京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20195号 / 理博第4280号 / 新制||理||1615(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

DNA methylation plays a pivotal role in governing cellular processes including genomic imprinting, gene expression, and development. Recently, the Tet family of methylcytosine dioxygenases(Tet1, Tet2 and Tet3) was found to catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate in the pathway of DNA demethylation. Tet enzymes catalyze this hydroxylation in a 2-oxoglutarate and Fe2+ dependent manner. We have recently reported significant levels of 5mC and 5hmC modification in immunoprecipitates of mammalian mitochondrial DNA(mtDNA). We provide the first evidence that a DNA Methyltransferase-1 isoform (mtDNMT1) translocates to the mitochondria using an N-terminal mitochondrial targeting sequence. mtDNMT1 expression is upregulated by NRF1 and PGC1α, master regulators of mitochondrial biogenesis and function, as well as by loss of p53. Altered mtDNMT1 expression asymmetrically affects mtDNA transcription. We are now pursuing the role of Tet proteins in generating 5hmC in mtDNA. Using an in vitro enzyme assay, we have successfully detected Tet activity in crude and percoll purified mitochondrial fractions of HCT116 cells. Mitoprot analysis on Tet family predicts that Tet1 may be translocated to the mitochondria. Immunoblot analysis indicates that a band of expected size(235kDa) is present on immunoblots of mitochondrial fraction from mouse embryonic stem cells with an antibody directed against Tet1. This band, however, is not protected from trypsin treatment of mitochondria indicating that Tet1 may not be transported to the mitochondrial matrix. The putative Tet1 mitochondrial targeting sequence (MTS) fails to carry heterologous protein to the mitochondria. Knock out of Tet1 in mouse ES cells also does not alter 5hmC signal in hydroxyMeDIP assay. We now seek to determine if Tet2/Tet3 may be involved in 5hmC generation. In the nucleus, 5hmC serves as an intermediate in the process of DNA demethylation through the combined action of cytidine deaminases and the base excision repair pathway. We plan to investigate if 5hmC holds the same functional significance in the mitochondria as it does in the nucleus. Our overall goal is to understand epigenetic regulation of normal mitochondrial function and changes that occur in diseases involving mitochondrial dysfunction such as ischemic heart disease, neurodegenerative diseases like Parkinsons disease, and cancer.

Small-molecule DNA interactive compounds are critical as both carcinogens and therapeutic agents. In this research, a synthetic precursor to a known carcinogen, (±)-N’-nitrosonicotine-5’-acetate was synthesized, and its interactions with DNA were evaluated by polyacrylamide gel electrophoresis and electrospray-ionization mass spectrometry. A library of skipped benzimidazolium aza-enediynes which selectively target unmethylated cytosines in presence of unmethylated cytosines were synthesized, and their biological properties were evaluated in a nicking assay and cytotoxicity study. Finally, a series of structural analogs to a antineoplastic agent, UK-1, were synthesized via a biaryl coupling at C2 on the benzoxazole. / text

Somatic mutations are the main triggers that initiate the formation of cancer. Large sequencing data sets in recent years revealed a substantial number of mutational processes, many of which are poorly understood or of completely unknown aetiology. These mutational processes leave characteristic sequence patterns, often called "signatures", in the DNA. Characterisation of the mutational patterns observed in cancer patients with respect to different genomic features and processes can help to unravel the aetiology and mechanisms of mutagenesis. Here, we explored the effects of DNA modifications and DNA replication on mutagenesis. The most common mutation type, C>T mutations in a CpG context, is thought to result from spontaneous deamination of 5-methylcytosine (5mC), the major DNA modification. Much less is known about the mutational properties of the second most frequent modification, 5-hydroxymethylcytosine (5hmC). Integrating multiple genomic data sets, we demonstrate a twofold lower mutagenicity of 5hmC compared to 5mC, present across multiple tissues. Subsequently, we show how DNA modifications may modulate various mutational processes. In addition to spontaneous deamination of 5mC, our analysis suggests a key role of replication in CpG > TpG mutagenesis in patients deficient in post-replicative proofreading or repair, and possibly also in other cancer patients. Together with an analysis of mutation patterns observed in cancers exposed to UV light, tobacco smoke, or editing by APOBEC enzymes, the results show that the role of DNA modifications goes beyond the well-known spontaneous deamination of 5mC. Finally, we explored which of the known mutational processes might be modulated by DNA replication. We developed a novel method to quantify the magnitude of strand asymmetry of different mutational signatures in individual patients followed by evaluation of these exposures in early and late replicating regions. More than 75 % of mutational signatures exhibited a significant replication strand asymmetry or correlation with replication timing. The analysis gives new insights into mechanisms of mutagenicity in multiple signatures, particularly the so far enigmatic signature 17, where we suggest an involvement of oxidative damage in its aetiology. In conclusion, our results suggest that DNA replication or replication-associated DNA repair interacts with most mutagenic processes.

