Globalement réprimés à la méiose (MSCI), les chromosomes sexuels sont partiellement réactivés dans les spermatides rondes avant l’arrêt général de la transcription dans les spermatozoïdes. Alors qu’il est clairement démontré que le MSCI est essentiel pour la poursuite de la spermatogenèse, la proportion de gènes réactivés ainsi que le mécanisme de régulation des chromosomes sexuels après la méiose demeurent un sujet de recherche et de débats. Chez la souris, la délétion du bras long du chromosome Y (MSYq) provoque la surexpression de plusieurs centaines de gènes, dont la majorité est portée par les chromosomes sexuels, associée à des modifications de la chromatine; ceci aboutit à la production de spermatozoïdes malformés et non-fécondants, présentant notamment une compaction anormale de leur chromatine. Sly est un des cinq gènes multicopies du MSYq et l’abolition de son expression chez la souris (souris Sly-KD) a récemment démontré qu’il est à la base de la dérégulation épigénétique des chromosomes sexuels et des problèmes de compaction de la chromatine des mâles MSYq-. De plus, les mâles avec délétion partielle de MSYq ainsi que les mâles Sly-KD produisent une descendance avec un excès de femelles, ce qui suggère l’existence d’un conflit intragénomique avec Slx, un gène multicopie homologue de Sly et porté par le chromosome X. Quel rôle pour SLY pendant la spermiogenèse ? Afin de répondre à cette question nous avons étudié les gènes cibles et les partenaires de SLY. Nous avons montré que SLY interagit avec TBL1XR1, membre inhérent au complexe répressif Ncor. De plus, localisée au niveau des promoteurs de gènes exprimés dans les spermatides et liés aux chromosomes sexuels et autosomaux, SLY contrôle des gènes impliqués dans la régulation génique et chromatinienne (e.g, variants H2A et DOT1L). Nous avons également détecté une baisse significative de la marque H3K79me2 accompagnée d’une rétention anormale des histones dans les spermatozoïdes des souris Sly-KD et proposons que DOT1L, la seule H3K79 méthyltransférase identifiée à ce jour, est essentielle au remodelage de la chromatine. Quels sont les mécanismes moléculaires du conflit intragénomique entre SLY et SLX ? Des expériences de co-immunoprécipitations ont démontré que SSTY, codée comme SLY par un gène multicopies du MSYq, interagit préférentiellement avec SLX in vivo. En outre, SLX et SLY sont capables toutes deux d’interagir avec SPIN1, homologue de SSTY et capable de se lier à H3K4me3. Ces différentes interactions entre SLX/SLY et SPIN1/SSTY pourraient participer au conflit intragénomique. Par la réévaluation de plusieurs jeux de données (RNA-Seq et ChIP-Seq) nous avons démontré que la répression des chromosomes sexuels ne persiste pas au-delà de la méiose et que le conflit intragénomique entre SLY et SLX représente une pression de sélection considérable, en partie responsable du paysage épigénétique spécifique des chromosomes sexuels et de leur enrichissement en gènes multicopies exprimés après la méiose. En conclusion, nos travaux ont permis de caractériser le mode d’action de SLX/SLY et d’identifier de nouveaux facteurs impliqués dans la régulation (épi)génétique pendant la spermiogenèse qui sont conservés chez l’homme. / Sex chromosomes in mammals are globally repressed during meiosis (MSCI ) and then partially reactivated in round spermatids prior to the transcriptional activity shut down occurring in spermatozoa. Whereas the MSCI is essential for spermatogenesis, the proportion of reactivated genes and the underlying mechanisms of the sex chromosomes regulation after meiosis is still a conundrum. In mice, deletions of the long arm of the Y chromosome (MSYq-) are responsible for the overexpression of more than hundred sex chromosome genes associated with epigenetic modifications that leads to impaired sperm functions and abnormal chromatin compaction. Sly is one of the five multicopy genes present on MSYq and Sly deficiency (Sly-KD) has recently been showed to be at the basis of the gene deregulation and sperm defects obrserved in MSYq- mice. Additionally, partially deleted MSYq males and Sly-KD mice produce offspring with a sex ratio distortion in favor of females; these observations suggest a postmeiotic intragenomic conflict involving Sly and its homolog Slx, an X-linked multicopy gene. What role for SLY during spermiogenesis? In order to decipher SLY mechanisms of action, we sought to study SLY target genes and partners. We showed that SLY interacts with TBL1XR1, an inherent member of the repressive Ncor complex. Meanwhile, we found that SLY is enriched at the promoter of spermatid expressed genes encoded both by sex chromosomes and autosomes. Additionally, SLY