Plants are sessile organisms and have developed varied mechanisms to tolerate stress. One such mechanism is DNA methylation, which plays a vital role within and across generational stress adaptation. To understand the role of DNA methylation in transgenerational stress adaptation, we exposed Arabidopsis thaliana for four generations of sub-lethal doses of glyphosate, trifloxysulfuron, clipping, and shading, which we further classified into the broader categories of stress ecological (shading and clipping) and herbicides (glyphosate and trifloxysulfuron). We analyzed phenotypic and whole-genome bisulfite sequencing data and found that the Arabidopsis phenotype adapts more rapidly to herbicide stress as compared to ecological stresses. DNA methylation changes for glyphosate were minimal after four generations of stress whereas the other three stresses showed dynamic change in the DNA methylation patterns. To understand within generation stress response, Sorghum bicolor was exposed to the same stresses at sub-lethal doses and we analyzed its phenotypic, whole genome bisulfite sequencing, and gene expression responses. Ecological stresses had higher negative impact on S. bicolor plant growth as compared to herbicide stresses. Similarly, we found higher differentially expressed genes for clipping as compared to both herbicides. All four stresses changed the methylome in a unique way; where we found 998 differentially methylated regions (DMR) for trifloxysulfuron, 193 for shading, 141 for clipping and 60 for glyphosate. Out of these DMR's some occurred genic region, which could potentially change gene expression and help plants withstand stress. Overall, DNA methylation can potentially help plants to withstand stress due to their dynamic and specific response to a variety of stresses both transgenerational and within generation. This information to better understand stress adaptation mechanisms in plants and used to develop stress-resilient crops. / Doctor of Philosophy / Environmental and anthropogenic stresses can negatively impact plant growth and development. Plants can have stress memory through epigenetic changes which helps them withstand stress in future generations. Epigenetics is the field of science where changes on the DNA and not sequence, that can be an addition or deletion of a methyl group, modification of histones, or production of small RNAs. We wanted to understand short and long-term effects of common anthropogenic and ecological stresses on how DNA methylation changes can help plants to withstand stress. We used the model plant Arabidopsis thaliana and the non-model crop/weed Sorghum bicolor. We exposed plants to sub-lethal doses of two herbicides, clipping, and shade stress, at levels high enough to cause significant visible injury but still allowed them to recover and reproduce for a single generation for S. bicolor and four generations for A. thaliana. We found that A. thaliana rapidly more adapts to herbicide stress as compared to ecological stresses. DNA methylation changes for glyphosate were minimal after four generations of stress whereas the other three stresses showed dynamic changes in the DNA methylation patterns. Each stressed impacted S. bicolor phenotype, DNA methylation, and gene expression in unique ways. We found ecological stresses greatly affected the phenotype of the S. bicolor plants as compared to herbicide stresses. Overall, our results showed that stress can cause DNA methylation changes and in transgenerational stress DNA methylation can potentially play a role in stress adaptation. This information could be useful for scientists to further understand stress resilience in plants.

Yes / Type 2 diabetes mellitus prevalence is growing globally, and the leading cause of mortality in these patients is cardiovascular disease. Epigenetic mechanisms such as microRNAs (miRs) and DNA methylation may contribute to complications of type 2 diabetes mellitus. We discovered an aberrant type 2 diabetes mellitus–smooth muscle cell phenotype driven by persistent up-regulation of miR-145. This study aimed to determine whether elevated expression was due to changes in methylation at the miR-145 promoter. Smooth muscle cells were cultured from saphenous veins of 22 non-diabetic and 22 type 2 diabetes mellitus donors. DNA was extracted, bisulphite treated and pyrosequencing used to interrogate methylation at 11 CpG sites within the miR-145 promoter. Inter-patient variation was high irrespective of type 2 diabetes mellitus. Differential methylation trends were apparent between non-diabetic and type 2 diabetes mellitus–smooth muscle cells at most sites but were not statistically significant. Methylation at CpGs −112 and −106 was consistently lower than all other sites explored in non-diabetic and type 2 diabetes mellitus–smooth muscle cells. Finally, miR-145 expression per se was not correlated with methylation levels observed at any site. The persistent up-regulation of miR- 145 observed in type 2 diabetes mellitus–smooth muscle cells is not related to methylation at the miR-145 promoter. Crucially, miR-145 methylation is highly variable between patients, serving as a cautionary note for future studies of this region in primary human cell types.

