京都大学 / 新制・課程博士 / 博士(医学) / 甲第24524号 / 医博第4966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 齊藤 博英, 教授 萩原 正敏, 教授 山崎 渉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

3D genome organization recapitulates genome function, typically at the level of transcriptional state or chromatin state. Ciliates like Oxytricha trifallax possess highly specialized germline genomes, which play a role in sexual development but are largely transcriptionally silent. During Oxytricha development, the germline undergoes genome-wide rearrangement, transforming into a structurally distinct, transcriptionally active somatic genome. This wholesale rearrangement is programmed by transgenerational RNA copies of the somatic genome, which guide the retention of a small portion of the germline. Retained and eliminated sequences must be distinguished from each other and processed separately, but how this dichotomy is effected spatially or by chromatin organization is unknown. The breadth of Oxytricha genome dynamics presents the possibility of a complex relationship between chromatin architecture and genome function. We present new tools for studying the Oxytricha germline and apply them to map the 3D organization of the genome’s vegetative and developmental states using Hi-C. We find that the vegetative conformation primes the germline for development, while a massive spatial reorganization of the genome during development differentiates between retained and eliminated regions and precedes rearrangement of DNA. Further experiments suggest a role for RNA-DNA interactions and chromatin remodeling in this reorganization. This evidence supports a critical role for 3D architecture in programmed genome rearrangement.

Phylogenetics is transitioning from a history of deep-time analyses with few genes to a future of full-genome data that allows species-level resolutions at deep and shallow time scales. Accompanying this transition is a new focus on demographic parameters like ancestral population sizes and gene flow events in addition to the bifurcating trees that are the cornerstone of the field. As access to more data has highlighted some shortcomings of traditional phylogenetic methods that do not account for the processes of recombination, selection, population size changes, and inter-species gene flow, the field is exploring new theory and methods to catch up with the data. My thesis focuses on signals of demographic processes in genomic data. In exploring these processes, we attempt to avoid biases involved in simply extending old phylogenetic methods -- which have typically been applied to just a handful of genes -- to genomic datasets. Chapter 1 introduces a tool, ipcoal, for simulating genomic data on phylogenetic trees within a framework that includes recombination and the ability to specify effective population sizes, gene flow events, recombination maps, and differences in generation times. This tool enables, to varying degrees, all further chapters. Chapter 2 studies the effects of species tree demographic parameters on the resulting linkage among nearby local genealogies, including implications for gene tree and species tree inference. Chapter 3 examines turnover in local histories along the genome using a theoretical framework, the MS-SMC, which links topological heterogeneity along the genome to species tree model. Chapter 4 introduces simcat, a machine-learning method that uses genome-wide SNP data to infer admixture events on a phylogeny without relying on gene tree inference. This is an important step toward decreasing gene tree estimation error over deep evolutionary time scales. Behind the scenes, simcat uses ipcoal to train a machine learning model to map patterns in SNP data to the demographic scenarios that produced them. These chapters demonstrate new phylogenetic theory and methods for refining our ability to infer historical processes at phylogenetic scales, while also illuminating the importance of population-scale processes like gene flow and recombination for shaping genomes sampled in the present day.

Third generation sequencing has revolutionized our understanding of genomics, and enabled the in-depth discovery of complex plant genomes. In this project I aimed to assemble and annotate the genome of Z. spina-christi, a native plant to Saudi Arabia, as part of the the Kingdom of Saudi Arabia Native Genome Project established at the Center for Desert Agriculture at KAUST. Initially, a voucher plant was selected from the Al Lith region of Western Saudi Arabia. Fresh leaf tissue was collected for high-molecular weight (HMW) DNA extraction, as well as seed for greenhouse propagation. After HMW DNA extraction, library construction and PacBio HiFi sequencing, I generated a de novo assembly of the Z. spina-christi genome using the Hifiasm assembler, which yielded a 1.9 Gbp long assembly with high levels of duplication. The assembled contigs were scaffolded using an in-house script based on the software RagTag, that yielded a 406 Mbp long scaffold with 331 gaps (85.45% of estimated genome size). A preliminary analysis of the assembly for transposable elements revealed a TE content of 32.36%, with Long Terminal Repeats retrotransposons (LTR-RTs) being the major contributor to the total TE content. Basline annotation was completed using Omicsbox revealing 18,330 functional genes. This work describes the first genomic resource for the desert plant Z. spina-christi. To improve the assembly, I suggest the use of scaffolding using optical mapping, long Nanopore reads and Hi-C data to capture the spatial organization of the genome. Further experimental, genetic and TEs analysis is needed to explore the plant’s resilience to abiotic stresses in extreme environments.

