京都大学 / 新制・課程博士 / 博士(理学) / 甲第23053号 / 理博第4730号 / 新制||理||1678(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 伊谷 原一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Development and application of genomic approaches based on 3C methods combined with increasingly powerful imaging approaches have enabled high-resolution genome-wide analysis of the spatial organization of chromosomes in genome function. In this thesis, I first describe an updated protocol for Hi-C (Hi-C 2.0), integrating recent improvements that significantly contribute to the efficient and high-resolution capture of chromatin interactions. Secondly, I present an assessment of the epigenetic landscape and chromosome conformation around the MYC gene in acute myeloid leukemia (AML) cells before and after small molecule, AI-10-49, treatment. MYC is up-regulated upon inhibition of the RUNX1 repressor by the fusion oncoprotein CBFβ-SMMHC. Treatment of AML cells with AI-10-49 blocks the RUNX1-CBFβ-SMMHC interaction, restoring RUNX1 at MYC regulatory elements. We demonstrate that the established loop is maintained and exchange between activating and repressive chromatin complexes at the regulatory elements, rather than altered chromatin topology, mediates disruption of target gene expression. Finally, Hi-C interaction maps represent the population-averaged steady-states. To understand the forces that promote and maintain the association of loci with specific sub-nuclear structures genome-wide, we developed liquid chromatin Hi-C. Detection of intrinsic locus-locus interaction stabilities and chromatin mobility are enabled by fragmenting chromosomes prior to fixation and Hi-C, thus removing strong polymeric constraints. Nuclear compartmentalization was found to be stable for average fragment lengths are 10-25 kb while fragmentation below 6kb led to a gradual loss of spatial genome organization. Dissolution kinetics of chromatin interactions vary widely for different domains and are analyzed in detail in the final chapter of this thesis., with lamin-associated domains being most stable, and speckle-associated loci most dynamic.

Comparing genomes in a computationally efficient manner is a difficult problem. Methods that provide the highest resolution are too inefficient and methods that are efficient are too low resolution. In this thesis, we show that the Colored de Bruijn Graph (CdBG) is a suitable method for comparing genomes because it is efficient while maintaining a useful amount of resolution. To illustrate the usefulness of the CdBG, the phylogenetic tree for 12 species in the Drosophila genus is reconstructed using pseudo-homologous regions of the genome contained in the CdBG.

<p>As part of development of a human pathogen suppressing in plantae model system within <i>Spinacia oleracea</i>, a known pathogen transmitting produce plants, the secretome of the <i>Stemphylium</i> strain PNW2016-02, an endophytic fungal isolate of spinach plants, was studied. This strain was previously isolated in Purdue University Northwest laboratories in the Biological Sciences Department. As the secondary metabolite secretions of PNW2016-02 were shown to have antibiotic properties against a broad range of bacteria, we sought to improve our understanding of these properties and other characteristics of PNW2016-02 by sequencing and annotating the genome of this strain. Chemical compound characterization was achieved using HPLC-MS, which provided the ability to identify and quantify chemical compounds contained within the PNW2016-02 secretome. Through multi-gene phylogenetic analysis and comparative genomics, we found that PNW2016-02 clusters with a clade representing <i>Stemphylium vesicarium</i>; however, genome annotation also uncovered several genes unique to PNW2016-02. To assist in more fully understanding the secondary metabolites PNW2016-02, a semi-automated bioinformatic pipeline was developed in the R statistical environment in order to reconstruct and characterize the metabolic pathways, especially those pertinent to antibiotic production. Analysis using the bioinformatic pipeline revealed the presence of a number of antimicrobial metabolites, such as flavonoids, and suggests these compounds are produced and transformed in pathways, such as the phenylpropanoid synthesis pathway. These findings also suggest an important vital role of the shikimate pathway for antimicrobial metabolite production within PNW2016-02. Overall, the findings presented here have implications for understanding the antimicrobial strategies employed by <i>Stemphylium</i> PNW2016-02 and its potential for use in the above-mentioned model system.</p>