Epigenetics comprises chemical modifications of the DNA and the proteins that the DNA is wrapped around them. These modifications play key roles in establishing and maintaining cellular identity throughout development and adulthood. In recent years, it has become increasingly clear that these actions are more dynamic than initially believed. The alteration of cellular identities during regeneration, ageing, and the formation of tumors is closely linked to systematic changes in epigenetic modifiers. The emergence of cutting-edge single-cell sequencing technologies has enabled thorough explorations of biological processes with high molecular precision. Nevertheless, the regulation of cellular behavior is intricately tied to collective processes occurring in both spatial and temporal dimensions, operating on the mesoscopic and macroscopic scales. However, these larger scales cannot be straightforwardly deduced from microscopic measurements along the DNA sequence. Consequently, the findings obtained from sequencing experiments stay at the descriptive level until they are coupled with methodologies capable of discerning collective degrees of freedom. Here, using statistical physics tools and sequencing technologies, we study the collective processes underlying epigenetic dynamics in cells that change their identity over time. Specifically, we investigate collective epigenetic processes during ageing and the reprogramming of cells after injury. In the first part of this thesis, we study the mechanistic basis of epigenetic modifications during ageing. Despite the accuracy of machine learning models in predicting the biological age based on epigenetic DNA methylation marks, these tools do not inform about the mechanistic basis of epigenetic ageing. We show that epigenetic ageing is reflected in systematic and collective changes in DNA methylation marks during ageing, which manifests in the stereotypical behavior of two-point correlation functions. We devise a stochastic theory that comprises competition of antagonistic enzymes at the boundaries of genomic regions with atypically high content of cytosine-guanine pairs. We systematically coarse-grain this theory to derive a macroscopic description in terms of a phase-field theory. This model predicts the changes in two-point correlation functions during ageing and explains diverse observations in the field of epigenetic ageing. In the second part of this thesis, we study the collective epigenetic processes during the regeneration of the liver after injury. In particular, we study the interplay between DNA methylation and the accessibility of chromatin and show the necessity for emergent memory of past injuries in the system. This memory is achieved by considering an effective projection between different scales of epigenetic modifications. In total, in this thesis, we derived theoretical descriptions of epigenetic processes that have so far only been studied descriptively. We showed that both epigenetic alterations during ageing and during reprogramming rely on an interplay between collective biochemical processes and the geometry of the DNA. With this work, we show how linear DNA sequencing can inform about collective epigenetic processes in space and time.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第25304号 / 工博第5263号 / 新制||工||2001(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 佐藤 啓文, 教授 森 泰生, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Multiple health issues are associated with obesity and numerous factors are causative of the disease. The role of genetic factors is well established, as is the knowledge that dietary and sedentary behavior promotes weight gain. Although there is strong suspicion towards the role of epigenetics as a driving force toward disease, this field remains l in the context of obesity. DNA methylation correlation networks were profiled from blood samples of 69 adolescents of two distinct weight-classes; obese (n=35) and normal-weight (n=34). The network analysis revealed major differences in the organization of the networks where the network of the obese had less modularity compared to normal-weight. This is manifested by more and smaller clusters in the obese, pertaining to genes of related functions and pathways, than the network of the normal-weight. Consequently, this suggests that biological pathways have a lower order of coordination between each other in means of DNA methylation in obese than normal-weight. Analysis of highly connected genes, hubs, in the two networks suggests that the difference in coordination between biological pathways may be derived by changes of the methylation pattern of these hubs; highly connected genes in one network had an intriguingly low connectivity in the other. In conclusion, the results suggest differential regulation of transcription through changes in the coordination of DNA methylation in overweight and normal weighted individuals. The findings of this study are a major step towards understanding the role of DNA methylation in obesity and provide potential biomarkers for diagnosing and predicting obesity.

