpG sites in the Avy metastable epiallele in male offspring, but not female offspring. Methylation changes at DMRs in Nnat, Peg3, and H19 also occurred in a dose-dependent manner. Furthermore, inhibition of the phenotypic and Avy methylation changes with an antioxidant-supplemented diet suggests that the mechanisms to induce epigenetic changes are mediated by oxidative stress. These results demonstrate that relevant, low doses of radiation can elicit epigenetic changes that lead to a persistent phenotype, but can be mitigated with dietary supplementation. The successful completion of this project has resulted in the first in vivo epigenetic characterization of LDIR exposure and will contribute to the development of more relevant risk assessment strategies for protecting human populations.</p> / Dissertation

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23611号 / 医科博第134号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 小川 誠司, 教授 山中 伸弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

La méthylation de l'ADN est fortement reprogrammée pendant le développement chez les mammifères, où elle semble jouer un rôle essentiel dans la répression génique et le maintien de l'identité cellulaire. Néanmoins, les cibles de la méthylation de l'ADN, la cinétique d'acquisition et les mécanismes de ciblage au cours du développement sont mal connus. Le premier objectif de ma thèse a donc été d'identifier les cibles de la méthylation de l'ADN pendant l'embryogenèse chez la souris. Sachant que plusieurs études dans les cellules ES ont mis en évidence un lien entre l'histone méthyltransférase G9a et l'établissement de la méthylation de l'ADN, le deuxième objectif de ma thèse a été de tester le rôle de G9a dans l'établissement de la méthylation de l'ADN au cours de l'embryogenèse. Pour cela, j'ai développé une technique d'analyse de la méthylation à l'échelle génomique à partir d'un petit nombre de cellules. Nous avons observé que la méthylation est essentiellement catalysée par DNMT3B et est mise en place principalement pendant l'implantation de l'embryon entre le blastoscyste et l'épiblaste. Pendant cette période, la méthylation cible préférentiellement les gènes de la lignée germinale et est indispensable à leur répression. La méthylation cible aussi des gènes spécifiques de différentes lignées somatiques telles que la lignée hématopoïétique, et peut être effacée ultérieurement pendant la différentiation. De manière surprenante, nous avons identifié des promoteurs de gènes non soumis à l'empreinte qui semblent résister à la reprogrammation de la méthylation de l'ADN et hériter la méthylation des gamètes parentaux. Enfin, nous avons montré que, contrairement à ce que suggèrent les études dans les cellules ES, G9a ne semble pas être indispensable à l'acquisition et au maintien de la méthylation de l'ADN au niveau des promoteurs pendant le développement in vivo. / DNA methylation is an epigenetic mark extensively reprogrammed during mammalian development. It is believed to play essential functions in gene regulation and the maintenance of cellular identity. However, the target genes of DNA methylation and the mechanisms that recruit DNA methylation during development remain poorly understood. The first aim of my PhD project was to identify the target genes of DNA methylation during early mouse development in vivo. In addition, because several studies show that G9a is required for DNA methylation establishment and maintenance during ES cells differentiation, the second aim was to determine whether G9a is required for the establishment of promoter DNA methylation patterns during early development in vivo.To address these questions, I developped a genomics approach to map DNA methylation starting from very small amount of cells. .We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Surprisingly, we identify nonimprinted genes that escape post-fertilization DNA methylation reprogramming and seem to inherit promoter DNA methylation from parental gametes. Finally we show that, unlike what it was shown in ES cells, the absence of G9a in an in vivo context does not have a drastic effect on the maintenance and the establishment of promoter DNA methylation during early development.

