付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24520号 / 医博第4962号 / 新制||医||1065(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 生田 宏一, 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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Inflammatory Bowel Disease (IBD) is a chronic disorder that affects millions of peopleworldwide. Although the etiology behind the disease is yet unknown, current theoriespropose a complex interplay between genetic susceptibility, exposure to environmentalfactors and exacerbated immune responses. While important efforts have been made to linkgenetics and environmental factors to IBD pathogenesis, a major challenge remains to assignthem a causative role. Particularly since most of the IBD-risk genetic polymorphisms arefound in non-coding regions (NCRs) with unknown regulatory activity, and for the lack ofknowledge about how environmental factors can modulate the function of these elements invivo . A main problem to address this challenge in IBD research is the lack of an appropriatemodel system in vivo that allows for high-throughput experiments with combinations ofdifferent IBD-risk factors, while keeping the in vivo context. In this work, we sought toovercome this issue by using a zebrafish reporter for a specific human IBD-risk NCR, inorder to investigate the modulation of this element by two groups of common environmentalfactors: pollutants, such as PolyFluoroAlkyl Substances (PFASs); and diet, by activation ofdietary sensors. We found that the activity of the WT-NCR in zebrafish larvae was increased in the presenceof PFAS, while the activation of the dietary sensor PPAR δ decreased the activity. These datalead us to suggest that the function of PFAS can be counteracted by PPARδ activation.Therefore, we propose zebrafish as a suitable in vivo model in which we can screen forpotentially harmful or beneficial effects of environmental factors in the activity of humannon-coding regions. / Inflammatorisk tarmsjukdom (IBD) är en kronisk störning som drabbar miljontals människorvärlden över. Även om etiologin bakom sjukdomen fortfarande är okänd, föreslår nuvarandeteorier ett komplext samspel mellan genetisk mottaglighet, exponering av miljöfaktorer ochförvärrat immunförsvar. Även om stora ansträngningar har gjorts för att koppla genetik ochmiljöfaktorer till IBD-patogenes, återstår en stor utmaning att tilldela dem en orsakande roll.Särskilt eftersom de flesta av IBD-riskgenetiska polymorfismer finns i icke-kodande regioner(NCR) med okänd reglerande aktivitet samt för bristen på kunskap om hur miljöfaktorer kanmodulera funktionen hos dessa element in vivo . Ett huvudproblem för att möta dennautmaning i IBD-forskning är avsaknaden av ett lämpligt modellsystem in vivo som möjliggörexperiment med hög kapacitet och kombinationer av olika IBD-riskfaktorer in vivo . I dettaarbete försökte vi få svar på denna fråga genom att använda en zebrafiskreporter för ettspecifikt humant IBD-risk icke-kodande område. Detta möjliggjorde att vi kunde undersökamodulering av två gemensamma miljöfaktorer: föroreningar, såsom PolyFluoroAlkyl-ämnen(PFASs); och diet, genom aktivering av dietsensorer. Vi fann att aktiviteten i WT-NCR hos zebrafisklarver ökade i närvaro av PFAS, medanaktiveringen av dietsensorn PPARδ minskade aktiviteten. Denna data leder till att vi antyderatt funktionen för PFAS kan motverkas genom PPARδ-aktivering. Därför föreslår vizebrafisk som en lämplig in vivo -modell, i vilken vi kan screena för potentiellt skadliga ellergynnsamma effekter av miljöfaktorer i mänskligt icke-kodande DNA.