Indiana University-Purdue University Indianapolis (IUPUI) / LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.

Indiana University-Purdue University Indianapolis (IUPUI) / Epigenetics is defined as “the study of stable, often heritable, changes that influence gene expression that are not mediated by DNA sequence” (Fingerman et al., 2013). Epigenetic marks such as covalent histone modifications and DNA methylation are important for maintaining chromatin structure and epigenetic inheritance. Several proteins have been found to bind and/ or regulate epigenetic marks. One such protein, CXXC finger protein 1 (Cfp1) is an important chromatin regulator that binds to unmethylated CpG islands. It has been found to be essential for mammalian development. Mice lacking Cfp1 exhibit an embryonic- lethal phenotype. However, the function of Cfp1 can be studied using Cfp1 Null mouse ES cells, which are viable. Thus far, Cfp1 has been shown to be important for cell growth, cytosine methylation, histone modifications, subnuclear localization of Set1A histone H3K4 methyltransferase, and cellular differentiation. When Cfp1 Null ES cells are induced to differentiate by removal of Leukemia Inhibitory Factor (LIF), the cells are not able to turn off pluripotency markers such as Oct4 and alkaline phosphatase and fail to express differentiation markers such as Gata4 and Brachyury. In this study, we used established protocols to further examine the differentiation capacity of Cfp1 Null cells. Specifically, we tested the ability of Cfp1 Null ES cells to retain stem cell properties in the absence of LIF, differentiate into cardiomyocytes in the presence of TGF-β2 and differentiate into neuron precursors in the presence of retinoic acid (RA). While the differentiation effects of RA were inconclusive, Null cells were able to start differentiating in the absence of LIF, either as individual cells or EBs, and the presence of TGF-β2 when seeded on gelatin coated tissue culture dishes. However, no difference was seen between cells treated without LIF and those treated with TGF-β2. In both conditions, only a small portion of cells were able to differentiate, while the majority of the cell population retained stem cell characteristics. Cell growth and the differentiation capacity of Cfp1 Null cells were also compromised in comparison to WT cells. Thus, further supporting the need for the correct epigenetic patterns maintained by Cfp1 during cellular differentiation.

Le palmier dattier (Phoenix dactylifera L.) est une espèce fruitière dioïque tropicale qui revêt une importance capitale sur le plan alimentaire, socio-économique et écologique pour les régions arides du globe. Malgré l'intérêt de disposer d'un outil moléculaire pour discriminer les plante s mâles et femelles pour les programmes d'amélioration génétique, aucun marqueur spécifique du sexe n'a été identifié et validé à ce jour. Afin de pouvoir étudier et comprendre le déterminisme sexuel du palmier dattier, nous avons entrepris la description et la caractérisation des processus cellulaires et moléculaires associés à la différenciation des organes sexuels. L'étude histologique du développement reproducteur a montré que le bourgeon floral est d'apparence bisexuelle jusqu'à l'initiation des primordia de l'androcée et du gynécée. Le premier dimorphisme sexuel observé à ce stade correspondant à un gynécée plus large dans les fleurs femelles résulterait d'une activité mitotique plus importante dans les cellules du gynécée fertile par rapport à son équivalent non fonctionnel. Les organes sexuels stériles, staminodes et pistillodes, cessent ensuite leur développement et présentent une différentiation incomplète. Des études d'hybridation in situ de l'expression du gène codant l'histone H4, marqueur de l'activité mitotique, ont montré que le blocage du développement des staminodes et des pistillodes serait dû à un arrêt des divisions cellulaires. Nos investigations de l'intégrité cellulaire par des observations en microscopie électronique à transmission et par coloration de l'ADN confirmeraient que l'avortement des organes stériles ne résulte pas d'un processus de dégradation cellulaire et nucléaire. De plus, l'étude de la méthylation de l'ADN par immunodétection des cytosines méthylées révèle que, par rapport aux organes fertiles, les pistillodes et les staminodes se distinguent par leur niveau plus élevé de méthylation. Ces résultats sont en cohérence avec la réversibilité du blocage de ces organes observés in planta ou in vitro en réponse à une induction hormonale. L'ensemble de ces données montrent que l'unisexualisation des fleurs de palmier dattier est associée à une hyperméthylation globale de l'ADN suivi d'un arrêt des divisions cellulaires dans les organes sexuels stériles. Cette