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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23803号 / 医博第4849号 / 新制||医||1058(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 村川 泰裕, 教授 滝田 順子, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chronic Obstructive Pulmonary Disease (COPD), which is characterized by limitation of pulmonary air flow, is now the third major cause of death worldwide. Barrero et al. have reported that the elevation of extracellular hyperacetylated histone H3.3 in the lungs of COPD patients is associated with cytotoxicity and disease progression. They found that extracellular hyperacetylated H3.3 was cytotoxic to lung structural cells and resistant to proteasomal degradation, and that mouse antibodies to either the C- or N- termini of H3.3 could partially reverse H3.3 toxicity in vitro. Thus, we hypothesize that human antibodies directed against H3.3 may be effective biotherapeutics useful to control progression of COPD in vivo. The discovery and development of human monoclonal antibodies (mAbs) is a fast growing field of biotherapeutics. In addition to full length mAbs, antibody fragments also have been used in antibody discovery research. We have used phage display technology in this project to discover human anti-H3.3 antibody Fab fragments. This technology utilizes genetically engineered phage particles containing genes encoding diverse Fab fragments displayed on the particles. The “Ylanthia” library from MorphoSys AG, a synthetic fully human Fab antibody phage display library with 1.3 x 1011 independent clones, was panned against purified recombinant human H3.3 immobilized on 96-well plates. Seven H3.3-binding Fab fragments with unique DNA sequences were isolated after four rounds of panning. Following their expression in E.coli and purification, Fab purities and electrophoretic mobilities were evaluated on SDS-PAGE. The concentration-dependent binding activities of all seven Fabs to human H3.3 were tested by ELISA. All seven Fabs were shown by ELISA to bind H3.3 but not histones 2A, 2B or 4. Since H3.3 is localized to the nucleus, western blotting was used to demonstrate that seven Fabs recognize purified, recombinant H3.3 and denatured natural histone(s) from nuclear extracts of human 293T cells. In order to characterize these molecules further, biological activity assays will be done to test their potential to reverse the toxic effects of H3.3 in cell culture. If these Fabs prove active in cell culture, they will be converted to IgGs and tested in animal models as potential biotherapeutics for COPD. / Pharmaceutical Sciences

Histone deacetylases (HDACs) are an ancient class of enzymes that have been conserved throughout evolution and are found in diverse organisms such as animals, plants, fungi, eubacteria and archaebacteria. In C. elegans, twelve HDACs have been identified so far. These HDACs have been grouped into four different classes (Class I, II, III and IV) based on their cofactor requirements and sequence homologies. hda-1 is one of the three Class I HDACs in C. elegans and plays a role in the morphogenesis of several organs including the vulva. This thesis focuses on the role of hda-1 in vulval morphogenesis. The hermaphrodite vulva has twenty-two cells which can be further divided into seven different cell types: VulAs, VulBls, VulB2s, VulCs and VulDs (secondary great granddaughters), YulEs and VulFs (primary great granddaughters). The analysis of expression pattern of hda-1 revealed that hda-1 is expressed in the progeny of both the primary and secondary vulval precursor cells (VPCs). To examine hda-1 mutant phenotype in detail, I examined the expression pattern of five different vulval cell-type specific markers (cdh-3, zmp-1, ceh-2, egl-17 and daf-6) in hda-1 animals. The results revealed that hda-1 is necessary for proper differentiation of multiple vulval cell types. To study the evolutionary conservation of hda-1 function, I examined the role of hda-1 ortholog in C. briggsae. C. briggsae is a close relative of C. elegans and has almost identical vulval morphology. Knocking down Cbr-hda-1 in C. briggsae animals resulted in defective vulval phenotype. Consistent with this result, the expression of two cell- fate specific markers (C. briggsae orthologs of zmp-1 and egl-17) was found to be altered in Cbr-hda-1 RNAi treated animals. Thus, hda-1 function in the vulva appears to be conserved in these two species. To identify the hda-1 targets in vulval morphogenesis in C. elegans, microarray approach was taken. Two genes fos-1 and lin-29 were identified as putative targets and were examined in some detail. Among the targets identified (these still need to be validated), I focused on fos-1 and lin-29 for detailed investigation. The RNAi-mediated knockdown of hda-1 caused alterations in the expression pattern ofthefos-1 transcript,fos-1b. To examine interaction between fos-1 and lin-29, I used double RNAi approach and examinedfos-1 (RNAi), lin- 29 (RNAi), hda-1 (cw2) animals. It was found that fewer animals exhibit defects in vulval morphology in these animals as compared to fos-1 (RNAi), hda-1 (cw2) animals. While this suggests a possible interaction between lin-29 and hda-1 in the vulva, these results need to be validated by doing additional experiments. In summary, the work described in this thesis demonstrates that hda-1 plays an important role in vulval morphogenesis and regulates the expression of several important genes. Also, the function of hda-1 in C. elegans and C. briggsae is evolutionarily conserved. / Thesis / Master of Science (MSc)