controls genes involved in genetic and chromatin regulation (e.g, H2A variants and DOT1L). We also observed a significant reduction of H3K79me2 levels associated with abnormal histone retention in Sly-KD spermatozoa. We propose that DOT1L, the principal H3K79 methyltransferase identified to date, is essential for chromatin remodeling in spermatids. What are the molecular mechanisms involved in the ongoing intragenomic conflict between SLY and SLX? We showed by co-immunoprecipitation that SSTY, another Y-linked multicopy gene, preferentially interacts with SLX in vivo. Furthermore, both SLX and SLY interact with SPIN1, a homolog of SSTY which is able to recognize H3K4me3. The interactions between SLX/SLY and SPIN1/SSTY could be part of the intragenomic conflict. By re-evaluating several RNA-Seq and ChIP-Seq datasets we demonstrated that MSCI does not persist beyond meiosis. We proposed that the intragenomic conflict between SLY and SLX constitutes a considerable selection pressure, partly responsible for the specific epigenetic landscape of sex chromosomes and their enrichment in multicopy genes expressed after meiosis. In conclusion, our work allowed a better understanding of the mode of action of SLX/SLY and the identification of new factors involved in the (epi)genetic regulation during spermiogenesis that are conserved in humans.

Understanding the mechanisms and nature of tumour heterogeneity is a key focus of current cancer research. Tumour heterogeneity can arise from clonal evolution and/or differentiation. This thesis investigated the role of methylation in dynamic regulation of CD24 based heterogeneity in colorectal cancer cell lines. First, E-Cadherin variation between the cell lines LS174T and LS180 was investigated to find out whether E-Cadherin had any causal role in the difference in lumen formation between these two cell lines derived from the same tumour. These studies found no evidence of a causal role of E-Cadherin. However, morphological heterogeneity in LS174T was observed during the E-Cadherin study, suggesting that this cell line might be a mixture of two different clones. Single cell sorting by FACS allowed to isolate and establish these clones which were stable in the culture for long enough (until passage ~30) to allow their characterisations. Between the two clones, the CD24⁺ clone (named the LS174T_Clone 1^CD24+) was found to have shorter doubling time (23 hours) than the CD24- clone (named the LS174T_Clone 2CD24-; 30 hours). When cultured in matrigel, the LS174T_Clone 1^CD24+ showed higher clonogenicity (Chapter 4). Microarray analysis further revealed differences in gene expression including LAPTM4B, CXCR4, TGFIB and IL8 between these clones. Interestingly, when maintained for a long time in culture (around passage 50, which is equivalent to ~7 months), CD24 expression went through a gradual change in these clones, which became more evident from subclones of LS174T_Clone 2^CD24- (Chapter 6) and clones from another CRC cell line SW480 (chapter 5 and 6). To understand the mechanism of this dynamic change in the expression of CD24, it was first shown that no mutation could be responsible for this phenomenon. This suggested that promoter methylation of CD24 might be the mechanism of the observed dynamic changes in CD24 expression. Bisulfite (BS) modification of DNA from the LS174T clones and CD24 differentially expressing CRC cell lines (such as CD24- cell lines: CC20, RKO vs CD24+ cell lines: DLD1, NCIH716) followed by Sanger sequencing showed that direct methylation of seven CpG positions in the CD24 promoter region Chr6:106975560-106975834 is strongly correlated with the expression of CD24. Further subcloning and sequencing revealed that changes in the methylation of only two out of the seven CpG positions might be the main contributor to the CD24 expression differences. This is the first evidence of direct methylation-mediated regulation of CD24, showing, more broadly, how methylation can contribute to and maintain dynamic heterogeneity in cancer cells. Finally, a mixed culture experiment with the CD24+ and CD24- clones was conducted to test a simple mathematical model, which aimed to explain the interaction between the clones that are stably present in the LS174T cell line (Chapter 7). Altogether, these experiments suggest that genes such as REG1A (an inducer of angiogenesis) might be expressed because of synergistic interactions between the clones, whereas CXCR4 and TFF2 might be involved in a receptor-ligand complementary relationship. These findings have set a ground for future studies to confirm such interactions between co-existing subpopulations within a heterogeneous milieu of cancer cells.

