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付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24534号 / 医科博第148号 / 新制||医科||10(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 寺田 智祐, 教授 川上 浩司, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

With the rapid progress of next-generation sequencing (NGS) technologies, new tools and methods have emerged to investigate the transcriptomics of various organisms. RNA sequencing (RNA-seq) employs NGS to evaluate the presence and abundance of RNA transcripts in biological samples. This technique offers a comprehensive snapshot of the RNA dynamics within cells. With the ability to profile the entire transcriptome of organisms rapidly and accurately, RNA-seq has become the state-of-the-art method for transcriptome profiling, surpassing the traditional microarray approach. Single-cell RNA sequencing (scRNA-seq) was introduced in 2009 to profile the single-cell gene expression in highly heterogeneous samples such as brain tissue and tumors. The advancement of scRNA-seq technologies enables the in-depth transcriptomic study in each cell subtype. When selecting an scRNA-seq method, researchers must weigh the trade-off between profiling more single cells versus obtaining more comprehensive transcripts per cell, while considering the overall costs. The throughput of full-length scRNA-seq methods is usually lower, as each single cell needs to be processed separately to produce scRNA-seq libraries. However, full-length methods enable the researchers to investigate the splicing variants and allele-specific expression. Non-full-length methods only capture the 3' or 5' ends of transcripts, which limits their application in isoform detection, but as cells are pooled after barcoding for cDNA synthesis, the throughput is 2–3 orders of magnitude higher than full-length methods. We developed a droplet-based platform for full-length single-cell RNA-seq, which enabled the efficient recovery of full-length mRNA from individual cells in a high-throughput manner. The developed platform can process ~8,000 single cells within 2 days and detect ~20% more genes compared to Drop-seq. Besides scRNA-seq technology development, we also applied a low-input RNA-seq method to study the transcriptomics in different biological samples. When handling precious biological samples, a low-input method is necessary to profile the transcriptome of homogeneous cell populations. We first studied the epigenomic and transcriptomic regulations in colorectal cancer (CRC) using MOWChIP-seq, a low-input high-throughput method, in conjunction with our low-input RNA-seq approach. Fusobacterium nucleatum (Fnn) is closely related to the progression of cancers like CRC and pancreatic cancer. However, the molecular mechanisms of how Fnn adjusts the tumor microenvironment (TME) and leads to poor clinical outcomes are still unclear. In this in-vitro study, we characterized how hypoxia, an important TME ignored by previous research, facilitates Fnn infection of CRC and corresponding alterations of global epigenome and transcriptome. We infer that hypoxia has similar effects as Fnn infection alone on the CRC cells. The Fnn infection under hypoxia further boosts the proliferation and progression of CRC. We then applied our low-input RNA-seq method to study brain neuroscience and immunology. Psychedelics like DOI show promising clinical efficacy in patients with psychiatric conditions. Although psychedelics exhibit rapid antidepression action and long-lasting effectiveness compared to conventional treatment, their acute psychotic symptoms and potential for drug abuse discourage their application in clinical practice. In this case, it is important to comprehend the molecular mechanisms responsible for psychedelics' clinical efficacy. This understanding can pave the way for the development of improved treatments that do not rely on psychedelics. After profiling the transcriptome of mouse brain samples exposed to psychedelics with different post-exposure times, we concluded that the psychedelic-induced transcriptomic variations are more transient than epigenomic changes. In the second brain neuroscience project, we first applied 3-color FACS sorting to differentiate four neuron and non-neuron subtypes in human postmortem prefrontal cortex tissues. Then we profiled the gene expression of the four subtypes and validated the FACS sorting by examining the expression of marker genes. Differentially expressed genes between each subtype and the others were extracted and proceeded to gene ontology analysis. We identified unique altered biological pathways related to each subtype. The immunology research focuses on revealing the difference between low-grade inflammation and monocyte exhaustion, as well as the unique biological pathways they regulate. Therefore, we profiled the transcriptome of bone marrow-derived monocytes stimulated by PBS control, a low- or high-dose LPS. In addition to wild-type mice, we also included TRAM-deficient and IRAK-M-deficient mice. We concluded that low-dose LPS specifically regulates the TRAM-dependent pathway of TLR4 signaling, and high-dose LPS exclusively upregulates exhaustion markers by impacting metabolic and proliferative pathways. / Doctor of Philosophy / Transcriptomics is the comprehensive study of RNA transcripts derived from an organism's genome. RNA plays a vital role in maintaining the fundamental functions of cells and organisms. In eukaryotes, the genetic information stored in the DNA of cells is transferred to messenger RNA (mRNA) molecules through a process called transcription. These mRNA molecules serve as a bridge between DNA and proteins, as they carry the instructions encoded in genes to ribosomes for protein synthesis. Studying mRNA transcripts reveals various cellular mechanisms and their impact on overall organism function, gene regulation, and disease pathways. With the aid of next-generation sequencing, various RNA-seq approaches have been developed to study mRNA transcripts quantitatively in the past decades. To better understand the gene expression regulations in biological samples, we first applied bulk RNA-seq to profile the transcriptome of various samples under different conditions. Our in-house bulk RNA-seq protocol has been proven to be both high-performance and cost-effective compared to commercial kits. To better understand cellular diversity and uncover rare cell types in heterogeneous biological samples, we developed a droplet-based scRNA-seq platform that can recover full-length mRNA transcripts in a high throughput manner. It can profile the transcriptome of thousands of single cells within two days. It combines the advantages of the droplet-based scRNA-seq method (high throughput) and the well plate-based scRNA-seq method (full-length mRNA recovery).