Southern African populations harbour great genetic diversity enhanced by  population migration to the area in the last two millennia. Africa is perhaps the least studied continent in regards to population genetics and is often underrepresented in global studies. Studying sex-biased admixture in admixed populations is a great tool to understand population demographic history as well as sex-biased admixture from past events. Various studies on sex-biased admixture in Southern Africa have shown male sex-biased admixture from the incoming Bantu-speaking populations. One study by Hollfelder (2018) shows female Bantu-speaking sex-biased admixture. Here I will try to determine if ascertainment bias is the cause of the unexpected results in Hollfelder (2018). I will do this by comparing the original results, genotyped using the Illumina Omni 2.5M Array, to overlapping SNPs in two different arrays, the Affymetrix Human Origin Array and the Infinium H3Africa Consortium Array. Additionally, I will use whole genome data containing same individuals and individuals from similar populations to form a hypothesis on how the sex-biased admixture should look like without ascertainment. Then extracting variants from the whole genome data to two array SNP panels, the Illumina 2.5M Array and the Infinium H3Africa Consortium Array. For both parts in my project a method by Goldberg and Rosenberg (2015) will be used to calculate female and male contribution from admixture proportions of the X-chromosome and the autosomes estimated using the software ADMIXTURE. The results obtained could not determine if ascertainment bias was the sole factor skewing the results. The overlap with the Affymetrix Human Origin Array showed results closest to expected results based on previous studies, suggesting that ascertainment bias likely affects the results. The results attained using the whole genome indicated that the genotype calls of individuals present in both parts of the study did not fully match and that was confirmed using a principal component analysis. Unfortunatly the data used and analytical limitations in this project did not yield answers to how ascertainment bias affects calculations on sex-biased admixture. The X-chromosome is difficult to work with, especially when using data from multiple publications, as there is no standard common best-practice pipeline available on how to process the data leading to different data sets having been treated differently, which possibly affects downstream analysis when combining data sets.

Much of Nature is composed of highly modular and composable nested multicomponent living systems. Synthetic biology and bioengineering exploit this modularity to understand and engineer living things. This thesis explores two projects coupled by these principles, the first utilizing a synthetic genomics approach to probe the evolutionary history, flexibility, and modularity of core metabolism, and the second adapting and engineering components of a living material to generate living architecture and embed add program new behaviors into the living biocomposite. Chapter 1 details the synthetic resurrection of a core metabolic pathway lost from the metazoan lineage millions of years ago. All metazoans are auxotrophic for 9 of the 20 amino acids, the so-called “essential” amino acids. The pressures behind the loss of the 9 are a deep evolutionary puzzle. To investigate this event and probe the limits of core metabolic flexibility, we generated a synthetic valine prototrophic mammalian cell line, restoring valine self-sufficiency to the metazoan lineage. The restoration of this pathway implies the modern mammalian metabolism is still compatible with autogenous valine production, suggests profound modularity in core metabolism, and underscores the potential usefulness of large-scale synthetic genomics approaches in a answering deep evolutionary questions. Chapter 2 describes the engineering of a hybrid fungal-bacterial biocomposite by adapting and leveraging existing behaviors and microbial constituents of a living material. Fungal biocomposites are composed of a particulate lignocellulosic feedstock bound together into a bulk biocomposite by a network of dense fungal mycelium. Using a bioprospecting approach, we designed architectural and design strategies that relied on the natural substrate flexibility and growth patterns of the fungal component of the biocomposite to form origami-inspired human scale folding structures. Similarly, we isolated, characterized, and engineered a natural microbial component of the biocomposite’s own microbiome and used its pre-adapted ability to engraft in the growing biomaterial to embed new genetic functionalities in biocomposite objects. We believe that the strategy of bioprospecting useful components and behaviors holds promise for the development of future biomaterials adapted from living systems.