Cytotoxicity is the capacity for immune cells to kill infected or malignant cells in order to eliminate pathogens and tumours through different mechanisms including the exocytosis of perforin-containing cytosolic granules. This crucial property is usually restricted to specialized innate and adaptive lymphocytes such as natural killer (NK) cells and CD8 T cells. T lymphocytes differentiate in the thymus and are delivered to the peripheral blood as naive T cells committed to either the CD8 or the CD4 lineage. CD8 T cells are programmed to acquire cytotoxic effector functions under the control of the transcription factor (TF) Runx3. The fate of CD4 T cells is to acquire multiple helper functions through the action of the TF ThPOK that promotes CD4 helper functions and restricts the CD8 cytotoxic program. However, this restriction is not absolute as cytotoxic CD4 (CD4CTX) T cells differentiate in vivo, indicating that the multipotency of human naive CD4 T cells includes the ability to acquire perforin expression and potent cytotoxicity in vitro and ex vivo. This cytotoxic potential correlates with outcome in human pathology and mediates protection against viral challenge and tumour eradication in murine models. CD4CTX T cells are terminally differentiated effector memory T cells that accumulate during cytomegalovirus chronic infection and ageing. They are phenotypically and functionally related to T helper type 1 (Th1)-effector memory cells. However, whether they belong to the Th1 pathway or constitute a separate specialized helper T cell subset is unknown. In this work, we show that CD4CTX T cell differentiation is an integral part of the Th1 pathway. Indeed, CD4 T cells acquire cytotoxic potential early in the memory differentiation process as central memory Th1 but not Th2 and Th17 cells are epigenetically primed to develop a cytotoxic program. The expression of perforin and other cytotoxic genes present a stepwise increase profile that is specific of the Th1 differentiation pathway. This profile has been recapitulated in an in vitro model of effector CD4 T cell differentiation in which naive CD4 T cells acquire cytotoxicity one to two weeks after polyclonal stimulation when cultured in presence of Th1 cytokines. The molecular regulation of CD4CTX T cells is poorly understood and most available data have been generated in mice. These data include the observation of intraepithelial CD4CTX T cells in the mouse gut after loss of ThPOK expression and subsequent up-regulation of a Runx3-dependent cytotoxic program. Other candidate regulators of CD4 T cell cytotoxic function include the TF regulating Th1 and CD8CTX T cells differentiation such as Runx3, T-bet and Eomesodermin (Eomes). We show that the transcriptional program of human CD4CTX T cells is enriched in CD8-lineage genes. However, by contrast to CD4CTX T cells from the mouse intestine, human circulating CD4CTX T cells maintain the expression of ThPOK and even up-regulate this TF upon differentiation from naive CD4 T cells. Surprisingly, this sustained expression of ThPOK was compatible with the establishment of a T-bet- and Runx3-dependent cytotoxic transcriptional program. The specific knockdown of T-bet or Runx3 but not Eomes resulted in impaired cytotoxic differentiation whereas ThPOK knockdown enhanced perforin expression and cytotoxicity. We propose that CD4CTX T cells constitute the terminal stage of Th1 memory differentiation and that ThPOK, Runx3 and T-bet co-regulate this process by instructing a cytotoxic transcriptional network largely shared with CD8CTX T cells. The modulation of this network is a potential target for novel immunotherapeutic strategies in viral infections and cancer. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

<div><div><div><p>Transcriptomic regulation of human liver is a tightly controlled and highly dynamic process. Genetic and environmental exposures to this process play pivotal roles in the development of multiple liver disorders. Despite accumulating knowledge have gained through large-scale genomics studies in the developed adult livers, the contributing factors to the interindividual variability in the pediatric livers remain largely uninvestigated. In the first two chapters of the present study, we addressed this question through an integrative analysis of both genetic variations and transcriptome-wide RNA expression profiles in a pediatric human liver cohort with different developmental stages ranging from embryonic to adulthood. Our systematic analysis revealed a transcriptome-wide transition from stem-cell-like to liver-specific profiles during the course of human liver development. Moreover, for the first time, we observed different genetic control of