京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22073号 / 農博第2365号 / 新制||農||1072(附属図書館) / 学位論文||R1||N5227(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 保川 清, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

In eukaryotic interphase cells, heterochromatin mostly localizes either at the nucleolar periphery or at the nuclear lamina. Genome localization studies are crucial due to evidence that spatial organization of the genome affects gene function. Nucleolus-associated domains (NADs) are mainly heterochromatic regions that have been mapped only in a handful of mouse and human somatic cells, and in plants. The extent to which changes in NAD localization occur during cellular differentiation remains unknown. In this thesis, we characterize a map of genome-wide NADs in F121-9 mouse embryonic stem cells (mESCs). We identified NADs by deep sequencing chromatin associated with biochemically purified nucleoli and using NADfinder software to call NAD peaks. F121-9 NADs are mostly comprised of genomic regions with inactive or lowly transcribed genes and overlap extensively with lamina-associated domains (LADs) and regions with late replication timing. Similar to somatic mouse embryonic fibroblasts (MEFs), where NADs have been previously characterized by our laboratory, F121-9 mESCs display abundant “Type I” NADs. This subset of NADs frequently associates with nuclear lamina and nucleolar periphery and resembles constitutive heterochromatin. Compared to MEFs, F121-9 mESCs have fewer “Type II” NADs; this subset of NADs is frequently found at the nucleolar periphery but not at the nuclear lamina. mESC NADs are also less enriched in H3K27me3 modified regions compared to MEF NADs. This suggests that Polycomb complex-mediated facultative vii heterochromatin expansion is part of NAD maturation during cellular differentiation. Comparison of MEF and mESC NADs also revealed enrichment of developmentally regulated genes in NADs specific to these cell types. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. Our F121-9 mESC NAD studies identified distinct features of stem cell NADs and will facilitate future studies of genome organization changes during mammalian development.

Transcription factors activated by exogenous or endogenous stimuli alter gene expression with major effects on chromatin accessibility and the epigenome. This thesis investigates that impact of environmental chemical and hormonal exposure on liver chromatin accessibility in a mouse liver model. Exposure to the constitutive androstane receptor (CAR)-specific agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) induces localized changes in chromatin accessibility at several thousand DNase hypersensitive sites (DHS). Activating histone marks, associated with enhancers and promoters, were induced by TCPOBOP and were highly enriched at opening DHS. Opening DHS were highly enriched for CAR binding sites and nuclear receptor direct repeat-4 motifs. These DHS were also enriched for the CAR heterodimeric partner RXRA, binding by CEBPA and CEBPB, and motifs for other liver-specific factors. Thus, TCPOBOP alters the enhancer landscape through changes in histone marks and by mechanisms linked to induced CAR binding. In other studies, the impact of pituitary growth hormone (GH) secretion patterns on chromatin accessibility changes associated with sex-biased liver gene expression was examined. In adult male liver, the transcription factor STAT5 is directly activated by each successive plasma GH pulse. In female liver, STAT5 is persistently activated by the near-continuous stimulation by plasma GH. A majority of the ~4,000 GH-regulated, sex-biased DHS have chromatin marks characteristic of enhancers and were enriched for proximity to sex-biased gene promoters. Chromatin accessibility is thus a key feature of sex-differential gene expression. Two major classes of male-biased DHS were identified: dynamic male-biased DHS, almost all bound by STAT5, which undergo repeated cycles of chromatin opening and closing induced by each GH pulse; and static male-biased DHS, whose accessibility is unaffected GH/STAT5 pulses and whose sex bias results from these chromatin sites being more closed in female liver. Sites with STAT5 binding showed greater chromatin opening, many of which also contain the STAT5 motif. Finally, the effect of a single GH pulse on hypophysectomized male mouse liver was investigated to identify DHS responsive to the male, pulsatile-GH, secretion pattern. These studies demonstrate that widespread epigenetic changes associated with target gene expression are induced by xenobiotics and hormones regulating liver gene expression. / 2022-01-31T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19917号 / 理博第4217号 / 新制||理||1606(附属図書館) / 33003 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 工藤 洋, 教授 長谷 あきら, 教授 鹿内 利治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.