abstract: Within the primate lineage, skeletal traits that contribute to inter-specific anatomical variation and enable varied niche occupations and forms of locomotion are often described as the result of environmental adaptations. However, skeletal phenotypes are more accurately defined as complex traits, and environmental, genetic, and epigenetic mechanisms, such as DNA methylation which regulates gene expression, all contribute to these phenotypes. Nevertheless, skeletal complexity in relation to epigenetic variation has not been assessed across the primate order. In order to gain a complete understanding of the evolution of skeletal phenotypes across primates, it is necessary to study skeletal epigenetics in primates. This study attempts to fill this gap by identifying intra- and inter-specific variation in primate skeletal tissue methylation in order to test whether specific features of skeletal form are related to specific variations in methylation. Specifically, methylation arrays and gene-specific methylation sequencing are used to identify DNA methylation patterns in femoral trabecular bone and cartilage of several nonhuman primate species. Samples include baboons (Papio spp.), macaques (Macaca mulatta), vervets (Chlorocebus aethiops), chimpanzees (Pan troglodytes), and marmosets (Callithrix jacchus), and the efficiencies of these methods are validated in each taxon. Within one nonhuman primate species (baboons), intra-specific variations in methylation patterns are identified across a range of comparative levels, including skeletal tissue differences (bone vs. cartilage), age cohort differences (adults vs. juveniles), and skeletal disease state differences (osteoarthritic vs. healthy), and some of the identified patterns are evolutionarily conserved with those known in humans. Additionally, in all nonhuman primate species, intra-specific methylation variation in association with nonpathological femur morphologies is assessed. Lastly, inter-specific changes in methylation are evaluated among all nonhuman primate taxa and used to provide a phylogenetic framework for methylation changes previously identified in the hominin lineage. Overall, findings from this work reveal how skeletal DNA methylation patterns vary within and among primate species and relate to skeletal phenotypes, and together they inform our understanding of epigenetic regulation and complex skeletal trait evolution in primates. / Dissertation/Thesis / Doctoral Dissertation Anthropology 2017

L’infertilité masculine est actuellement considérée comme un problème majeur qui pose une situation alarmante sur la santé publique. L’oligozoospermie, l’asthénozoospermie et la tératozoospermie sont les trois anomalies les plus connues des spermatozoïdes. Elles affectent, respectivement, la densité, la motilité et la morphologie des spermatozoïdes. Un spermatozoïde anormal est très souvent corrélé à des altérations génétiques et épigénétiques qui peuvent affecter considérablement le transcriptome. Dans ce sens, le séquençage aléatoire du transcriptome entier des spermatozoïdes ou RNA-seq constitue un outil puissant pour caractériser ces maladies. Jusqu’à présent, il n’existe aucune étude exploitant des données RNA-seq chez des hommes présentant de telles anomalies spermatiques. L’objectif principal de notre étude fût d’identifier des profils distincts des modifications du transcriptome de chaque phénotype d’infertilité pour ainsi révéler des gènes-signatures qui tamponnent une spermatogenèse pathologiquePour ce faire, les transcriptomes des spermatozoïdes de 60 sujets infertiles atteints soit d’oligozoospermie, d’asthénozoospermie ou de tératozoospermie ont été comparés à ceux de 20 patients fertiles. Ces analyses supervisées nous ont conduit à identifier: (i) les gènes clés spécifiques aux différentes anomalies des spermatozoïdes (ii) les voies de signalisation associées, (ii) les différents longs ARNs non codants dérégulés dans ces anomalies. Au niveau de l’oligozoospermie, les transcrits de spermatozoïdes dérégulés étaient associées à divers stades de la spermatogenèse, y compris le cycle cellulaire méiotique, l’assemblage du complexe synaptonémal, la cohésion des chromatides sœurs, les processus métaboliques de piRNA, le processus catabolique protéique dépendant de la voie de l’ubiquitine, à la réponse aux dommages de l'ADN et particulièrement le processus de fécondation. Quant à l’asthenozoospermia, la spermatogenèse, l’assemblage du cil, des voies métaboliques reliées à la spermatogenèse, la chimiotaxie et la physiologie des cellules immunitaires ont été significativement dérégulés. De plus, ce qui nous a intéressé au plus était l’analyse des transcrits sous-exprimés qui a permis l’identification de nombreux transcrits associées aux modifications des histones. Nous avons aussi mis en évidence une sous expression des gènes différentiellement exprimés qui définit la tératozoospermie. Cette sous expression est associée au système ubiquitine-protéasome, à l’organisation du cytosquelette, au cycle cellulaire, à la SUMOylation en réponse aux dommages de l'ADN et aux protéines de réparation ainsi qu’à de nombreux modulateurs épigénétiques. Les gènes signature de l'oligozoospermie ont été liés au processus de fécondation et les composants de la matrice extracellulaire, tandis que ceux de la tératozoospermie sont liés à la spermatogenèse et la morphogenèse cellulaire, alors que les gènes signature de l'asthénozoospermie sont impliqués dans l'assemblage du ribosome et du flagelle. En complément de cette étude, nous avons réalisé une étude très globale du paysage épigénétique du sperme des hommes infertiles. Nous avons, ainsi comparé les niveaux des espèces réactives de l’oxygène (ERO), de méthylation de l’ADN, ainsi que l’intégrité de la chromatine dans les spermatozoïdes de 30 individus infertiles avec ceux de 33 individus fertiles. Nos analyses montrent des niveaux élevés d’ERO chez les individus infertiles. Ces niveaux sont d’une part négativement corrélés avec les niveaux de méthylation globale de l’ADN et d’autre part négativement corrélés avec ceux de la 