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Die Beschreibung genregulatorischer Ereignisse ist entscheidend um Zelldifferenzierung und -entwicklung zu verstehen. Dynamische Vernderungen der Chromatinstruktur, Histonmodifikationen und das Binden von Transkriptionsfaktoren an Enhancer und Promotoren, koennen mit Hilfe von genomweiten Hochdurchsatz-Sequenziertechniken wie ChIP-Seq, DNase-Seq, ATACSeqund RNA-Seq untersucht werden. In dieser Arbeit entwickele ich mehrere probabilistische Modelle fuer die Analyse von genomweiten Sequenzierungsdaten. Diese umfassen 1. einen Peak-Finder fuer ChIP-/DNase-/ATAC-Seq-Daten, der sich Replikate zunutze macht und praezise Peak-Weiten berechnet, 2. eine Pipeline um das Genom in hoher Aufloesung in eindeutige Klassen von Kombinationen von Histonmodifikationen zu segmentieren, 3. ein Bayes-Netzwerk-Modell welches multiple zeitlich aufgelste Histonmodifikations-ChIP-seq-Daten kombinatorisch clustert Klassen von regulatorischen Elementen zu identifizieren. Mit Hilfe dieser Modelle untersuchen wir die Promotorumgeben und zeigen einen Zusammenhang zwischen Chromatinstruktur und Promotordirektionalitaet. Darueber hinaus verwenden wir ein Modell zur direkten Reprogrammierung von Stammzellen in Motorneuronen durch die gezielte Expression von Transkriptionsfaktoren und analysieren die dadurch induzierten zeitlichen Vernderungen der Chromatinstruktur und Transkriptionsfaktorbindedynamik. Wir beobachten, dass Promotoren verschiedenen Chromatin-Dynamiken zur Aktivierung und Repression folgen, die mit den Chromatin-Dynamiken von Enhancer-Elementen korrelieren. Enhancer hingegen werden durch kooperatives Verhalten direkt induzierter Transkriptionsfaktoren und anderen Faktoren, die in den Stammzellen zu Beginn vorhanden waren oder im Verlaufe der Differenzierung aktiviert wurden, kontrolliert. Diese Arbeit zeigt wie wichtig Chromatin-Dynamik und ihre Beziehung zur Logik von Transkriptionsfaktoren ist, um die Veraenderungen der Genexpression zu verstehen. / Delineating transcription regulatory events is crucial to understand cell differentiation and development. Dynamic changes of chromatin structure, histone modifications and transcription factor binding to enhancers and promotors can be investigated with the aid of genome-wide high-throughput sequencing technologies such as ChIP-Seq, DNase-Seq, ATAC Seq and RNA Seq. In this thesis, I develop several probabilistic models for the analysis of genome-wide sequencing data. These include: 1. a peak finder for ChIP-Seq, DNase-Seq and ATAC Seq data, which exploits biological replicates and accurately demarcates peak widths, 2. a pipeline for high-resolution genome segmentation into unique classes of combinations of histone modifications and 3. a Bayesian network model that can co-cluster multiple time-course histone modification ChIP-Seq data sets into distinct classes of regulatory elements. With the aid of these models we investigate the promoter chromatin environment and show a link between chromatin state and transcription initiation directionality. In addition, we use a system for direct reprogramming of stem cells in motor neurons by the targeted expression of transcription factors to analyse changes in chromatin state and transcription factor dynamics during differentiation. We observe that promoters follow different chromatin dynamics for activation and repression that correlate with the chromatin dynamics of enhancer elements. Enhancers are controlled by cooperative behavior of directly induced transcription factors and other factors present in the stem cells initially, or activated in the course of differentiation. Overall, this work demonstrates the importance of understanding chromatin dynamics and their relationship to transcription factors logic in order to better explain changes in gene expression.