Previous studies have shown that genes can be switched on or off by age, environmental factors, diseases, and lifestyles. The open or compact structures of chromatin is a crucial factor that affects gene expression. Epigenetics refers to hereditary mechanisms that change gene expression and regulations without changing DNA sequences. Epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA interaction, play critical roles in cell differentiation and disease processes. The conventional approach requires the use of a few million or more cells as starting material. However, such quantity is not available when samples from patients and small lab animals are examined. Microfluidic technology offers advantages to utilize low-input starting material and for high-throughput. In this thesis, I developed novel microfluidic technologies to study epigenomic regulations, including 1) profiling epigenomic changes associated with LPS-induced murine monocytes for immunotherapy, 2) examining cell-type-specific epigenomic changes associated with BRCA1 mutation in breast tissues for breast cancer treatment, and 3) developing a novel microfluidic oscillatory hybridized ChIRP-seq assay to profile genome-wide lncRNA binding for numerous human diseases. We used 20,000 and 50,000 primary cells to study histone modifications in inflammation and breast cancer of BRCA1 mutation, respectively. In the project of whole-genome lncRNA bindings, our microfluidic ChIRP-seq assay, for the first time, allowed us to probe native lncRNA bindings in mouse tissue samples successfully. The technology is a promising approach for scientists to study lncRNA bindings in primary patients. Our works pave the way for low-input and high-throughput epigenomic profiling for precision medicine development. / Doctor of Philosophy / Traditionally, physicians treat patients with a one-size-fits-all approach, in which disease prevention and treatment are designed for the average person. The one-size-fits-all approach fits many patients, but does not work on some. Precision medicine is launched to improve the low efficiency and diminish side effects, and all of these drawbacks are happening in the traditional approaches. The genomic, transcriptomic, and epigenomic data from patients is a valuable resource for developing precision medicine. Conventional approaches in profiling functional epigenomic regulation use tens to hundreds of millions cells per assay, that is why applications in clinical samples are restricted for several decades. Due to the small volume manipulated in microfluidic devices, microfluidic technology exhibits high efficiency in easy operation, reducing the required number of cells, and improving the sensitivity of assays. In order to examine functional epigenomic regulations, we developed novel microfluidic technologies for applications with the small number of cells. We used 20,000 cells from mice to study the epigenomic changes in monocytes. We also used 50,000 cells from patients and mice to study epigenomic changes associated with BRCA1 mutation in different cell types. We developed a novel microfluidic technology for studying lncRNA bindings. We used 100,000-500,000 cells from cell lines and primary tissues to test several lncRNAs. Traditional approaches require 20-100 million cells per assay, and these cells are infected by virus for over-producing specific lncRNA. However, our technology just needs 100,000 cells (non-over-producing state) to study lncRNA bindings. To the best of our knowledge, this is the first allowed us to study native lncRNA bindings in mouse samples successfully. Our efforts in developing microfluidic technologies and studying epigenomic regulations pave the way for precision medicine development.

Introduction: The epigenetic modifications can significantly affect and alter the gene activity by regulating their expression, having direct impact on various processes in human body. Epigenetic processes are involved in ethiopathogenesis of many diseases. From this point of view, MHC genes are very important as they were linked to many autoimmune disorders, for example type 1 diabetes mellitus. In general autoimmune diseases appear to be connected to certain MHC class II genes. Aims: The aim of this thesis is to determine the relationship between expression levels and histone modifications present in the promoter area of MHC class II gene, DQA1. Moreover, we also analyze and compare the DQA1 gene mRNA expression depending on the QAP promoter allele. Methods: We isolated both nucleic acids (DNA and RNA) and leukocytes from peripheral blood samples collected from voluntary donors. DNA was utilized for genotypization of individuals. RNA was subjected to reverse transcription and the quantitative PCR was performed in order to determine the level of expression. Leukocytes were used for chromatin immunoprecipitation, which was evaluated using quantitative PCR. Results: The expression level of QAP allele 3.1 was found to be higher than for the rest of the alleles Allele 4.1A showed, on the other hand,...

As we gain increased understanding of genetic disorders and gene regulation more focus has turned towards complex interactions. Combinations of genes or gene and environmental factors have been suggested to explain the missing heritability behind complex diseases. Furthermore, gene activation and splicing seem to be governed by a complex machinery of histone modification (HM), transcription factor (TF), and DNA sequence signals. This thesis aimed to apply and develop multivariate machine learning methods for use on such biological problems. Monte Carlo feature selection was combined with rule-based classification to identify interactions between HMs and to study the interplay of factors with importance for asthma and allergy. Firstly, publicly available ChIP-seq data (Paper I) for 38 HMs was studied. We trained a classifier for predicting exon inclusion levels based on the HMs signals. We identified HMs important for splicing and illustrated that splicing could be predicted from the HM patterns. Next, we applied a similar methodology on data from two large birth cohorts describing asthma and allergy in children (Paper II). We identified genetic and environmental factors with importance for allergic diseases which confirmed earlier results and found candidate gene-gene and gene-environment interactions. In order to interpret and present the classifiers we developed Ciruvis, a web-based tool for network visualization of classification rules (Paper III). We applied Ciruvis on classifiers trained on both simulated and real data and compared our tool to another methodology for interaction detection using classification. Finally, we continued the earlier study on epigenetics by analyzing HM and TF signals in genes with or without evidence of bidirectional transcription (Paper IV). We identified several HMs and TFs with different signals between unidirectional and bidirectional genes. Among these, the CTCF TF was shown to have a well-positioned peak 60-80 bp upstream of the transcription start site in unidirectional genes.