hepatic gene expression in different developmental stages. Motivated by the critical roles of genetics variations and development in regulating hepatic gene expression, we constructed robust predictive models to impute the virtual liver gene expression using easily available genotype and demographic information. Our model is promising in improving both PK/PD modeling and disease diagnosis for pediatric patients. In the last two chapters of the study, we analyzed the genomics data in a more liver disease- related context. Specifically, in the third chapter, we identified Macrophage migration inhibitory factor (MIF) and its related pathways as potential targets underlying human liver fibrosis through an integrative omics analysis. In the last chapter, utilizing the largest-to-date publicly available GWAS summary data, we dissected the causal relationships among three important and clinically related metabolic diseases: non-alcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), and obesity. Our analysis suggested new subtypes and provided insights into the precision treatment or prevention for the three complex diseases. Taken together, through integrative analysis of multiple levels of genomics information, we improved the current understanding of human liver development, the pathogenesis of liver disorders, and provided implications to precision medicine.</p></div></div></div>

<p>Triple Negative Breast Cancer (TNBC) is the most aggressive type of breast cancer. TNBC patients are resistant to virtually all target therapies and suffer a higher post-chemotherapy relapse with a worse overall survival compared with other types of breast cancers. Therefore, the development of an effective therapy is urgently needed. PKM2 plays a prominent role in mediating<b> </b>tumor glycolysis and PKM2 is often overexpressed in human cancers. However, whether PKM2 mediated glycolysis is necessary for cancer cell growth is questionable. Here, I have found that inhibition of PKM2 does not affect TNBC cell growth due to a metabolic switch from glycolysis to fatty acid oxidation (FAO). We show that PKM2 directly interacts with EZH2 to coordinately mediate epigenetic silencing of SLC16A9, transporter of a key player in FAO, Carnitine. Inhibition of either PKM2 or EZH2 increases levels of SLC16A9 and intracellular Carnitine to promote FAO and thereby sustains cancer cell growth. Direct inhibition of EZH2 using a clinically tested EZH2 inhibitor, GSK126, is able to elicit a previously unidentified vulnerability to a clinically tested FAO inhibitor, Etomoxir. As a result, combined GSK126-Etomoxir treatment synergistically abolishes TNBC xenograft tumor growth in vivo. Together, this study uncovers PKM2-EZH2 mediated metabolic reprogramming that leads to a new drug combination therapy by dual targeting of EZH2 and FAO for effective treatment of TNBC.<b> </b></p> <p> </p> <p>Furthermore, Dendritic Cell (DC) vaccination has shown promise in treating cancer patients. However, the <i>in vitro</i> generation of a fully functional DC remains a big challenge in this field. EZH2 inhibition has shown to be able to create an immunologically ‘hot’ tumors. Nonetheless, the role of EZH2 in regulation of DC function is still unclear. I found that the expression levels of EZH2 and its functional maker, H3K27Me3, are enhanced following maturation from immature DC (iDC) into two functional DCs, α-type 1-polarized-DC (αDC) and gold standard DC (sDC). Moreover, inhibition of EZH2 by GSK126 treatment elicits a dependency of sDC on FAO. These results suggest that EZH2 plays a role in maturation of DC through metabolic reprogramming, which may also provide new DC based immunotherapy of TNBC. </p>

RNA interference (RNAi) is an important pathway that regulates gene expression in several organisms. The role of RNAi in post-transcriptional gene silencing in the cytoplasm is well characterized. In contrast, the role of RNAi components in the nucleus remains to be elucidated. Previous reports have indicated that RNAi components (Dicer and Argonaute proteins) and small RNAs act in the nucleus to regulate various pathways including heterochromatin formation, transposable elements repression, RNA Pol II processivity and alternative splicing. Nuclear Ago1 and Dicer have also been found to associate with active promoters and enhancers in mammalian cells, however their functional roles and mechanisms remain elusive. In this work, I investigated the functional role of nuclear RNAi components in gene expression regulation during skeletal muscle differentiation. To address this question, I undertook genomic and biochemical approaches applied to myogenic cells (C2C12) as a model system. I found that Ago1 and Dicer are present