5-hydroxyméthylcytosine et de la 5-formylcytosine (intermédiaire dans le processus de déméthylation active). Ces derniers suggèrent qu’une infertilité associée au stress oxydatif conditionne l’épigénome du sperme. En conclusion, l’ensemble de notre travail apporte des ressources précieuses et originales dans la compréhension des pathologies de sperme. / Male infertility is actually considered as a public alarming health problem. The sperm pathologies spectrum ranges between different phenotypes including oligozoospermia, asthenozoospermia and teratozoospermia depending on the sperm conventional parameters abnormalities. Abnormal sperm is characterized by genetic alterations and epigenetic alterations which can affect the transcriptome extensively. These alterations in RNA profiles are retrospectively indicative of aberrant spermatogenic events. RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome. To date, RNA-seq analysis of sperm from infertile men has not been reported. Our objectives are: (i) recognize key clusters, key pathways and specific gene transcripts for different sperm abnormalities; (ii) catalog the spermatozoal lncRNAs in different sperm pathologies; (iii) identify signature genes which are mechanistically important in the cascade of events driving a pathological spermatogenesis; (iii) portray the global epigenetic landscape in sperm from infertile men. Expression data from 60 sperm samples from 3 groups of infertile men (oligozoospermia, asthenozoospermia, and teratozoospermia) were generated on Illumina HiSeq platform, compared to 20 fertiles, and the resulting gene expression patterns were analyzed for functional enrichment. Our supervised analyses identified numerous differentially expressed genes between fertile and infertile men. In oligozoospermia, the deregulated spermatozoal transcripts were associated with various stages of spermatogenesis including meiotic cell cycle, synaptonemal complex assembly, sister chromatid cohesion, piRNA metabolic process, ubiquitin-dependent protein catabolic process, cellular response to DNA damage stimulus and interestingly fertilization. As for asthenozoospermia, spermatogenesis, cilium assembly, metabolic-related pathways, chemotaxis and immune cell physiology were most significantly differentially expressed. Interestingly, numerous transcripts associated with histone modifications were highly down-regulated. With regards to teratozoospermia, we evidenced sperm-specific differentially expressed genes which are involved in the ubiquitin-proteasome, cytoskeleton organization, the cell cycle pathway, SUMOylation of DNA damage response and repair proteins, as well as many putative epigenetic modulators of gene expression.. We also attempted to identify distinct patterns of gene expression changes that were definite to the different abnormal sperm phenotypes in infertile men relative to controls. Signature genes of oligozoospermia were over-enriched by genes involved in fertilization and extracellular matrix components, while signature genes of teratozoospermia were enriched by genes involved in spermatogenesis and cellular components involved in morphogenesis, whilst signature genes of asthenozoospermia were enriched by genes implicated in ribosome and cilium assembly.We complemented this work by a parallel epigenetic analysis of the global epigenetic landscape in infertile men. We compared the levels of reactive oxygen species (ROS), DNA integrity and global epigenetic parameters in sperm from 33 infertile subjects with abnormal semen parameters compared to fertile individuals. We pointed out that infertile men are characterized by strikingly high levels of reactive oxygen species (ROS) which were in part negatively correlated with the global DNA methylation, and positively correlated with the levels of 5-hydroxymethylcytosine and 5-formylcytosine (active demethylation intermediates). These findings suggest that male infertility associated with oxidative stress shapes the sperm epigenetic landscape. In summary, this original work yielded a transcriptional portrait of sperm abnormalities and provided valuable resources that would further elucidate sperm pathologies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17951号 / 医博第3835号 / 新制||医||1000(附属図書館) / 30781 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 生田 宏一, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The field of cancer genomics is currently being enhanced by the power of Epigenome-wide association studies (EWAS). Over the last couple of years comprehensive sequence data sets have been generated, allowing analysis of genome-wide activity in cohorts of different individuals to be increasingly available. Finding associations between epigenetic variation and phenotype is one of the biggest challenges in biomedical research. Laboratories lacking dedicated resources and programming experience require bioinformatics expertise which can be prohibitively costly and time-consuming. To address this, we have developed a collection of freely available Galaxy tools (Poterlowicz, 2018a), combining analytical methods into a range of convenient analysis pipelines with graphical user-friendly interface.The tool suite includes methods for data preprocessing, quality assessment and differentially methylated region and position discovery. The aim of this project was to make EWAS analysis flexible and accessible to everyone and compatible with routine clinical and biological use. This is exemplified by my work undertaken by integrating DNA methylation profiles of melanoma patients (at baseline and mitogen-activated protein kinase inhibitor MAPKi treatment) to identify novel epigenetic switches responsible for tumour resistance to therapy (Hugo et al., 2015). Configuration files are publicly published on our GitHub repository (Poterlowicz, 2018b) with scripts and dependency settings also available to download and install via Galaxy test toolshed (Poterlowicz, 2018a). Results and experiences using this framework demonstrate the potential for Galaxy to be a bioinformatics solution for multi-omics cancer biomarker discovery tool.