The first part of this work describes the synthesis of a new histone deacetylase (HDAC) inhibitor (HDI). HDAC enzymes modify core histones, influence nucleosome structure and change gene transcription by removing the acetyl groups from lysine residues on proteins. HDIs are showing exciting potential as a new class of drugs for cancer and a variety of other diseases. A new HDAC inhibitor based on the hydroxamic acid motif has been synthesized. Two characteristic structural features were incorporated into the design of the novel inhibitor. A cyclic peptide mimetic of known structure was fused to a hydroxamic acid moiety through an aliphatic chain. The HDAC inhibitor provided significant inhibitory activity against HDACs with an IC50 value of 46 ± 15 nM, and against HDAC8 with an IC50 value of 208 ± 20 nM. The potent HDAC inhibitory activity of the HDAC inhibitor demonstrates the importance of the rim recognition region in the design of HDIs. The hydrophobic cyclic turn mimic allows the formation of a tight complex between HDI and HDAC enzymes. The second part of this work is to synthesize secondary structure mimics and incorporate them into the helix-turn-helix (HTH) motif. One of the important methods to study the conformation of the biologically active peptides is to incorporate the rigid peptidomimetics into the relevant peptides. Important information can be obtained from the study of conformationally constrained peptides. HTH proteins are well characterized and found in many organisms from prokaryotes to eukaryotes. The relatively small size, simple structure, and significance in stabilizing tertiary structures make the HTH peptide an attractive target to mimic. Both a Gly HTH turn mimic and a Ser HTH turn mimic were synthesized using stereoselective hydrogenation and macrocyclization starting from unnatural amino acids in a yield of 33% and 14%, respectively. The synthesis of Fmoc protected HTH turn mimics allowed incorporation into HTH peptides using Fmoc chemistry on solid phase. The incorporation of the HTH turn mimics into the peptides proved to be challenging, either by sequential elongation or by segment condensation. Alternative peptide synthesis strategies were employed in attempts to solve the problems. / Ph. D.

The developing brain is highly sensitive to environmental influences. Unfavorable nutrition is one kind of stress that can cause acute metabolic disorders during the neonatal period [1,2,3] and severe diseases in later life [4,5]. These early life experiences occurring during heightened periods of brain plasticity help determine the lifelong structural and functional aspects of brain and behavior. In humans, for example, weight gain during the first week of life increased the propensity for developing obesity several decades later [5]. This susceptibility is, if not all, related to the dynamic reversible epigenetic imprints left on the histones [6,7,8], especially during the prenatal and postpartum period [9]. Histones are highly dynamic and responsive towards environmental stress [10,11]. Through covalent modification of the histone tail, histones are able to direct DNA scaffolding and regulate gene expression [10,12]. Thus far, various types of post translational modifications have been identified on various histones tails [12]. Among them, the methylation and acetylation on lysine residue (K) 27 on histone 3 (H3) has been tightly linked to gene repression [13,14] and activation [15], respectively. EZh2 (enhancer of zeste 2) in the polycomb repressive complex 2 (PRC2) is the only methyltransferase that has been linked to catalyze this methylation reaction. In addition, SUZ (suppressor of zeste) and EED (embryonic ectoderm development) are two other key proteins in PRC2 function core that help EZH2. As previous reported, increased H3K27 methylation was monitored after fasting stress during neonatal period in chicks' paraventricular nucleus (PVN). In this study, we investigated the detailed mechanism behind changes in H3K27 methylation following fasting stress. After 24 hours fasting on 3 days-of-age (D3), chicks exhibited elevated mRNA levels of PRC2 key components, including EZH2, SUZ and EED, in the PVN on D4. Western blots confirmed this finding by showing increased global methylation status at the H3K27 site in the PVN on D4. In addition, until 