The enzyme carbonic anhydrase (CA) contributes to multiple physiological processes by catalysing the reversible hydration of carbon dioxide. However, regulation of CA activity in response to homeostatic challenges remains poorly understood. The objectives of this thesis were to investigate whether CA is transcriptionally regulated by cortisol in fish and whether post-translational modification (PTM) of CA occurs in fish. The results of an in vivo reporter assay used to investigate potential transcriptional regulation of zebrafish, Danio rerio, cytoplasmic CA (CAc) were inconsistent, and it remains unclear whether zebrafish CAc is regulated transcriptionally by cortisol. Phosphorylation of rainbow trout, Oncorhynchus mykiss, CAc was predicted from in silico analysis of the putative amino acid sequence and confirmed by Western analysis of phosphoprotein levels following in vitro incubation of CA, purified from trout gill, under conditions designed to potentiate endogenous kinases. Again using in vitro incubations designed to potentiate endogenous kinases and phosphatases, changes to the phosphorylation state of CAc were found to modulate its enzymatic properties. These findings suggest that CA activity may be regulated by signalling pathways that activate cellular protein kinases, and future work should focus on identifying these pathways.

La génération de la diversité morphologique n’implique pas seulement l’émergence de nouveaux caractères morphologiques, mais aussi la modification de caractères pre-existants. Des changements dans l'expression génique sous-tendent l’apparition et la divergence de morphologies au cours de l'évolution. Les variations de l'expression d'un gène peuvent résulter des modifications dans la séquence nucléotidique de ses éléments cis-régulateurs (ECRs) et/ou dans la séquence codante des facteurs de transcription (FTs) qui le régulent. En conséquence, la compréhension de l'évolution d'un nouveau trait exige l'élucidation des modifications en cis et en trans expliquant les différences d'expression des gènes. Les études sur la pigmentation des drosophiles ont considérablement contribué à notre compréhension du rôle joué par la modification des ECRs dans la diversification morphologique. Pour comprendre les mécanismes génétiques et moléculaires qui sous-tendent l’émergence et la diversification des traits morphologiques, nous avons décidé d’étudier l'évolution des motifs de pigmentation de l’aile chez les mouches du groupe d'espèces Drosophila melanogaster. Nous constatons que la diversification des modes de pigmentation de l’aile dans ce groupe est en corrélation avec la divergence d'expression d'un gène de pigmentation, yellow. Nous avons choisi de disséquer la régulation transcriptionnelle de yellow dans l’une des espèces tachetées, Drosophila biarmipes, avec pour objectif d'élucider les mécanismes responsables de la diversification de l'expression de yellow.Drosophila biarmipes, une espèce proche de Drosophila melanogaster, a développé une nouvelle tache de pigmentation sur aile qui est absente chez D. melanogaster. L'évolution de ce caractère est corrélée au changement d’expression de yellow. Une précédente étude a identifié, dans la région 5' du promoteur de yellow de D. biarmipes, un ECR qui suffit à reproduire une partie de l'expression de yellow lorsqu’il est transformé chez D. melanogaster. Avec un crible RNAi, j’ai identifié cinq FTs qui peuvent réguler l’ECR de yellow. Un de ces candidats, Distal-less, est exprimé d’une façon qui correspond à celle de l'expression de yellow et au profil de la pigmentation de l’aile chez D. biarmipes. Une analyse comparative de Distal-less et yellow a mis en évidence que les profils d'expression de ces gènes sont également corrélés avec les différents patrons de pigmentation dans les ailes d’autres espèces de drosophiles. J’ai aussi pu montrer que la surexpression de Distal-less dans l’aile de D. biarmipes est suffisante pour induire de la pigmentation ectopique. Ceci suggère que Distal-less ne régule pas seulement yellow, mais l’ensemble des gènes de pigmentation nécessaires à l’apparition de tache chez D. biarmipes. Mon travail montre que l'évolution de nouveaux motifs de pigmentation a commencé par le recrutement de Distal-less sur une batterie de gènes pigmentation. A partir de ces constatations, nous proposons un modèle expliquant les mécanismes de régulation conduisant à l'émergence et la diversification des motifs de pigmentation de l'aile au sein du groupe d'espèces melanogaster. Nous suggérons que dans un ancêtre commun de ce groupe, Distal-less a été recruté par une batterie des gènes de pigmentation, qui conduisent à l'émergence d'un motif de pigmentation. Par ailleurs, nous faisons l’hypothèse qu’une fois un lien régulatoire a été formé entre Distal-less et les gènes de pigmentation, alors la divergence d'expression de Distal-less pourrait entraîner des changements dans la régulation spatiale de tous les gènes de pigmentation donnant lieu à la diversification des motifs de pigmentation. / The generation of morphological diversity involves not only the emergence of novel morphological traits but also the modification of pre-existing ones. To understand the genetic and molecular mechanisms underlying the gain and diversification of morphological traits, we chose to study the evolution of wing pigmentation patterns in a group of flies in the melanogaster species group. We find that the diversification of wing pigmentation patterns in this group is mirrored by the divergence of expression of a pigmentation gene, yellow. To decipher the regulatory mechanisms underlying the diversification of yellow expression, we chose to dissect the transcriptional regulation of yellow in one spotted species, D. biarmipes. A functional dissection of the yellow spot enhancer, situated at the 5’ of the D. biarmipes yellow promoter, shows that a homeobox transcription factor, Distal-less (Dll), is a direct activator of yellow. Moreover, we show that ectopic expression of Dll is sufficient to induce ectopic pigmentation in D. biarmipes, which suggests that Dll is regulating yellow and other pigmentation genes. Furthermore, we find that the divergence of Yellow expression pattern in the melanogaster species group is correlated with the divergence in expression of Dll. Based on our findings we propose a model to explain the regulatory mechanisms that lead to the emergence and diversification of wing pigmentation patterns within the melanogaster species group. We suggest that in a common ancestor of all spotted species within the Oriental lineage, there was a gain of regulatory links between Dll and terminal pigmentation genes, such as yellow, which lead to the emergence of an ancestral pigmentation pattern. We further propose that once a regulatory link was formed between Dll and terminal pigmentation genes, divergence of Dll expression could lead to changes in the spatial regulation of all downstream target genes, which would give rise to the diversification of pigmentation patterns. Our study illustrates how key regulators of terminal genes could be targets for mutations that are more likely to lead to morphological diversification.

