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京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20285号 / 医科博第76号 / 新制||医科||5(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 篠原 隆司, 教授 浅野 雅秀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Although the understanding of genetic predisposition to prostate cancer (PCa) has been improved through genome-wide association studies (GWAS), little is known about the biological implication of germline variants residing in coding or non-coding regions in cancer development and progression. Our hypothesis is that inherited variants may predispose to specific early recurrent genomic events observed in PCa adenocarcinomas, possibly in the context of variable androgen receptor (AR) signaling that changes during a man’s lifetime. Recent in silico analysis by our group on potential association between germline variants and PCa specific somatic lesions identified a non-coding polymorphic regulatory element at the 7p14.3 locus associated with DNA repair and hormone regulated transcript levels and with an early recurrent prostate cancer specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene (OR=5.54, P=1.22e-08) in human prostate tissue data. In order to functionally characterize the polymorphic 7p14.3 locus (rs1376350, single nucleotide polymorphism, G>A), we set up to establish isogenic cell lines harboring the minor allele by using the CRISPR/Cas9 system. In parallel, CRISPR/Cas9 system was used to knock out different portion of the region encompassing the 7p14.3 variant and to eliminate transcription factors (TFs) binding sites that were identified from previous in silico analysis (i.e. AR and CCAAT/Enhancer Binding Protein (C/EBP) beta (CEBPβ)). The transcriptomes of edited pools and edited single clones from macrodeletion (731 bp), microdeletion (50 bp) and alterations of TFs binding sites were analyzed and compared to the transcriptomes of isogenic cells heterozygous (A/G) and homozygous (A/A) for the minor allele A of the risk variant rs1376350 (with or without AR overexpression). These data identified a set of genes scattered throughout the genome with the same pattern of deregulation suggesting the implication of the variant on the regulation of genes residing in different chromosomes. Additionally, ChIP-qPCR experiments for histone modification supported the identification of the 7p14.3 locus with enhancer activity. Furthermore, ChIP-qPCR of histone mark associated with transcriptional activation or repression in isogenic cells harboring the minor allele A upon AR overexpression showed that the activity of the locus is higher for the minor allele A compared to G, independently from AR activation. Despite the limitations of our model and the current lack of validation in other cells, we confirmed that some of the differentially expressed genes that emerged from the comparative analysis of edited cells are deregulated in human normal and tumor prostate samples as well. This work is a proof of concept of germline predisposition to molecularly distinct cancer subclasses and has the potential to nominate new mechanisms of cancer development. Future work aims to elucidate the mechanisms implicated in the deregulation of the transcriptome by combining the information obtained until now with potential new players that we expect to identify by Mass Spectrometry experiments. To clarify the link between the 7p14.3 variant and the somatic mutations in SPOP, we plan to express mutant SPOP in isogenic cells harboring the minor allele and to asses DNA damage response upon overexpression or silencing of TFs binding at and around the rs1376350 variant. My work is an example of how the CRISPR/Cas9 system can be used to develop a technical framework with convergent approaches to functionally characterize polymorphic regulatory regions including but not limited to the establishment of isogenic cells upon single nucleotide editing.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24089号 / 医博第4865号 / 新制||医||1059(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 武藤 学, 教授 万代 昌紀, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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La divison cellulaire asymétrique est un processus essentiel qui permet aux cellules souches de s’auto-renouveller et de produire une cellule fille destinée à la différenciation. La lignée germinale de C. elegans, totipotente et immortelle, est une lignée de cellules souches qui contient des organites ribonucléoprotéiques appelés granules P. Au cours du développement ces derniers sont toujours localisés spécifiquement dans les cellules précurseurs de la lignée germinals, suggérant qu’ils sont des déterminants de la lignée germinale. De façon intéressante, des granules ribonucléoprotéiques, comme les P bodies impliqués dans le contrôle post-transcriptionnel, ont été observés chez tous les organismes. Néanmoins, la fonction précise des granules P de C. elegans est inconnue. Récemment, notre laboratoire a montré que NHL-2, un homologue de Mei-P26 de Drosophile, colocalise avec les granules P dans des embryons précoces et joue un rôle dans la division cellulaire asymétrique et dans la polarité cellulaire. Tous les granules P contiennent NHL- 2, ce qui nous a mené à poser l’hypothèse que NHL-2 régule la biogenèse et la fonction des granules P. Nous avons testé cette hypothèse par imagerie et quantification de l'intensité de PGL-1, un composant essentiel des granules P, dans des embryons fixés. Nos résultats montrent que dans des embryons mutants pour nhl-2 il y a une réduction du nombre de granules P, de l'intensité de fluorescence moyenne (IFM) et de l'intensité de fluorescence total (IFT) de PGL-1. Une analyse plus poussée a montré qu'il existe deux populations distinctes d’embryons mutants pour nhl-2 : l’une présente une intensité de PGL-1 comparable à celle d’une population sauvage alors que le second groupe présente une forte réduction des quantités de PGL-1 et est comparable à des mutants pour pgl-1. Cette variabilité est aussi observée dans le phénotype de stérilité de nhl-2 mutant à des températures élevées. Globalement, nos résultats suggèrent que la perte de fonction de NHL-2 perturbe la prolifération des cellules germinales ainsi que la formation et/ou la stabilité des granules P au cours des étapes précoces du développement des précurseurs de la lignée germinals. D’autre part, ils suggèrent que la fonction de NHL-2 pourrait être partiellement redondants avec les autres régulateurs de la stabilité des granules P. Mots-clés : Granules P, NHL-2, Cellules germinals. / Asymmetric cell division is a process that enables stem cells to simultaneously self-renew and generate progeny committed to differentiation. For instance the totipotent and immortal germ lineage in C. elegans is a stem cell lineage that contains ribonucleoprotein organelles called P granules. P granules are present specifically in germ cells and have been proposed to function as germ line determinants. Interestingly, various RNP granules, such as P bodies that regulate post-transcriptional control have been also observed in all organisms. Nevertheless, the precise function of C. elegans P granules remains elusive. Recently our lab showed that NHL-2, a C. elegans homologue of Drosophila Mei-P26, localizes to P granules in early embryos and plays a role in asymmetric cell division and cell polarity. All P granules contain NHL-2, raising the possibility that NHL-2 plays a role in regulating P granule biogenesis and function. We investigated this possibility by imaging and quantifying the intensity of PGL-1, a core component of P granules, in fixed embryos. This analysis revealed that there is a reduction in: 1) the number of P granules and 2) the mean fluorescence intensity (MFI) and total fluorescence intensity (TFI) of PGL-1 staining in nhl-2 null mutant embryos compared to wild type. Our further analysis of nhl-2 loss of function demonstrates that NHL-2, similar to P granule core components such as DEPS-1, GLH-1 and PGL-1, is required for proper germ cell proliferation and fertility at elevated temperature. One aspect of the nhl-2 loss of function phenotype is that nhl-2 mutants are distributed in different groups based on both their P granules number and PGL-1 intensity: one population has wild type PGL-1 intensity while the second group has severely reduced amounts of PGL-1. Strikingly, such variability is observed even in the sterility phenotype of nhl-2 mutants at elevated temperatures. Taken together, our results suggests that NHL-2 loss of function disrupts germ cell proliferation as well as P granule formation or stability during early stages of germ cell precursor development and that NHL-2 function may be partially redundant with other regulators of P granule stability. Keywords : P granules, NHL-2, Germline.