Orientador: Prof. Dr. Alexandre Hiroaki Kihara / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, São Bernardo do Campo, 2018. / A determinação do destino das células neuronais é um processo dinâmico regulado pela expressão de centenas de genes simultaneamente. A modificação pós-transcricional das caudas N-terminais das histonas é uma forma dinâmica de regulação gênica. Várias evidências sugerem que a modulação das modificações das histonas desempenha um papel importante na regulação da determinação do destino neuronal e atuam em muitos processos do desenvolvimento. Entre os diferentes moduladores, as enzimas modificadoras de histonas, que possuem como alvos as caudas das histonas, estão no centro da atenção. As histonas demetilases (HDMs) são uma grande família de enzimas que possuem atividade catalítica seletiva contra sítios específicos de metilação de histonas. Jmjd3 é uma HDM específica para histona H3K27 cuja atividade enzimática torna o ambiente propício para aumentar a taxa de transcrição gênica. Para investigar o provável papel da Jmjd3 no desenvolvimento da retina em ratos na fase pós-natal, realizamos o bloqueio desta enzima com o composto farmacológico GSK-J1. Como primeira abordagem, determinamos a localização de Jmjd3 na retina de ratos neonatos e adultos, o que foi consistente com a localização em neurônios diferenciados, incluindo células ganglionares na retina de ratos neonatos. Nesta idade do desenvolvimento, também observamos a presença de Jmjd3 em células indiferenciadas. Injeções subretinianas de GSK-J1 causaram a diminuição do nível proteico de H3k27me3 em retinas de ratos neonatos. Curiosamente, a injeção de GSK-J1 aumentou simultaneamente o número de células proliferativas e apoptóticas. Além disso, mais células imaturas foram detectadas na camada plexiforme externa, com processos neuronais mais longos. Finalmente, a influência da GSK-J1 na citogênese retiniana pós-natal foi examinada. Fomos capazes de determinar que a GSK-J1 especificamente causou uma diminuição significativa no número de células PKC-positivas que, quando localizadas na parte externa da camada nuclear interna, é um marcador confiável de células bipolares de bastonete. Estes dados fornecem as primeiras evidências dos efeitos do bloqueio farmacológico in vivo das histonas demetilases durante o desenvolvimento inicial da retina pós-natal, com impacto sobre processos como proliferação celular, maturação, indução de apoptose e determinação celular específica. / Neuronal cell fate determination is dynamic process regulated by expression of hundreds of genes simultaneously. The posttranslational modification of the N-terminal tails of the histone proteins is dynamic way of gene regulation. Countless numbers of the evidences propose that regulation of histone modification play principal role in various developmental process such as neuronal fate determination. Among different modulators the histone modifying enzymes that are targets histone tails are in the center of attraction. The histone demethylases (HDMs) family is comprised of several enzymes that have selective catalytic activity against specific sites of histone methylation. The enzymatic activity of the histone H3K27-specific demethylase Jmjd3 leads to transcriptionally permissive chromatin environments. To investigate the probable role of Jmjd3 in early postnatal rat retinal development, we tried to block this enzyme with pharmacological compound GSK-J1. As a first approach, we determined the localization of Jmjd3 in neonate and adult rat retina, which is consistent with localization in differentiated neurons, including ganglion cells in the retina of neonate rats. At this developmental age, we also observed the presence of Jmjd3 in undifferentiated cells. Subretinal injection of GSK-J1 caused the decrease of the global level of H3k27me3 in retinas of neonate rats. Interestingly, injection of GSK-J1 simultaneously increased the number of proliferative and apoptotic cells. In addition, more immature cells were detected in outer plexiform layer, with longer neuronal processes. Finally, the influence of GSK-J1 on postnatal retinal cytogenesis was examined. We were able to determine that GSK-J1 specifically caused significant decrease in the number of PKCa-positive cells, which when located in the outer part of the inner nuclear layer is a reliable marker of rod-on bipolar cells. These data provide the first evidence of in vivo pharmacological blocking of histone demethylases during early postnatal retinal development. In summary, we were able to show that application of GSK-J1 can influence on cell proliferation, maturation, apoptosis induction, and specific cell determination.