Alzheimer’s disease (AD) is the most common form of dementia and the sixth leading cause of death in the United States, with no therapeutic option to slow or halt disease progression. Development of two characteristic pathologic lesions, amyloid beta plaques and neurofibrillary tangles, in the brain are associated with synaptic dysfunction and neuron loss leading to memory impairment and cognitive decline. Although mutations in genes involved in amyloid beta processing are linked to increased plaque formation in the inherited familial form of AD, the more common idiopathic form, termed sporadic AD, develops in the absence of gene mutations. In contrast, alterations in gene expression and transcription occur in plaque and tangle susceptible brain regions of sporadic AD subjects, even in the earliest stages of development of pathologic burden, and may give insight into the pathogenesis of AD. Epigenetic modifications to cytosine are known to alter transcriptional states and gene expression in embryonic development as well as in cancer studies. With the discovery of enzymatically oxidized derivatives of 5-methylcytosine (5-mC), the most common epigenetic cytosine modification, a probable demethylation pathway has been suggested to alter transcriptional states of DNA. The most abundant 5-mC derivative, 5-hydroxymethylcytosine (5-hmC), while expressed at low concentrations throughout the body, is expressed at high concentrations in brain cells. To determine the role cytosine modifications play in AD, this study was directed at the quantification of epigenetic modifications to cytosine in several stages of AD progression using global, genome-wide, and gene-specific studies. To determine global levels of each cytosine derivative in brain regions relevant to AD progression, a gas chromatography/mass spectrometry quantitative analysis was utilized to analyze cytosine, 5-mC, and 5-hmC in tissue specimens from multiple brain regions of AD subjects, including early and late stages of AD progression. To determine the genome-wide impact of 5-hmC on biologically relevant pathways in AD, a single-base resolution sequencing analysis was used to map hydroxymethylation throughout the hippocampus of late stage AD subjects. Finally, to determine gene-specific levels of cytosine, 5-mC, and 5-hmC, a quantitative polymerase chain reaction (qPCR) protocol was paired with specific restriction enzyme digestion to analyze target sequences within exons of genes related to sporadic AD. Results from these studies show epigenetic modifications to cytosine are altered on the global, genome-wide, and gene-specific levels in AD subjects compared to normal aging, particularly in early stages of AD progression, suggesting alterations to the epigenetic landscape may play a role in the dysregulation of transcription and the pathogenesis of AD.

No description available.

