La spermatogénèse, processus de production des gamètes mâles, représente un modèle physiologique pertinent pour l’étude de la dynamique de la chromatine. En effet, une réorganisation drastique du génome est observée en fin de spermatogénèse, lors des étapes post-méiotiques du développement de la lignée germinale mâle. Ces réorganisations visent d’une part à compacter le génome afin de le protéger avant et lors de la fécondation et d’autre part à établir un épigénome mâle spécifique nécessaire pour le développement embryonnaire précoce. La chromatine organisée en nucléosomes est alors totalement remaniée de manière à ce que les protamines remplacent les histones dans les spermatozoïdes. Cette réorganisation est initiée par une vague d’acétylation des histones à l’échelle du génome, suivie par un remplacement massif des histones par de petites protéines basiques, les protéines de transition et les protamines. Les mécanismes moléculaires responsables de cette réorganisation de la chromatine sont très mal connus. Mon travail de thèse vise à explorer ces mécanismes en utilisant une approche d’invalidation de gènes chez la souris correspondant à des acteurs épigénétiques exprimés spécifiquement en post-méiose : le facteur nucléaire Nut (Nuclear protein in Testis) et le variant de l’histone H2A, H2A.L.2. Nous avons démontré que la protéine Nut recrute l’acétyltransferase p300 et induit l’hyperacétylation de l’histone H4 précisément au niveau des résidus lysines en position 5 et 8. Cette acétylation est nécessaire à l’interaction avec le premier bromodomaine de Brdt initiant le processus de remplacement des histones par les protamines. Ainsi, le facteur Nut est l’élément majeur de la vague d’acétylation des histones. Nous avons également mis en évidence le rôle crucial de H2A.L.2 dans “ l’ouverture ” de la structure du nucléosome permettant ainsi son invasion par les protéines de transition. Ces protéines vont à leur tour générer une plateforme pour le recrutement et la maturation des protamines et induire la formation de structures transitoires avant l’étape de compaction finale du génome des spermatozoïdes matures. Ces études nous ont ainsi permis d’établir pour la première fois un modèle moléculaire cohérent permettant de comprendre la programmation épigénétique post-méiotique du génome mâle et son impact sur la fertilité masculine. / Spermatogenesis, the process of producing male gametes, represents a relevant physiological model for the study of chromatin dynamics. Indeed, a drastic reorganization of the genome is observed at the end of spermatogenesis, during post-meiotic stages of the development of the male germ cells. These reorganizations are intended both to compact the genome to protect it before and during fertilization and to establish a specific male epigenome necessary for early embryonic development. In spermatozoa, during these post-meiotic stages, chromatin is completely reorganized so that the protamines replace the histones. This reorganization is initiated by a wave of genome-wide histone acetylation, followed by massive replacement of histones by small basic proteins, transition proteins and protamines. The molecular mechanisms responsible for this reorganization of the genome remain very poorly known today. My thesis aims to explore these mechanisms by using gene inactivation in mouse of epigenetic actors specifically expressed in post-meiotic germ cells : the nuclear factor Nut (Nuclear protein in Testis) and the histone H2A variant, H2A.L.2. We have demonstrated that the Nut protein interacts and stimulates the p300 acetyltransferase activity and induces the hyperacetylation of histone H4 precisely on the lysine residues at 5 and 8 positions. This acetylation is necessary for the interaction with the first bromodomain of Brdt initiating the process of histone replacement by protamines. Thus, the Nut factor is the main element of the histone acetylation wave. We have also deciphered the crucial role of H2A.L.2 in the “opening ” of nucleosomal structures in post-meiotic germ cells thus allowing its invasion by the transition proteins. These transition proteins will in turn generate a platform for the recruitment and maturation of protamines and induce the formation of transient structures before the final compaction of the mature sperm genome. These studies allowed us to establish for the first time a coherent molecular model for understanding the post-meiotic epigenetic programming of the male genome and its impact on male fertility.