La transition fleur-fruit, appelée nouaison, est déclenchée par la pollinisation des fleurs et ce processus est essentiel pour cycle reproducteur des plantes, la formation des semences et le rendement de production. Les mécanismes moléculaires contrôlant cette importante transition développementale ont été peu explorés. Les marques histones et la méthylation de l'ADN sont les deux principaux modes de régulation épigénétique, mais à ce jour, leurs contributions respectives à la reprogrammation transcriptionnelle qui est associée au programme d’initiation des fruits charnus n’ont pas fait l’objet d’aucune étude sur aucune espèce de plante. Afin d’explorer l’importance dans la transition fleur-fruit de ces deux types de régulation épigénétique, des approches de transcriptomique "genome-wide", de ChIP-seq se et de séquençage bisulfite d'ADN ont été mises en place chez la tomate, une espèce économique majeure et un modèle d’étude pour les fruits charnus. Les résultats révèlent une corrélation étroite entre le repositionnement des marques histones et les changements observés de l'expression génique globale. L’étude montre aussi que les marques H3K9ac et H3K4me3 agissent en synergie pour activer la transcription génique, alors que la marque H3K27me3 a un effet répressif. A l’inverse, il n’y a pas de corrélation entre les variations de la méthylation de la cytosine et l’évolution des profils transcriptomiques. Il ressort donc que ce sont les changements au niveau des marques histones plutôt que de la méthylation de l'ADN qui constituent le moteur principal de la reprogrammation génétique associée au processus de transition fleur-fruit chez la tomate. En concordance avec cette idée, le niveau d'expression des gènes associés à l’initiation du fruit, tels que ceux liés au métabolisme hormonal, à la division cellulaire ou au développement embryonnaire, est corrélé avec les modifications des marques H3K9ac ou H3K4me3, mais pas avec la méthylation de l'ADN. En outre, l'étude comparative des profils transcriptomiques associés à la formation du fruit dépendant et indépendant de la pollinisation révèle l'intervention complexe de multiples voies de signalisation hormonales. Au total, notre étude présente un nouvel aperçu du contrôle de la reprogrammation génétique nécessaire à l’initiation du développement du fruit et révèle le rôle important du contrôle épigénétique dans ce processus de transition développementale. Dans le même temps, l’étude identifie un groupe de gènes impliqués dans la régulation épigénétique qui offrent des cibles potentielles pour les programmes d’amélioration de la nouaison des fruits, un processus majeur affectant le rendement de production / The flower-to-fruit transition, so-called fruit setting, is triggered by flower pollination and this process is essential for plant reproductive success, seed formation and crop yield. The underlying molecular mechanisms controlling this developmental transition remain unclear. Histone marking and DNA methylation are the main epigenetic modes for genetic reprogramming, however, their respective contribution to the fruit set-associated transcriptomic reprogramming is also unknown. To address the contribution of the two types of epigenetic regulation to fruit set, genome-wide transcriptomic profiling, ChIP-sequencing and DNA bisulfite sequencing were applied to tomato, a major economic crop and a model system for fleshy fruit. The study emphasizes the tight correlation between histone repositioning and gene expression changes revealing that H3K9ac and H3K4me3 histone marks synergistically promote gene transcription, whereas H3K27me3 marking has a repressive effect. We concluded that changes in histone marks rather than in DNA methylation are the main drivers of genetic reprogramming associated with the fruit set transition in tomato, and H3K9ac and H3K4me3 marking is the primary players in this control mechanism. Consistently, the expression level of fruit set-associated genes such as those related to hormone metabolism, cell division, and embryo development correlated with changes in H3K9ac or H3K4me3 marking, but not with DNA methylation. In addition, comparative study of transcriptomic profiling between pollination-dependent and -independent fruit set, uncovered the complex intervention of multiple hormone signaling pathways involved in the flower-to-fruit transition. Auxin appears as the central hormone triggering the extensive transcriptomic reprogramming associated with the initiation of early fruit growth. Altogether, the study provides new insight into the control of gene reprogramming underlying fruit the shift from flower to fruit and uncovers a set of genes encoding modifiers of epigenetic marks which may provide new targets for breeding programs aiming to improve fruit setting, a major process impacting crop yield.