The genome of the Atlantic sheepshead minnow, <u>Cyprinodon variecratus</u> (cyprinodontidae) contains at least two highly repetitive satellite DNA sequences. The major satellite sequence is present at roughly 10⁶ copies per haploid genome. The basic repeating unit averages 170 bp in length, tandemly arranged, and is bracketed by a Hind III restriction site. The Hind III satellite has little or no sequence similarity with other fish satellite DNAs reported in the 1iterature. Comparative hybridization experiments detect related sequences in the genomes of several species of pupfish of the genus <u>Cyprinodon</u> as well as in some cyprinodontid species of the genera <u>Jordanella</u> and <u>Floridichthys</u>. No significant hybridization was detected with the New World species <u>Cualac tesselatus</u> and Megupsilon aporus or within species of the Old World genera <u>Valencia</u> and <u>Aphanius</u>. / Ph. D.

RNA, which serves as a pivotal intermediary between DNA and proteins, plays a crucial role in shaping cellular functions and morphology. Generations of different techniques have been developed to enhance the understanding of RNA with respect to its sequence, function and spatial localization. While recent advancements have produced RNA detection methods capable of providing high-resolution insights into various RNA species, the disassociation process of these methods sacrifices the spatial arrangement of cells and native cell morphology. This calls for the need of in situ detection of RNA in its natural states, which would retain the constitution of cells. Multiplexed in situ RNA detection methods have evolved rapidly in the past decade. These techniques enable optical profiling and decoding of RNA, allowing for the acquisition of both transcriptomic and spatial features of the cells. This thesis focuses on the study of two in situ RNA detection methods: RNAmap and CRISPRmap. These approaches significantly increase signal level, and the cyclical readout probe hybridization scheme enables multiplexed readout, with the additional benefit of not being dependent on third-party sequencing reagents. The first chapter of this thesis sets forth an overview of different RNA detection methods, with an emphasis on RNAmap and CRISPRmap. The second chapter details the optimization of RNAmap in a novel organism, Oxytricha trifallax. RNAmap is able to detect different mRNA species in Oxytricha with robust signal intensity and specificity. Fluorescent in situ hybridization detection of telomeres in Oxytricha cells at different developmental time points also unveil a series of genome arrangement activities and nuclear morphology changes, which introduces novel biological questions for future research. The third chapter outlines the application of CRISPRmap, a pooled optical screening assay developed in our lab, to screen for novel modulators of WRN protein, an interesting synthetic lethal target of microsatellite unstable cancers. A pilot screen perturbed known modulators, and demonstrated expected effects on WRN’s function. After testing out CRISPRmap’s efficacy, we ventured from perturbing known modulators and expanded our screen to the entire kinome, exploring kinase regulation of WRN’s diverse function in DNA damage repair and cell viability.