Parmi tous les animaux domestiques, le cheval est sans aucun doute celui ayant le plus influencé l’histoire des peuplements humains. Le cheval domestique a d’abord fourni à de nombreuses civilisations des ressources primaires essentielles telles que la viande et le lait. Utilisé pour sa force physique et comme moyen de transport, il a eu de profondes conséquences sur les mouvements de personnes et de biens ainsi que sur la diffusion de cultures et d’idées à travers l’Eurasie. Le cheval a ainsi fortement contribué à l’expansion de sociétés et d’empires pendant des millénaires, et ce jusqu’au vingtième siècle. Les différentes étapes de la domestication du cheval restent cependant mal comprises d’un point de vue archéologique et sont complexes à retracer à partir des données génétiques recueillies sur les races chevalines actuelles. L’émergence de la génomique ancienne au début des années 2010 a révolutionné la biologie de l'évolution, en donnant un accès direct à l’histoire des populations anciennes et actuelles. Elle est donc particulièrement adaptée pour étudier la transition historique induite par la domestication du cheval. En s'appuyant sur les dernières avancées en matière d’extraction d'ADN ancien et des technologies de séquençage d’ADN à haut débit, ce travail de doctorat vise à décrypter les modifications génétiques sous-jacentes au processus de domestication du cheval. Pour se faire, nous avons généré le plus grand jeu de données génomiques anciennes jamais rassemblées sur un organisme non humain. Celles-ci ont révélé que les chevaux domestiqués pour la première fois à Botai, dans le nord du Kazakhstan, il y a environ 5 500 ans, ne sont pas les ancêtres des chevaux domestiques ayant vécu pendant ces dernières ~4 100 années. Ce sont les ancêtres des chevaux de Przewalski, que l’on pensait jusqu’alors totalement sauvages. Cette découverte inattendue suggère qu'un remplacement majeur de la population de chevaux domestiques a eu lieu au cours du troisième millénaire avant notre ère, contribuant probablement à faire entrer l'humanité dans l'âge du Bronze. En outre, ces trois années de recherche ont permis d'identifier les signatures génétiques associées à différentes stratégies d’élevage du cheval et ont révélé les dynamiques évolutives en jeu lors des étapes clés de la domestication. En particulier, il ressort des analyses de génomes anciens que les chevaux ibériques n’ont contribué que marginalement à la création du cheval domestique tel qu’on le connaît aujourd'hui. Ce travail de thèse a par ailleurs permis de détecter une influence croissante des chevaux perses dès le début du Moyen Age. / Among all domesticates, the horse can confidently be considered as the animal that most impacted the history of human dynamics. Once they domesticated the horse, human civilizations got hold of essential domestication products including meat and milk, but also invaluable secondary products, such as fast transportation and powerful workforce. The horse thus deeply enhanced the circulation of people, goods, culture and ideas, promoting the spread of vast military and political units across Eurasia up until the 1900s. The various steps underpinning horse domestication are however difficult to track in the archaeological record and still poorly understood based on patterns of DNA variation among modern breeds. In the last decade, the advent of ancient genomics has revolutionized evolutionary biology by providing a direct window into the past history of populations. Ancient genomics therefore provides the necessary time travel machine to investigate the key historical transition in the history of humankind that was induced by the horse domestication. Leveraging the latest advances in ancient DNA recovery and High-Throughput sequencing technologies, this PhD project aimed at deciphering the genetic changes underlying the horse domestication process by generating the largest ancient genome dataset for a non-human organism, spanning the whole temporal and geographic range of horse domestication. This dataset revealed that horses first herded at Botai in Northern Kazakhstan ~5,500 years ago are not the ancestors of modern domestic horses but instead of modern Przewalski’s horses, previously thought to represent last true wild population on Earth. This major discovery also suggests that a swift genomic replacement in the domestic stock took place in the third millennium BCE, probably contributing to precipitating humankind into a new metal era, the Bronze Age. Additionally, this PhD work identified the genetic signatures associated with different management strategies and the evolutionary dynamics at play within distinct domestication stages. In particular, we were able to rule out Iberia as a major contributor to the modern domestic stock and moving towards more recent times, we characterized the growing influence of Persian-like horses starting in the early Middle Ages.