in the nucleus of C2C12 cells and expressed during differentiation. Chromatin Immunoprecipitation (ChIP) coupled with high throughput sequencing and quantitative real-time PCR indicate that Ago1 and Dicer are enriched at promoters and enhancer regions of myogenic genes. Interestingly, I found that depletion of Ago1 and Dicer reduces enhancer RNAs (eRNAs) levels at enhancer regions and expression of MyoD during differentiation. I observed that loss of Ago1 impacts differentiation, whereas, loss of Dicer leads to cell death and has severe effects on C2C12 cells. Moreover, using Chromosome Conformation Capture (3C), I revealed that Ago1 is involved in enhancer-promoter interaction at MyoD locus. The knockdown of Ago1 destabilizes these interactions and decreases the expression of MyoD. Finally, I demonstrated that Ago1 binds to eRNAs and interacts with CBP Acetyl-transferase in the nucleus of myotube cells. Ago1 depletion leads to loss of eRNA-CBP interaction and consequent impairment of CBP acetyltransferase activity and failure of MyoD mediated activation of the myogenic program. Taken together, these finding indicate that nuclear Ago1 together with eRNAs and CBP regulates MyoD expression by stimulating histone acetylation during differentiation. This study uncovered a novel function of chromatin associated Ago1 in gene expression regulation during mammalian skeletal muscle differentiation.

La télomérase est présente dans un nombre limité de cellules, telles que la plupart des cellules cancéreuses où son activité est indispensable pour l’immortalisation de ces dernières. C’est pourquoi cette enzyme a été proposée comme cible prometteuse pour des thérapies anticancéreuses. Ainsi, il est d’un intérêt certain d’identifier les mécanismes par lesquels elle est régulée, notamment via sa sous-unité catalytique, hTERT. Les rétinoïdes sont des inducteurs bien connus de la maturation granulocytaire associée à une répression de hTERT dans les blastes de leucémie aiguë promyélocytaire (LAP). Dans une lignée cellulaire LAP résistant à la maturation induite par les rétinoïdes (NB4-LR1), a précédemment été identifiée une nouvelle voie de répression transcriptionnelle de hTERT en absence de différenciation. De plus, mon laboratoire d’accueil a isolé un variant de la lignée NB4-LR1 résistant à cette répression transcriptionnelle de hTERT, la lignée NB4-LR1SFD. Ensemble, ces lignées cellulaires, qui se comportent différemment face à un traitement à l’ATRA, fournissent un outil unique et puissant pour obtenir plus d’informations sur plusieurs problèmes de la régulation de la biologie moléculaire de la télomérase. Dans cette étude, en utilisant plusieurs approches complémentaires (immunoprécipitation de la chromatine combinée à une technique d’analyse à haut débit du positionnement des nucléosomes et de la méthylation de l’ADN et une approche de FISH), j’ai pu obtenir une vue intégrée des événements épigénétiques qui promeuvent la transition du promoteur de hTERT d’un état silencieux à un état actif et inversement. Cette information sera cruciale pour le développement de stratégies anticancéreuses ciblant l’expression de hTERT. / Telomerase is present in a limited number of cells, most of them cancerous where its activity is crucial for their immortalisation. Thus, that enzyme has been proposed as a promising target for anticancer therapies. Therefore, it is of great interest to identify the mechanisms by which it is regulated, notably through its catalytic subunit hTERT. Retinoids are well-known inducers of granulocytic maturation associated with hTERT repression in acute promyelocytic leukemia (APL) blasts. However, in NB4-LR1, a maturation-resistant APL cell line, was previously identified a new pathway of retinoid-induced hTERT transcriptional repression independent of differentiation. Furthermore, my host lab reported the isolation of a variant of NB4-LR1 cells resistant to this repression: NB4-LR1SFD. These two cell lines, which behave distinctly in response to ATRA treatment, provide a unique and interesting tool to gain further insights into the molecular biology of telomerase regulation. In this thesis project, using several complementary approaches (chromatin immunoprecipitation combined to a high-resolution, single-molecule nucleosome positioning assay and DNA methylation, and a FISH approach) allowed me to shed more light on the integrated epigenetic events that lead to hTERT promoter transition from its silent to its active state and vice versa. The information obtained could be crucial for the development of anticancer strategies targeting hTERT expression.

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

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