Background: Oesophagogastric cancers generally present late in life with advanced disease and carry a poor prognosis. Few patients receive curative treatment. Polo-like Kinase 1 (PLK1) is a mitotic kinase with regulatory functions at the G2/M cell cycle phase transition. In mammalian cells, PLK1 phosphorylates and activates FOXM1, a forkhead transcription factor at the G2/M cell cycle phase transition. FOXM1 then promotes transcription of multiple gene products, including PLK1 and CCNB1, which then act individually or in complexes to further phosphorylate FOXM1 generating a positive feedback loop driving the cell into M phase. Aims: We aimed to assess the expression of PLK1 and FOXM1 in oesophageal and gastric cancer patients. Secondly we aimed to investigate the expression and inter- relationship of PLK1 and FOXM1 in oesophageal cell lines during the cell cycle. Results: FOXM1 and PLK1 are expressed in oesophageal cell lines and demonstrate cross-regulatory interactions. Inhibition of PLK1 leads to the decreased expression of FOXM1 and it’s target gene in oesophageal cell lines. FOXM1 and PLK1 are also concomitantly overexpressed in a large proportion of oesophageal and gastric carcinoma’s at both the protein and mRNA level. Other FOXM1 target genes including, CCBN1, AURKB and CKS1 are co-expressed in a similar manner. In a homogenous cohort of patients who underwent surgery, the expression of PLK1 and AURKB was prognostic for overall survival. Conclusions: This study has demonstrated that FOXM1 and a number of target genes including PLK1 are coordinately expressed in a proportion of oesophageal and gastric carcinomas. This suggests that chemotherapeutic treatments that target this pathway may be of clinical utility.

Although pathological alterations in gene expression and mitochondria function in response to cardiac ischemia are well recognized, the mechanisms driving these changes are incompletely understood. Nuclear to mitochondrial communication regulating gene expression and mitochondrial function is a critical process following cardiac ischemic injury. Here we determine that cyclin C, a component of the transcriptional regulator, Mediator complex, directly regulates cardiac and mitochondrial function by modifying mitochondrial fission. We tested the hypothesis that cyclin C has a binary function as a transcriptional cofactor in the nucleus and acute regulation of cardiac energetics in ischemia by enhancing mitochondrial fission in the cytoplasm. In response to stress, cyclin C translocates to the cytoplasm enhancing mitochondria fission in part through interactions with Cdk1. Using cardiac specific cyclin C knockout and overexpression mouse models, we determined cyclin C regulates mitochondria morphology under basal and ischemic conditions in vivo. Furthermore, pretreatment with a Cdk1 inhibitor followed by ischemia in vivo results in reduced mitochondrial fission. Together, our study reveals that cyclin C regulates both hypertrophic gene expression and mitochondrial fission providing new insights into the regulation of cardiac energy metabolism following acute ischemic injury.

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Deformed epidermal autoregulatory factor 1 (DEAF1) encodes a transcription factor essential in early embryonic and neuronal development. In humans, mutations in the DNA binding domain of DEAF1 cause intellectual disability together with clinical characteristics collectively termed DEAF1-associated neurodevelopmental disorders (DAND). The objective of this study is to 1) assess the pathogenicity of newly identified variants using established functional assays, and 2) confirm and map the interaction domain of DEAF1 with HDAC1 and evaluate the importance of DEAF1-HDAC1 interaction on DEAF1-mediated transcriptional repression. Exome sequencing analysis identified six de novo DEAF1 mutations (p.D200Y, p.S201R, p.K250E, p.D251N, p.K253E, and p.F297S). Promoter activity experiments indicate DEAF1 transcriptional repression activity was altered by p.K250E, p.K253E, and p.F297S. Transcriptional activation activity was altered by p.K250E, p.K253E, p.F297S, and p.D251N. Combined with clinical phenotype of the patients, this work establishes the pathogenicity of new DEAF1 variants. Previous studies identified a potential protein interaction between DEAF1 and several proteins of the nucleosome remodeling and deacetylating (NuRD) complex including Histone Deacetylase 1 (HDAC1), Retinoblastoma Binding Protein 4 (RBBP4), Methyl CpG Binding Domain Protein 3 (MBD3). GST pull-down and co-immunoprecipitation (CoIP) assays confirmed and mapped the interaction with HDAC1 between amino acids 113 – 176 of DEAF1. To determine whether DEAF1-mediated repression requires HDAC1 activity, HEK293t wild type or CRISPR/Cas9-mediated DEAF1 knockout cells were treated with the HDAC inhibitor Trichostatin A (TSA). Interestingly, this study demonstrates that the requirement of HDAC1 activity on DEAF1-mediated transcriptional repression activity is target gene specific and expands our understanding of DEAF1 mediated transcriptional repression.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23609号 / 医科博第132号 / 新制||医科||9(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 濵﨑 洋子, 教授 森信 暁雄, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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