38 days post fasting, SUZ and EZH2 remained inhibited. A newly identified anorexigenic factor, Brain-derived neurotrophic factor (BDNF), was used as an example of multiple hormones expressed in PVN to verify this finding. Both BDNF protein and mRNA exhibited compatible changes to global changes of tri- (me3) and di-methylated (me2) H327. Furthermore, by using chromatin immunoprecipitation assays (ChIP), we were able to monitor the changes of H3K27me2/me3 deposition along the Bdnf gene. Fasting significantly increased H3K27me2/me3 as well as EZH2 at the Bdnf's promoter, transcription start site and 3'-untranslated region. These data show that fasting stress during the early life period could leave epigenetic imprinting in PVN for a long time. Next, we tried to understand the function of this epigenetic imprinting in the chicks' PVN. Thus, we compared naive chicks (never fasted) to chicks that received either a single 24 hour fast on D3 or two 24 hour fast on both D3 and 10 days-of-age (D10). We found that the D3 fasted group significantly increased the level of PRC2 key components and its product H3K27me2/me3 compared to the naive group. However, D3 fasting and D10 fasting together decreased the surges of H3K27me2/me3, SUZ and EED (not EZH2) compared to the naive group. We called this phenomenon "epigenetic memory". The Western blot, qPCR and CHIP assay results from BDNF all confirmed the existence of "epigenetic memory" for PRC2. These data suggested that fasting stress during the early period of brain development could leave long term epigenetic modifications in neurons. These changes could be beneficial to the body, which keeps homeostasis of inner environment and prevent massive response to future same stress. The EZH2 protein was knocked down and the H3K27 methylation status changes were monitored after applying the same treatment. We first confirmed that EZH2 antisense oligonucleotides (5.5 ug), but not EZH2 siRNA and artificial cerebrospinal fluid (ACSF), inhibit EZH2 protein by 86 % in the PVN. Then, on D3, chicks were subjected to a 24 hour fasting stress (D3-fasting) post either EZH2 antisense or ACSF injection. The EZH2 antisense blocked the surge of both EZH2 mRNA and H3K27 methylation after D3-fasting. At the same time, BDNF exhibited elevated expression levels and less methylated H3K27 deposition along the Bdnf gene. In addition, we were also interested in the changes of "epigenetic memory" post EZH2 antisense injection. We found that after EZH2 antisense injection, chicks' PVN no longer exhibited any "epigenetic memory" to repetitive fasting stress. While EZH2 mRNA was constantly inhibited, SUZ, EED and H3K27me2/3 levels were unpredictable. These findings suggested that neurons in the PVN utilized PRC2 as a major H3K27 methylation tool. Knockdown of EZH2 in the PRC2 impaired the proper response in PVN to fasting stress and PVN's ability to acclimate to repetitive fasting stresses. Thus, EZH2 is an important H3K27 methyltransferase inside chicken hypothalamus to maintain homeostasis. In conclusion, fasting stress during the early life period could leave epigenetic markers on chromosomes of neurons in the feeding regulation center. These epigenetic markers will be left on chromosomes for a long period of time and have a beneficial role in keeping homeostasis when individuals face future fasting stress again. H3K27 methylation is one of these epigenetic markers and inhibits expression of various genes inside neurons. EZH2 is so far the only detected methyltransferases for H3K27 that form the PRC2. Thus EZH2 plays a key function in the body's response to fasting. / Ph. D.

Le complexe Histone Acétyltransféranse (HAT) NuA4 s'inscrit comme un élément clef dans le contrôle de plusieurs fonctions cellulaires essentielles chez les eucaryotes. L'implication maintenant connu de NuA4 dans la transcription et dans la réponse aux dommages à l'ADN nous ont poussé à approfondir la caractérisation fonctionnelle des diverses sous-unités de NuA4, notamment au niveau des rôles que peuvent occuper les différents domaines protéiques retrouvés au sein de ce complexe. Une première série d'analyses a démontré l'importance de plusieurs résidus du chromodomaine de Esa1, la sous-unité catalytique de NuA4. La mutation de ces résidus engendre des défauts majeurs d'acétylation de la chromatine, suggérant ainsi un rôle du chromodomaine dans l'activité catalytique de Esa1. Parallèlement, d'autres études ont permis d'approfondir la fonction du domaine SANT de la protéine Eaf2, du PHD finger de Yng2 et du domaine PI-3 kinase de Tra1. Ce dernier domaine intéragit avec le complexe MRX, un complexe de levure recruté directement au site de cassure de l'ADN. Des recherches menées autour de l'étude de l'activité kinase de cette protéine ont permis de suggérer l'implication de NuA4 dans les événements précoces survenant suite à un bris double brin, précisant ainsi le rôle de ce complexe dans la réparation de l'ADN.