Orientador: Paulo Eduardo Martins Ribolla / Resumo: Introdução: O desenvolvimento de estratégias para redução no custo do sequenciamento de genoma completo (WGS) é importante para projetos que demandam por grandes quantidades de amostras. Uma estratégia de baixo custo é o sequenciamento de baixa cobertura aliado a técnicas de imputação para genotipagem eficiente e de confiabilidade adequada. A malária é uma das principais doenças transmitidas por artrópodes no mundo e o Brasil é considerado um país com alta incidência de malária, principalmente na região Amazônica, sendo principal vetor o mosquito Anopheles darlingi. Objetivo: O objetivo do presente estudo foi desenvolver estratégia para analisar dados de WGS de baixa cobertura de mosquitos Anopheles darlingi coletados no município de Mâncio Lima no Acre e verificar associação entre dados genéticos e dados de importância epidemiológica, tais como comportamento de picada, horário de atividade e distanciamento em escala microgeográfica. Materiais e métodos: Amostras de mosquitos Anopheles darlingi foram coletadas no município de Mâncio Lima - AC, entre 2016 e 2017. As bibliotecas foram preparadas com Nextera™ XT e sequenciadas no NextSeq500 da Illumina. Foi realizado genotipagem por sequenciamento e aplicado imputação. Estudos de associação ampla do genoma foram realizados com comportamento de picada e horário de atividade. Sinais de estratificação na população foram investigados por FST amplo no genoma e teste de permutação para significância. Resultados: Sinais fracos porém si... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Strategy development to reduce the cost of whole genome sequencing (WGS) is important for projects that demand large quantities of samples. A low-cost strategy is low-coverage sequencing combined with imputation techniques for efficient genotyping and sufficient confiability. Malaria is one of the main diseases transmitted by arthropods in the world and Brazil is considered a country with a high incidence of malaria, especially in the Amazon region with the main vector being the Anopheles darlingi mosquito. Objective: The objective of the present study was to develop a strategy to analyze low-coverage WGS data from Anopheles darlingi mosquitoes collected in the municipality of Mâncio Lima in Acre State and verify associations between genetic data and data of epidemiological importance, such as biting behavior, time of activity and distance on a microgeographic scale. Materials and methods: Samples of Anopheles darlingi mosquitoes were collected in the municipality of Mâncio Lima - AC, between 2016 and 2017. The libraries were prepared with Nextera ™ XT and sequenced on Illumina's NextSeq500. Genotyping by sequencing was performed and imputation was applied. Genome wide association studies were performed with biting behavior and time of activity. Population stratification signals were investigated by genome-wide FST and permutation test applied for significance. Results: Weak but significant stratification signals were identified considering distances of 2 to 3 k... (Complete abstract click electronic access below) / Mestre

Hedgehogs from the genus Erinaceus are an important model organism for studying the postglacial recolonisation of Europe and the processes that take place in the secondary contact zones of their areas of distribution. In this study, five individuals of white-breasted hedgehog (Erinaceus roumanicus), four individuals of western hedgehog (Erinaceus europaeus) and one estimated hybrid were analysed. Geographical distribution of individuals used in the study covers the region of the Central Europe, however in the further research expansion of analsysed individuals will be needed and the whole Palearct should be sampled. The main goal was to implement novel methods in research of hedgehogs, which will enable to map the population-genomic structure of the genus Erinaceus in western Palearct. The method RADSeq (Restriction site associated DNA sequencing) enables to obtain polymorphic markers, e.g., SNPs which we used (Single Nucleotide Polymorphisms) across the genome. In this work it was analyzed 16382 SNPs. Using the binary data which indicates the presence and absence of SNPs for each species, hypotheses raised under classical analyzes of genetic markers from previous studies have not been fully confirmed. In further research it will be necessary to verify possible occurrence of biases connected with...

Alzheimer’s disease (AD) is a complex neurodegenerative disease characterized by progressive memory loss and caused by a combination of genetic, environmental, and lifestyle factors. AD susceptibility is highly heritable at 58-79%, but only about one third of the AD genetic component is accounted for by common variants discovered through genome-wide association studies (GWAS). Rare variants may contribute to some of the unexplained heritability of AD and have been demonstrated to contribute to large gene expression changes across tissues, but conventional analytical approaches pose challenges because of low statistical power even for large sample sizes. Recent studies have demonstrated by expression quantitative trait locus (eQTL) analysis that changes in gene expression could play a key role in the pathogenesis of AD. However, regulation of gene expression has been shown to be context-specific (e.g., tissue and cell-types), motivating a context dependent approach to achieve more precise and statistically significant associations. To address these issues, I applied a strategy to identify new AD risk or protective rare variants by examining mutations occurring only in cases or only controls, observing that different mutations in the same gene or variable dose of a mutation may result in distinct dementias. I also evaluated the impact of rare variation on expression at the gene and gene pathway levels in blood and brain tissue, further strengthening the rare variant findings with functional evidence and finding evidence for a large immune and inflammatory component to AD. Lastly, I identified cell-type specific eQTLs in blood and brain tissue to explain underlying genetic associations of common variants in AD, and also discovered additional evidence for the role of myeloid cells in AD risk and potential novel blood and brain AD biomarkers. Collectively, these findings further explain the genetic basis of AD risk and provide insight about mechanisms leading to this disorder. / 2022-01-21T00:00:00Z