Ancient DNA analyses are reliant on reference genomes to authenticate and identify endogenous genomes. While this has lead to many successful studies involving proboscidians, hominids, and ancient pathogens such as Yersinia pestis, our reliance on at most a small number of genomes greatly limits our ability to functionally describe the genome of interest. Further, given the existence of open bacterial genomes and horizontal gene transfers it is likely that reference biases have been incorporated and cited in following studies as representative of past gene diversity. By implementing and standardizing the use of bacterial pan-genomes the effect of these biases are greatly diminished while also revealing the relative capabilities of the target genome compared to the modern diversity. Describing an ancient strain by both its phylogenetic and functional similarities to modern strains allows for a more nuanced analysis of the species evolutionary history. Incongruencies between the phylogeny and genetic function are ripe for deeper analyses and the implications of its findings resonate beyond the characterization of an ancient genome. A pan-genome centric approach to ancient bacterial studies offers significant improvements compared to the current paradigm. / Dissertation / Doctor of Philosophy (PhD)

Yes / Several bacterial families are known to be highly abundant within the human microbiome, but their ecological roles and evolutionary histories have yet to be investigated in depth. One such family, Lachnospiraceae (phylum Firmicutes, class Clostridia) is abundant in the digestive tracts of many mammals and relatively rare elsewhere. Members of this family have been linked to obesity and protection from colon cancer in humans, mainly due to the association of many species within the group with the production of butyric acid, a substance that is important for both microbial and host epithelial cell growth. We examined the genomes of 30 Lachnospiraceae isolates to better understand the origin of butyric acid capabilities and other ecological adaptations within this group. Butyric acid production-related genes were detected in fewer than half of the examined genomes with the distribution of this function likely arising in part from lateral gene transfer (LGT). An investigation of environment-specific functional signatures indicated that human gut-associated Lachnospiraceae possess genes for endospore formation, whereas other members of this family lack key sporulation-associated genes, an observation supported by analysis of metagenomes from the human gut, oral cavity, and bovine rumen. Our analysis demonstrates that adaptation to an ecological niche and acquisition of defining functional roles within a microbiome can arise through a combination of both habitat-specific gene loss and LGT. / Canadian Institute for Health Research (grant number CMF-108026), Genome Atlantic and the Canada Research Chairs program to R.G.B.

Yes / Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil. / CNPq (scholarships 207422/2014-1, 500769/2014-1, 311554/2013-0; grants 407624/2012-0, 459100/2014-9).

The genome of bovine parvovirus (BPV) has been cloned by blunt end ligation of double-stranded virion DNA into the plasmid pUC8. The resulting genomic clones were infectious after transfection into bovine fetal lung (BFL) cells. Sequencing of the plasmids demonstrated that deletions were common at both ends of the cloned BPV genome. Deletions of up to 34 bases at the 3’ end lowered but did not abolish infectivity, while a deletion of 52 bases eliminated infectivity, End label analysis demonstrated the repair of deletions of up to 34 bases at the 3’ end or 35 bases at the 5’ end to the wild type length. Mutually inverted sequence orientations of the palindromic termini, known as the flip and flop forms, can occur during replication of parvovirus DNA. Cloning of BPV terminal sequences permitted the identification of the 3’ flop sequence inversion as a natural component of BPV DNA. This is the first report of sequence inversions within the 3’ end of an autonomous parvovirus. Clones with the 3’ flop or flip conformations were equally infectious. Wild type virion DNA was shown to have predominantly the 3’ flip conformation but a significant amount of 3’ flop was also detected. At the 5’ end, both the flip and flop sequence conformations were identified in nearly equal amounts. The progeny virion DNA from transfection of genomic clones had the same ratio of flip to flop as did wild type at both the 3’ and 5’ ends, regardless of the starting terminal conformations of the genomic clone. These data suggest that, while sequence inversion occurs at both termini during BPV DNA replication, some mechanism exists for the preferential replication of the 3’ flip conformation. Replicative form DNA from BPV infected cells had the same ratio of flip and flop at each end and the same termini as virion DNA. A set of deletion and frameshift mutants affecting each of the coding regions of BPV was constructed using one of the genomic clones. None of these mutants was infectious when transfected into BFL cells, which demonstrates that all three of the major open reading frames are essential for the production of infectious virus. / Ph. D. / incomplete_metadata