Les facteurs de transcription induits par l’hypoxie (HIF) sont responsables de la transcription de nombreux gènes impliqués dans la réponse à l’hypoxie. En plus de réguler de nombreux processus cellulaires et physiologiques, ces facteurs sont impliqués dans plusieurs pathologies. Hétérodimères constitués d’une sous-unité β constitutive et d’une sous-unité α sensible à l’oxygène, ces facteurs sont majoritairement régulés par l’hydroxylation et la dégradation de la sous-unité α. En situation d’hypoxie, ce mécanisme de dégradation est inhibé, ce qui favorise la formation de complexes HIF. Les travaux présentés dans cette thèse visent à élucider les mécanismes régulant l’activation de HIF en situation d’hypoxie ou de normoxie. Dans la section Résultats, vous retrouverez une section consacrée à l’activation de HIF par l’angiotensine II (AngII) chez les cellules musculaires lisses vasculaires. Plus précisément, le rôle de la transactivation de récepteurs à activité tyrosine kinase suivi de l’implication de HIF dans la biologie de ces cellules seront abordés. Dans un deuxième temps, un inhibiteur des métalloprotéases, le BiPS, vous sera présenté comme étant un puissant inducteur des protéines HIF-α. En effet, le BiPS est un puissant inhibiteur des enzymes responsables de la dégradation des protéines HIF-α. En outre, le BiPS permet l’activation des complexes HIF ainsi formés. Ces résultats inattendus pourraient avoir des répercussions importantes dans l’utilisation de cet agent à des fins angiostatiques dans le traitement du cancer en plus de présenter un nouvel agent ayant un potentiel thérapeutique important dans le traitement de pathologies ischémiques. Finalement, vous retrouverez une section consacrée à l’étude d’un nouveau répresseur de HIF, l’histone acétyltransférase HBO1. De façon étonnante, HBO1 réprime l’activité des complexes HIF par un mécanisme indépendant de la stabilisation des sous-unités α mais dépendant du remodelage de la chromatine. En conclusion, ces résultats mettent en lumière de nouveaux mécanismes de régulation de l’activité des facteurs de transcription HIF. Considérant les rôles physiologiques importants de ces complexes ainsi que leurs implications dans diverses maladies, ces résultats permettront d’accroître les connaissances disponibles quant aux fonctions de ces complexes et mèneront vers le développement d’outils thérapeutiques efficaces. / Hypoxia-inducible transcription factors (HIF) are decisive elements in the transcriptional regulation of numerous genes expressed in conditions of hypoxic stress. In addition to their roles in many physiological and cellular processes, HIF are also involved in diverse pathological situations. Obligate heterodimers composed of a constitutive β subunit and of an oxygen tension-regulated α subunit, these transcription factors are mainly regulated by the hydroxylation and subsequent degradation of the α subunit. In hypoxia, this degradation mechanism is inhibited, resulting in HIF complex formation and binding to specific DNA sequences. The work presented in this thesis aims to elucidate regulatory mechanisms involved in HIF activation during hypoxia or in normal oxygen conditions. In the Results section, you will find a study devoted to HIF activation by angiotensin II (Ang II) in vascular smooth muscle cells. Specifically, the role of receptor tyrosine kinase transactivation on HIF activation was evaluated along with a description of HIF-1’s role in smooth muscle cells biology. Next, an inhibitor of matrix metalloproteases, BiPS, will be presented as a novel and potent HIF activator. This unexpected effect may have important implications for the use of this compound for its angiostatic potential in cancer treatment. In addition, BiPS and derivative molecules could also have strong therapeutic potential in ischemic diseases. Finally, you will find a section devoted to the study of a new transcriptional repressor of HIF complexes, the histone acetyltransferase bound to ORC-1, HBO1. Surprisingly, HBO1 represses the activity of HIF complexes by a mechanism independent of the availability of the α subunits, but dependent on a chromatin remodelling event. In conclusion, this thesis highlights new regulatory mechanisms responsible for HIF activation. Considering the important physiological roles of HIF complexes and their implications in the pathogenesis of different diseases, these studies increase the available knowledge concerning the biological functions of these complexes and could contribute to the development of more effective and safe therapeutic tools.