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19917号 / 理博第4217号 / 新制||理||1606(附属図書館) / 33003 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 工藤 洋, 教授 長谷 あきら, 教授 鹿内 利治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13139号 / 論医博第2139号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 中川 一路, 教授 原田 浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Le neuroblastome pédiatrique est un des cancers extra-crâniens des plus fréquents chez les enfants. Malgré une amélioration du taux de survie avec les thérapies actuellement disponibles, les stades avancés de neuroblastome ou en rechute présentent un très mauvais pronostic. De nouvelles approches thérapeutiques doivent donc être développées afin d'augmenter la survie des patients. Une de ces approches est la thérapie épigénétique. Le neuroblastome, comme plusieurs autres cancers pédiatriques, contient plusieurs altérations épigénétiques au niveau de la méthylation de l'ADN et des modifications des histones. Lors d'un criblage de médicaments déjà approuvés par la FDA, nous avons découvert quelques molécules ayant des caractéristiques de médicaments épigénétiques jusqu’alors jamais découvertes. Notre étude cherche donc à démontrer l'efficacité de ces molécules dans le traitement de lignées cellulaires de neuroblastome. Suite à des tests préliminaires, une des molécules approuvées par la FDA s'est démarquée : le disulfirame, un médicament approuvé pour le traitement de l’alcoolisme chronique. Nous avons donc traité des lignées cellulaires de neuroblastomes (IMR-32, N91, SK-N-DZ, SK-N-SH et SK-N-AS ) pendant 48 heures avec du disulfirame à des concentrations pertinentes sur le plan clinique (10nM à 50 µM). Nos résultats démontrent une inhibition de croissance de 50 % (IC50) d'environ 80 nM pour les lignées cellulaires testées. De plus, après analyse par cytométrie de flux, on observe un blocage du cycle cellulaire en G2/M. Nous avons également observé une diminution du facteur de transcription MYCN ainsi qu’une baisse d’acétylation de plusieurs marques d’histones (H3K9ac, H3K14ac, H3K27ac). Une analyse par séquençage d'ARN a confirmé le bloc en G2/M par une baisse d'expression de gènes associés à cette phase ainsi que la diminution de MYCN par une baisse de gènes cible de MYC. Des travaux sont en cours afin de déterminer le mécanisme d’action du disulfirame. Cette recherche permettra d’évaluer l’efficacité du disulfirame dans le traitement du neuroblastome / Pediatric neuroblastoma is one of the most common extracranial cancer in children. Despite an improvement in survival with the currently available therapies, neuroblastoma with an amplification of the transcription factor MYCN has a very poor prognosis. New therapeutic approaches must be developed to increase the survival of patients. One such approach is epigenetic drug therapy. Neuroblastoma, like many other pediatric cancers, contains several epigenetic alterations at the level of DNA methylation and histone modifications. In a screening of FDA-approved drugs, we discovered some molecules having characteristics of epigenetic drugs that were unknown until now. Our study seeks to demonstrate the efficacy of these molecules in the treatment of neuroblastoma cell lines. Following preliminary tests, one of the molecules approved by the FDA stood out: disulfiram, a medication approved for the treatment of chronic alcoholism. We treated neuroblastoma cell lines (MYCN amplified: IMR-32, N91 and SK-N-DZ; MYCN non-amplified: SK-N-AS and SK-N-SH) for 48 hours with disulfiram at clinically relevant concentrations (from 10 nM to 20 µM). Our results demonstrate a 50% growth inhibition (IC50) of 80nM for the cell lines tested. In addition, after analysis by flow cytometry, we found a cell cycle block in G2/M. RNA sequencing also revealed that disulfiram affects a many genes (downregulated n=508, upregulated n=207). We also observed a decrease in the transcription factor MYCN and a reduction in acetylation of several histone marks by Western blot’s analysis. Further studies are underway to determine the mechanism of action of disulfiram. This study shows the potential of disulfiram in the treatment of neuroblastoma.