Les profils de méthylation de l’ADN sont des marques épigénétiques essentielles contrôlant l'expression génétique spécifique des tissus. Ces profils sont fidèlement reproduits par l’enzyme DNMT1 qui est dirigée par la protéine UHRF1 vers les sites CpG hémiméthylés (HM). La spécificité élevée d’UHRF1 vis-à-vis de ces sites CpG HM est liée à la capacité de son domaine SRA de basculer sélectivement les résidus méthylcytosine (mC). Par conséquent, la compréhension de la capacité d’UHRF1 à lire les séquences d'ADN et de basculer leurs résidus mC est une question importante en épigénétique moléculaire. Dans le présent travail, nous avons utilisé des analogues de nucléobases sensibles à l'environnement pour étudier le basculement de base induit par SRA. Nous avons découvert qu’un étiquetage par la 2-thiényl-3-hydroxychromone (3HCnt) à proximité de la cible CpG méthylée, permet le suivi de ce basculement SRA-induit de mC et de sa dynamique. Les spectroscopies de fluorescence à l'état stationnaire et de "stopped flow" ont montré des différences significatives entre les ADNs HM et non méthylé (NM) vis-à-vis de la reconnaissance et la cinétique de liaison du SRA. Effet, nous avons montré que SRA est capable de se lier et de glisser avec une cinétique rapide sur le duplex NM, en accord avec le rôle de lecteur d’UHRF1. Par contre, la cinétique de basculement de mC s’avère beaucoup plus lente, ce qui augmente sensiblement la durée de vie d’UHRF1 lié à un site CpG hémi-méthylé et donc la probabilité de recruter DNMT1 afin de dupliquer fidèlement le profil de méthylation de l’ADN. Nous avons ainsi obtenu pour la première fois un test capable de suivre le basculement de la base induit par UHRF1, ce qui nous a permis de proposer un mécanisme pour le recrutement de DNMT1 par UHRF1 sur les sites HM. / DNA methylation patterns are key epigenetic marks which control tissue specific gene expression. These patterns are faithfully replicated by the DNMT1 enzyme which is directed by the UHRF1 protein to hemi-methylated (HM) CpG sites. The high specificity of UHRF1 to HM CpG sites is related to the ability of its SRA domain to selectively flip methylcytosine (mC) residues. Therefore, the understanding of how UHRF1 reads DNA sequences and flips mC residues is an important question in molecular epigenetics. In the present work, we apply environment-sensitive nucleobase analogues to study the SRA-induced base flipping. We found that only labelling by 2-thienyl-3-hydroxychromone (3HCnt) outside but close to the target methylated CpG allows monitoring the SRA-induced mC-flipping and its dynamics. Fluorescence steady-state spectroscopy and stopped flow measurements showed significant differences in the recognition and binding kinetics of SRA for HM and non-methylated (NM) DNA. Indeed, SRA was found to bind and slide with fast kinetics on NM duplexes, in line with the reader role of UHRF1. In contrast, the kinetics of mC flipping was found to be much slower, substantially increasing the lifetime of UHRF1 bound to a CpG site in HM duplexes and thus, the probability of recruiting DNMT1 in order to faithfully duplicate the DNA methylation profile. Therefore, we proposed for the first time an assay able to sensitively monitor the UHRF1-induced base flipping, which helped us to provide a possible mechanism for the UHRF1 directing function on DNMT1.