La protéine Haspine est une sérine / thréonine kinase mitotique conservée, connue pour fonctionner par la phosphorylation de l'histone H3 (H3pT3). Bien que H3T3 soit le seul substrat bien connu de Haspine, il se peut que H3pT3 ne suffise pas à expliquer toutes les fonctions de Haspine au cours de la mitose. Fait intéressant, l’homologie de la portion N-terminale de H3 avec la portion Cterminale de H2B suggère que la thréonine 119 de H2B (H2BT119) est un candidat potentiel fort pour être un substrat majeur de Haspine. Ainsi, l’objectif de ce projet était d’étudier la phosphorylation de H2BT119 dépendante de Haspine pendant la mitose. La phosphorylation de H2B recombinant sur la position T119 par Haspine a été confirmée par un dosage de kinase en utilisant la radioactivité. En outre, le signal H2BpT119 sur la protéine recombinante H2B a été détecté par un anticorps anti-H2BpT119, confirmant la phosphorylation de H2B dépendante de Haspine sur ce site dans le test de kinase in vitro. En outre, une augmentation de la taille moléculaire de H2B a été observée après le dosage de la kinase. Les résultats des expériences in vivo, incluant l'analyse par Western-blot d'extrait d'histones mitotiques et la microscopie à fluorescence avec l'anti-H2BpT119, suggèrent une phosphorylation de H2B au site T119, qui s'est également avérée dépendant de l'activité de Haspine. Cependant, en raison de la potentielle réactivité croisée de l’anti-H2BpT119 avec H3pT3, une forme marquée de H2B a été utilisée lors de l’étude du signal H2BpT119 au cours de la mitose. Le marquage augmente la taille moléculaire de H2B et aide à reconnaître H2BpT119 loin de H3pT3. Plusieurs systèmes de marquage ont été utilisés, mais toutes les tentatives ont échoué, en raison du faible niveau de H2B exogène. Cependant, les résultats de ce projet suggèrent que le signal H2BpT119 pendant la mitose pourrait révéler un nouveau mécanisme dépendant de Haspine pour la régulation de la ségrégation des chromosomes. Par conséquent, il reste important d'étudier cette marque d'histone au cours de la mitose. / Haspin is a conserved mitotic serine/threonine kinase that is known to function through histone H3 phosphorylation (H3pT3). Despite this, H3T3 is the only well-known substrate for Haspin, H3pT3 may not be enough to explain all Haspin functions during mitosis. Interestingly, homology of H3 N-terminus with H2B C-terminus suggests that H2BT119 is a strong potential candidate to be a major substrate of Haspin. Thus, the aim was to investigate Haspin-dependent phosphorylation of H2BT119 during mitosis. Phosphorylation of recombinant H2B at T119 by Haspin was confirmed by a radioactivity-based kinase assay. Also, H2BpT119 signal on recombinant H2B was detected by an anti-H2BpT119 antibody, confirming Haspin-dependent phosphorylation of H2B at this site in the in vitro kinase assay. Also, an upshift of H2B was observed following the kinase assay. Results from in vivo experiments, including Western blot analysis of mitotic histone extract and immunofluorescence microscopy with the anti-H2BpT119, support phosphorylation of T119 in H2B, which also was found to depend on Haspin activity. However, due to the potential anti-H2BpT119 cross-reactivity with H3pT3, while exploring H2BpT119 signal during mitosis, tagged H2B was used. The tag increases H2B molecular size and helps to distinguish H2BpT119 from H3pT3. Several tagging systems was used, but all attempts failed, because of the low level of the exogenous tagged H2B. However, the results of this project suggest H2BpT119 signal during mitosis that may reveal a novel Haspindependent mechanism for chromosome segregation regulation. Therefore, it remains important to study this histone mark during mitosis.