The self-organisation of cells into complex tissues relies on the tight regulation of molecular processes governing their behaviour. Understanding these processes is a central questions in cell biology. In recent years, technological breakthroughs in single-cell sequencing experiments have enabled us to probe these processes with unprecedented molecular detail. However, biological function relies on collective processes on the mesoscopic and macroscopic scale, which do not necessarily obey the rules that govern it on the microscopic scale. Insights from these experiments on how collective processes determine cellular behaviour consequently remain severely limited. Methods from nonequilibrium statistical physics provide a rigorous framework to connect microscopic measurements to their mesoscopic or macroscopic consequences. In this thesis, by combining for the first time the possibilities of single-cell technologies and tools from nonequilbrium statistical physics, we develop theoretical frameworks that overcome these conceptual limitations. In particular, we derive a theory that maps measurements along the linear sequence of the DNA to mesoscopic processes in space and time in the cell nucleus. We demonstrate this approach in the context of the establishment of chemical modifications of the DNA (DNA methylation) during early embryonic development. Drawing on sequencing experiments both in vitro and in vivo, we find that the embryonic DNA methylome is established through the interplay between DNA methylation and 30-40 nm dynamic chromatin condensates. This interplay gives rise to hallmark scaling behaviour with an exponent of 5/2 in the time evolution of embryonic DNA methylation and time dependent, scale-free connected correlation functions, both of which are predicted by our theory. Using this theory, we successfully identify regions of the DNA that carry DNA methylation patterns anticipating cellular symmetry breaking in vivo. The primary layer determining cell identity is gene expression. However, read-outs of gene-expression profiling experiments are dominated by systematic technical noise and they do not provide “stochiometric” measurements that allow experimental data to be predicted by theories. Here, by developing effective spin glass methods, we show that the macroscopic propagation of fluctuations in the concentration of mRNA molecules gives direct information on the physical mechanisms governing cell states, independent of technical bias. We find that gene expression fluctuations may exhibit glassy behaviour such that they are long-lived and carry biological information. We demonstrate the biological relevance of glassy fluctuations by analysing single-cell RNA sequencing experiments of mouse neurogenesis. Taken together, we overcome important conceptual limitations of emerging technologies in biology and pioneer the application of methods from stochastic processes, spin glasses, field and renormalization group theories to single-cell genomics.

No description available.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression via inhibiting mRNA translation or degrading mRNA. Since the discovery of miRNAs, dysregulated miRNAs have been identified in human patients with various diseases. Moreover, the role of miRNAs in biological processes, including immune homeostasis and autoimmunity pathogenesis, has been widely investigated. Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease that causes systemic damage to multiple organs and is characterized by the production of pathogenic autoimmune antibodies. In previous work in my lab, a set of commonly upregulated miRNAs in splenic lymphocytes of three lupus-prone mouse models was identified, including the miR-183-96-182 cluster (miR-183C) and miRNAs located at DLK1-DIO3 region. The work presented in this dissertation focuses on comparing the dysregulation pattern of miRNAs from different cell sources of lupus-prone mice and investigating the potential role of miR-183C in the pathogenesis of SLE and inflammation. The first goal was to test whether dysregulated miRNAs initially identified in the spleen of MRL/lpr mice, a standard model for SLE, is also reflected in the peripheral blood mononuclear cells (PBMCs) as PBMC is the primary source of lymphocytes in human patients. In MRL/lpr mice, we found that dysregulated miRNAs in PBMCs were overall comparable to those identified in the splenic lymphocytes. Further, comparing dysregulated miRNAs between mice and humans showed a similarity in the dysregulation of miRNAs in PBMCs of murine and human lupus. Among the upregulated miRNAs, the expression of three miRNAs of miR-183C was found to be commonly upregulated. To investigate the role of miR-183C, we developed miR-183C in CD2+ cells of C57BL/6 Faslpr/lpr (miR-183C-/-B6/lpr) mice. In miR-183C-/-B6/lpr mice, we observed a significantly reduced level of anti-dsDNA in the serum and IgG immunocomplex deposition in the kidney. Importantly, in vitro inhibition of miR-183C in activated splenic lymphocytes led to reduced production of the proinflammatory cytokine, IFN, and Foxo1, a transcription factor that is a target of miR-183C miRNAs. I also tested for miRNA changes in C57BL/6 Faslpr/lpr mice with conditional deletion of Early Growth Response-2 (EGR2) (Egr2-/- B6/lpr), another knockout mouse developed in our laboratory. Egr2 has recently been shown to regulate immunity and autoimmunity and play a role in lupus. An unexpected observation is that Egr2-/-B6/lpr mice had significantly reduced expression of a group of lupus-related miRNAs that are