L'hormone thyroïdienne (T3) contrôle à la fois les processus développementaux et physiologiques. Elle agit via les récepteurs de l'hormone thyroïdienne (TR), membres de la famille des récepteurs hormonaux nucléaires. Ils agissent comme des facteurs de transcription dépendants du ligand. La méthylation de l'ADN en position 5 de la cytosine est une modification épigénétique importante qui affecte la structure de la chromatine et l'expression des gènes. Des études récentes ont établi un rôle important des protéines de la famille TET (Ten-eleven translocation) dans la régulation de la dynamique de la méthylation de l'ADN. Elles convertissent la 5-méthyl-cytosine (5mC) en 5-hydroxyméthylcytosine (5hmC). D’autres études ont démontré que les protéines TET (TET1, TET2 et TET3) possèdent des fonctions de régulation transcriptionnelle dépendantes et indépendantes de leur activité catalytique. Notre étude a identifié TET3 comme une nouvelle protéine interagissant avec TR. Le domaine AF2 de TR ainsi que le domaine catalytique et le domaine CXXC de TET3 sont responsables de cette interaction. Celle-ci permet la stabilisation de TR lié à la chromatine, entraînant une potentialisation de son activité transcriptionnelle. L'effet de modulation de TET3 sur TR présenté ici est indépendant de son activité hydroxylase de TET3. Ainsi, cette étude met en évidence un nouveau mode d'action de TET3 en tant que régulateur non classique de TR, modulant sa stabilité et son accès à la chromatine plutôt que son activité de transcription intrinsèque. Des mutations du gène codant pour TRα provoquent le symptôme RTHα dont la gravité varie en fonction de la mutation. Les différentes capacités d’interaction des mutants TRα, pertinents pour la maladie de RTHα humaine, avec TET3 pourraient expliquer les différences d’effet dominant négatif. La fonction de régulation de TET3 pourrait s’appliquer plus généralement aux facteurs de transcription des récepteurs nucléaires, car différents membres de la superfamille des récepteurs nucléaires présentent la même interaction avec TET3, tels que AR (récepteur des androgènes), ERR (récepteur des œstrogènes) et RAR (récepteur de l'acide rétinoïque). L'interaction entre TET3 et RAR implique le domaine de liaison ADN de RAR. La pertinence fonctionnelle de l'interaction TET3 / RAR a été étudiée plus en détail dans les cellules souches embryonnaire (cellules ES). L’absence combinée des trois TET a entraîné la diminution de 5hmC et la dérégulation des gènes impliqués dans la différenciation des cellules ES. Parmi les gènes dérégulés, nous avons identifié un sous-ensemble de gènes cibles de l’acide rétinoïque, suggérant que les RAR (récepteurs d'acide rétinoïque) et les TET pourraient travailler ensemble pour réguler la différenciation des cellules ES. Une étude supplémentaire a révélé que les protéines TET peuvent jouer un rôle dans la facilitation du recrutement de RAR aux régions promotrices de ses gènes cibles. En outre, nos résultats montrent un rôle potentiel de l'activité hydroxylase des protéines TET dans la modulation de l'activité transcriptionnelle des RAR. En conclusion, notre travail a identifié les protéines TET comme nouveaux régulateurs des récepteurs nucléaires. Les mécanismes exacts impliqués doivent être étudiés plus avant. / Thyroid hormone (T3) controls both developmental and physiological processes. Its nuclear receptors, thyroid hormone receptors (TRs), are members of the nuclear hormone receptor family which act as ligand-dependent transcription factors. DNA methylation at the fifth position of cytosine is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (TET) family proteins in regulating DNA methylation dynamics by converting 5-methyl-cytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Studies demonstrated that TETs proteins (including TET1, TET2 and TET3) possess catalytic activity dependent and independent transcriptional regulatory functions. Our study identified TET3 as a new TR interacting protein. The AF2 domain of TR and the catalytic domain and CXXC domain of TET3 are responsible for their interaction. This interaction allows the stabilization of chromatin bound TR, resulting in a potentiation of its transcriptional activity. The modulation effect of TET3 on TR presented here is independent of its hydroxylase activity. Thus this study evidences a new mode of action for TET3 as a non-classical regulator of TR, modulating its stability and access to chromatin rather that its intrinsic transcriptional activity. Mutations in TR cause the RTH symptom which severity varies with the particular mutation. The differential ability of different TRα mutants, relevant for the human RTHα disease, to interact with TET3 might explain their differential dominant negative activity. The regulatory function of TET3 might be more general towards the nuclear receptor transcriptional factors since different members of the superfamily present the same interaction with TET3, such as AR (androgen receptor), ERR (Estrogen-related receptor) and RAR (retinoic acid receptor). The interaction between TET3 and RAR involves the DNA binding domain of RAR. The functional relevance of TET3/RAR interaction was further studied in ES cells. Combined deficiency of all three TETs led to depletion of 5hmC and deregulation of genes involved in ES differentiation. Among the deregulated genes, a subset of RA response genes was identified, suggesting that RARs (retinoic acid receptors) and TETs might work together to regulate ES cell differentiation. Further dissection revealed that TET proteins may have a role in facilitating RAR recruitment to the promoter regions of these RAR target genes. Moreover, our results indicated a potential role of the hydroxylase activity of TET proteins in modulating RAR transcriptional activity. Altogether, our work identified TET proteins as new regulators of NR (Nuclear Receptors). The exact mechanisms involved need to be further studied.