The canonical model of transcriptional regulation in prokaryotes restricted binding site locations to promoter regions and suggested that the binding sequences serve as the main determinants of binding. In this dissertation, I challenge these assumptions. As a member of the TB Systems Biology Consortium, I analyzed and validated ChIP-Seq and microarray experiments for over 100 transcription factors (TFs). In order to study the transcriptional functions of predicted binding sites, I integrated binding and expression data and assigned potential regulatory roles to 20% of the binding sites. Stronger binding sites were more often associated with regulation than weaker sites, suggesting a correlation between binding strength and regulatory impact. Seventy-six percent of the sites fell into annotated coding regions and a significant proportion was assigned to regulatory functions. To study the importance of binding sequences, I compared experimental sites with computational motif predictions. Although a conservative binding motif was found for most TFs, only a fraction of the observed motifs appeared bound in the experiment. Some low-affinity binding sites appeared occupied by the corresponding TF while many high-affinity binding sites were not. Interestingly, I found exactly the same nucleotide sequences (up to 15 residues long) bound in one area of the genome but not bound in another area, pointing to DNA accessibility as an important factor for in vivo binding. To investigate the evolutionary conservation of binding-site occupancy, sequence, and transcriptional impact, I analyzed ChIP-Seq and expression experiments for five conserved TFs for two-to-four Mycobacterial relatives. The regulon composition showed significantly less conservation than expected from the overall gene conservation level across Mycobacteria. Despite expectations, sequence conservation did not serve as a good indicator of whether or not a computationally predicted motif was bound experimentally; and in some cases, a fully conserved motif was bound in one relative but not in the other. Conservation of genic binding sites was higher than expected from the random model, adding to the evidence that at least some genic sites are functional. Understanding the evolutionary story of binding sites allowed me to explain unusual site configurations, some of which indicated a role for DNA looping.

Promoter-enhancer interactions are essential for gene regulating, Capture Hi-C is a chromosome conformation capture method to map promoter-enhancer interactions at high resolution. We have Capture Hi-C data forGM12878 cells, immortalized primary B lymphocytes, in three replicates. Although Capture Hi-C maps enhancer elements together with the promoters they regulate, the overlap between enhancer datasets produced by other methods such as ChIP-seq and Capture Hi-C is lower than expected. In order to understand the reasons for lower overlap, we investigated the enhancer potential of replicated and non-replicated Capture Hi-C interactors, as well as enhancer overlapping and non-overlapping Capture Hi-C interactors. We performed a systematic comparison between our interactor and experimental regulatory and transcriptomic datasets to determine the extent of enhancer mapping. The results show replicated interactors have higher enhancer potential than non-replicated ones. However, there is evidence that interactors not overlapping with experimental validated regulatory datasets can also potentially be true enhancers.