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Two problems hampering efforts to produce salt-tolerant plants through constitutive expression of transgenes include: 1. Spatial control. Particular cell-types must respond specifically to salt stress to minimise the amount of Na⁺ delivered to the shoot; and, 2. Temporal control. Transgenes are typically expressed in plants at similar levels through time, irrespective of the stress encountered by the plant, which may exacerbate pleiotropic effects and means that, particularly in low-stress conditions, costly and/or detrimental metabolic processes may be active, thus reducing yield. To address these issues, Gateway® destination vector constructs were developed combining the GAL4 UAS (upstream activating sequence) with the ethanol-inducible gene expression system to drive inducible cell-specific expression of Na⁺ transporter transgenes (or to silence salt transporter transgenes inducibly and cell-specifically). Rice (Oryza sativa L. cv. Nipponbare) GAL4-GFP enhancer trap lines (Johnson et al., 2005: Plant J. 41, 779-789) that express GAL4 and GFP specifically in either the root epidermis or xylem parenchyma (and therefore ‘trap’ cell-type specific enhancer elements) were transformed with this GAL4 UAS – ethanol switch construct, thereby allowing both spatial and temporal control of transgenes. In preliminary experiments, the expression system successfully limited the expression of RFP to specific cell-types after induction with ethanol. Other genes expressed using this system include PpENA1, a Na⁺-extruding ATPase from the moss, Physcomitrella patens, and AtHKT1;1, a Na ⁺ transporter from Arabidopsis thaliana. The two enhancer trap rice lines were also transformed with the GAL4 UAS driving stable expression of AtHKT1;1 and PpENA1 specifically in root epidermal or xylem parenchyma cells. Expression of AtHKT1;1 in root epidermal cells reduced Na⁺ accumulation in the shoots, while expression in the root xylem parenchyma appeared to have little effect on shoot Na⁺ accumulation. Using cryo-scanning electron microscopy (SEM) X-ray microanalysis, the outer cells of the roots of the line expressing AtHKT1;1 in the epidermal cells were found to accumulate higher levels of Na⁺ than the parental enhancer trap line. Additionally, this line had decreased unidirectional ²²Na⁺ influx. Similar results were observed for plants expressing AtHKT1;1 driven by the CaMV 35S / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325289 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Two problems hampering efforts to produce salt-tolerant plants through constitutive expression of transgenes include: 1. Spatial control. Particular cell-types must respond specifically to salt stress to minimise the amount of Na⁺ delivered to the shoot; and, 2. Temporal control. Transgenes are typically expressed in plants at similar levels through time, irrespective of the stress encountered by the plant, which may exacerbate pleiotropic effects and means that, particularly in low-stress conditions, costly and/or detrimental metabolic processes may be active, thus reducing yield. To address these issues, Gateway® destination vector constructs were developed combining the GAL4 UAS (upstream activating sequence) with the ethanol-inducible gene expression system to drive inducible cell-specific expression of Na⁺ transporter transgenes (or to silence salt transporter transgenes inducibly and cell-specifically). Rice (Oryza sativa L. cv. Nipponbare) GAL4-GFP enhancer trap lines (Johnson et al., 2005: Plant J. 41, 779-789) that express GAL4 and GFP specifically in either the root epidermis or xylem parenchyma (and therefore ‘trap’ cell-type specific enhancer elements) were transformed with this GAL4 UAS – ethanol switch construct, thereby allowing both spatial and temporal control of transgenes. In preliminary experiments, the expression system successfully limited the expression of RFP to specific cell-types after induction with ethanol. Other genes expressed using this system include PpENA1, a Na⁺-extruding ATPase from the moss, Physcomitrella patens, and AtHKT1;1, a Na ⁺ transporter from Arabidopsis thaliana. The two enhancer trap rice lines were also transformed with the GAL4 UAS driving stable expression of AtHKT1;1 and PpENA1 specifically in root epidermal or xylem parenchyma cells. Expression of AtHKT1;1 in root epidermal cells reduced Na⁺ accumulation in the shoots, while expression in the root xylem parenchyma appeared to have little effect on shoot Na⁺ accumulation. Using cryo-scanning electron microscopy (SEM) X-ray microanalysis, the outer cells of the roots of the line expressing AtHKT1;1 in the epidermal cells were found to accumulate higher levels of Na⁺ than the parental enhancer trap line. Additionally, this line had decreased unidirectional ²²Na⁺ influx. Similar results were observed for plants expressing AtHKT1;1 driven by the CaMV 35S / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325289 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008