located at the genomic imprinted DLK1-DIO3 locus. Given that the upregulation of DLK1-DIO3 miRNAs in lupus is subjected to DNA methylation regulation and that the epigenetic regulatory role of EGR2 is emerging in recent studies, reduced representative bisulfite sequencing (RRBS) was performed to evaluate the methylation changes induced by Egr2 deletion. Global DNA hypomethylation and methylation changes at specific sites at DLK1-DIO3 region were noticed in CD4+ T cells of Egr2-/-B6/lpr mice. Overall, our research suggested a therapeutic effect of inhibiting the miR-183C expression on SLE. The interplay between epigenetic factors could help expand the possibility of controlling epigenetic regulators in autoimmune disease treatment. / Doctor of Philosophy / Systemic lupus erythematosus (SLE) is an autoimmune disease that causes damage to multiple organs. Same with other autoimmune diseases, the exacerbated immune reaction to self-antigen and auto-reactive adaptive immune cells were described in SLE. Currently, the treatment of lupus mainly uses immunosuppressive drugs to inhibit the global immune reaction. Thus, the innovative drug is desperately needed for SLE patients. MicroRNAs (miRNAs) are small RNAs that inhibit the expression of genes by binding to mRNAs in a complimentary manner. Since the discovery of the first microRNA, the pivotal role of microRNAs in immunity and autoimmunity was vigorously investigated. Our lab was the first to describe a set of miRNAs that are commonly upregulated in three murine lupus models. Among these miRNAs, miR-183, miR-96, and miR-182 belong to the miR-183-96-182 cluster (miR-183C). The aim of the study in this dissertation focused on illuminating the dysregulation pattern of miRNAs in different cell sources in the murine lupus model and the role of miR-183C in the pathogenesis of SLE. We found that miRNAs are similarly dysregulated in peripheral blood mononuclear cells and splenic lymphocytes of MRL/lpr mice. Then we conditionally knocked out the miR-183C in B6/lpr mice and investigated the effect of miR-183C loss on the pathogenesis of autoimmunity. Importantly, we found that the deletion of miR-183C led to a reduced production level of autoantibodies and ameliorated the deposition of immune complexes in the kidney. Moreover, the production of proinflammatory cytokines of splenic lymphocytes was regulated by miR-183C as well. Besides miR-183C, I also investigated the effect of early growth response 2 (EGR2), a transcription factor, on the expression of a set of lupus-related miRNAs and the methylation change at the genome location of these miRNAs. In summary, miR-183C can be a potential therapeutic target for lupus treatment while clinical human studies are needed to better clarify the effectiveness and efficiency.

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24534号 / 医科博第148号 / 新制||医科||10(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 寺田 智祐, 教授 川上 浩司, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Infection with the single-celled parasite Toxoplasma gondii (phylum Apicomplexa) is usually benign in normal healthy individuals, but can cause congenital birth defects, ocular disease, and also life-threatening infection in immunocompromised patients. Acute infection caused by tachyzoites is controlled by a healthy immune response, but the parasite differentiates into a latent cyst form (bradyzoite) leading to permanent infection and chronic disease. Current therapies are effective only against tachyzoites, are highly toxic to the patient, and do not eradicate the encysted bradyzoites, thus highlighting the need for novel therapeutics. Inhibitors of histone deacetylases have been shown to reduce parasite viability in vitro demonstrating that chromatin remodeling enzymes, key mediators in epigenetic regulation, might serve as potential drug targets. Furthermore, epigenetic regulation has been shown to contribute to gene expression and differentiation in Toxoplasma. This dissertation focused on investigating the physiological role of a Toxoplasma GCN5-family histone acetyltransferase (HAT), termed TgGCN5-B. It was hypothesized that TgGCN5-B is an essential HAT that resides within a unique, multi-subunit complex in the parasite nucleus. Studies of TgGCN5-B have revealed that this HAT possesses a unique nuclear localization signal (311RPAENKKRGR320) that is both necessary and sufficient to translocate the protein to the parasite nucleus. Although no other protein motifs have been identified in the N-terminal extension of TgGCN5-B, it is likely that this extension plays a role in protein-protein interactions. All GCN5 homologues function within large multi-subunit complexes, many being conserved among species, but bioinformatic analysis of the Toxoplasma genome revealed a lack of many of these conserved components. Biochemical studies identified several potential TgGCN5-B associating proteins, including several novel apicomplexan transcription factors. Preliminary evidence suggested that TgGCN5-B was essential for tachyzoites; therefore, a dominant-negative approach was utilized to examine the role of TgGCN5-B in the physiology of Toxoplasma. When catalytically inactive TgGCN5-B protein was over-expressed in the parasites, there was a significant decrease in tachyzoite growth and viability, with initial observations suggesting defects in nuclear division and daughter cell budding. These results demonstrate that TgGCN5-B is important for tachyzoite development and indicate that therapeutic targeting of this HAT could be a novel approach to treat toxoplasmosis.