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Deux phénomènes liés au TFIIH ont été étudiés : l'influence des mutations spécifiques dans la sous-unité XPD de TFIIH sur la réponse transcriptionnelle de certains gènes après l'irradiation UV, et l'interaction entre le TFIIH et la transcription des gènes de classe III. Une analyse détaillée de la dynamique du transcriptome a été effectuée pour la réponse des cellules humaines mutantes XP-D/CS à l'UV. Il a été démontré que la dysrégulation sélective observée de l’expression des gènes était liée à l'incapacité pour la ré-initiation transcriptionnelle et à l'hétérochromatinisation suivante, où l'histonedésacétylase SIRT1 a été identifiée comme le principal facteur. Son inhibition a permis de recouvrer l'expression normale d'un nombre substantiel des gènes affectés. Une étude de la participation pangénomique du coeur de TFIIH dans latranscription a découvert son association avec les gènes actifs de classe III. Cette association a été démontrée être indépendante de Pol II. Le coeur de TFIIH a été montré participer directement à la transcription effectuée in vitro par Pol III. / In this work, two TFIIH-related phenomena were investigated : the influence of specific mutations in TFIIH XPD subunits on the transcriptional response of different genes on UV irradiation and the interaction between TFIIH and transcription of class III genes. For the first time the detailed investigation of transcriptome dynamics was carried out for the response of XP-D/CS mutant human cells to UV-irradiation. The transcription regulation nature of the observed selective gene expression dysregulation was clearly observed. Its relation to failure of transcription re-initiation and consequentheterochromatisation was demonstrated. SIRT1 histone deacetylase was identified as the main driver of the repressive chromatin establishment on the certain genes upon UV. Inhibition of SIRT1 was found to recover normal expression of substantial number of affected genes. SIRT1 mediated mechanism was shown to be XP-D/CS specific. A potential link between this longevity related protein and progeria features of XP-D/CS mutants was hypothesised. Genome-wide study of the involvement of the core TFIIH in transcription revealed its association with active class III genes, not described previously. This association was demonstrated to be Pol II-independent. The core TFIIH was shown to be directly involved in Pol III mediated transcription in vitro.

Notre génome est constamment attaqué par des facteurs endogènes et exogènes qui menacent son intégrité et conduisent à différents types de dommages. Les cassures double brins (CDBs) font partie des dommages les plus nuisibles car elles peuvent entraîner la perte d'information génétique, des translocations chromosomiques et la mort cellulaire. Tous les processus de réparation se déroulent dans le cadre d'une chromatine hautement organisée et compartimentée. Cette chromatine peut être divisée en un compartiment ouvert transcriptionnellement actif (euchromatine) et un compartiment compacté transcriptionnellement inactif (hétérochromatine). Ces différents degrés de compaction jouent un rôle dans la régulation de la réponse aux dommages à l’ADN. L'objectif de mon premier projet était de comprendre l'influence de l'organisation 3D du génome sur la réparation de l'ADN. Pour cela, j’ai utilisé deux approches complémentaires dans le but d’induire et de cartographier les CDBs dans le génome de souris. Mes résultats ont mis en évidence un enrichissement de γH2AX, facteur de réparation des dommages à l’ADN, sur différentes régions du génome de cellules souches embryonnaires de souris, et ont également montré que les dommages persistent dans l’hétérochromatine, contrairement à l’euchromatine qui est protégée des dommages. Pour mon deuxième projet, j'ai cartographié l'empreinte génomique de 53BP1, facteur impliqué dans la réparation des CDBs, dans des cellules U2OS asynchrones et des cellules bloquées en G1 afin d’identifier de nouveaux sites de liaison de 53BP1. Mes résultats ont permis d’identifier de nouveaux domaines de liaison de 53BP1 couvrant de larges régions du génome, et ont montré que ces domaines de liaison apparaissent dans des régions de réplication moyenne et tardive. / Our genome is constantly under attack by endogenous and exogenous factors which challenge its integrity and lead to different types of damages. Double strand breaks (DSBs) constitute the most deleterious type of damage since they maylead to loss of genetic information, translocations and cell death. All the repair processes happen in the context of a highly organized and compartmentalized chromatin. Chromatin can be divided into an open transcriptionally active compartment (euchromatin) and a compacted transcriptionally inactive compartment (heterochromatin). These different degrees of compaction play important roles in regulating the DNA damage response. The goal of my first project was to understand the influence of 3D genome organization on DNA repair. I used two complementary approaches to induce and map DSBs in the mouse genome. My results have shown that enrichment of the DNA damage repair factor γH2AX occurs at distinct loci in the mouse embryonic stem cell genome and that the damage persists in the heterochromatin compartment while the euchromatin compartment is protected from DNA damage. For my second project, I mapped the genomic footprint of 53BP1, a factor involved in DSBs repair, in asynchronous and G1 arrested U2OS cells to identify novel 53BP1 binding sites. My results have identified novel 53BP1 binding domains which cover broad regions of the genome and occur in mid to late replicating regions of the genome.