étude a permis d'améliorer nos connaissances sur les mécanismes qui gouvernent la différenciation des organes sexuels et permettra d'ouvrir des perspectives pour l'identification de marqueurs moléculaires du sexe chez le palmier dattier. / The date palm (Phoenix dactylifera L.) is a dioecious tropical fruit crop plant which has vital dietary, socio-economic and ecological importance in arid regions of the world. Despite the interest of developing molecular tools to discriminate male and female plants for the benefit of biodiversity preservation and genetic improvement programs, no sex-specific markers have been identified and validated to date. To study and understand the sex determination of date palm, we undertook to characterise the cellular and molecular processes underlying sex organ differentiation in this plant.A histological study of date palm reproductive development showed that the immature flower is bisexual in appearance until the initiation of the androecium and gynoecium. The first sign of sexual dimorphism is observed at this stage, namely a wider gynoecium in female flowers resulting from greater mitotic activity in the functional gynoecium of female flowers compared to the pistillode of male ones. The sterile sex organs (pistillode and staminodes) were observed to cease their development by progressive loss of cell proliferation and ultimately displayed incomplete differentiation.Cell division patterns and the nuclear integrity of reproductive organs were investigated respectively by RNA in situ hybridization to a histone H4 gene probe and by DNA coloration combined with scanning electron microscopy. The results obtained revealed an absence of cell cycle activity and nuclear degradation in the residual sex organs. In addition, a study of DNA methylation, by immunodetection of methylated cytosines revealed that compared to the fertile reproductive organs, staminodes and pistillodes displayed relatively high levels of global DNA methylation. These results are consistent with the observed reversibility of sterile organ developmental arrest observed in planta or in vitro in response to hormonal induction. Overall, these data demonstrate that the floral unisexuality of date palm is characterized by cell cycle arrest, higher DNA methylation in sterile sexual organs and an absence of cell degeneration rather than a cell death process. This study has improved our understanding of the mechanisms that govern the differentiation of sex organs and forms a useful starting point for research on the identification of molecular markers of sex determination in date palm.Kewords: Date palm - flower - sex determination - cell cycle - DNA methylation

Worldwide obesity has more than doubled since 1980 and at least 2.8 million people die each year as a result of being overweight or obese. An elevated body weight is the result of the interplay between susceptibility gene variants and an obesogenic environment, and recent evidence shows that epigenetic processes are likely involved. The growing availability of high-throughput technologies has made it possible to assess quickly the entire epigenome of large samples at a relatively low cost. As a result, vast amounts of data have been generated and researchers are now confronted to both bioinformatic and biostatistic challenges to make sense of such data in the context of obesity and its complications. In this doctoral thesis, we explored associations between the human blood methylome and obesity-associated gene variants as well as dietary fat quality and quantity. We used well described preprocessing techniques and statistical methods, along with publicly available data from consortiums and other research groups, as well as tools for pathway enrichment and chromatin state inference. We found associations between obesityassociated SNPs and methylation levels at proximal promoters and enhancers, and some of these associations were replicated in multiple tissues. We also found that contrary to dietary fat quantity, dietary fat quality associates with methylation levels in the promoter of genes involved in metabolic pathways. Then, using a gene-targeted approach, we looked at the impact of an acute environmental stress (sleep loss) on the methylation and transcription levels of circadian clock genes in skeletal muscle and adipose tissue of healthy men. We found that a single night of wakefulness can alter the epigenetic and transcriptional profile of core circadian clock genes in a tissue-specific manner. Finally, we looked at the effects of chronic maternal obesity and subsequent weight loss on the transcription of epigenetic machinery genes in the fetus and placenta of mice. We found that the transcription of epigenetic machinery genes is highly sensitive to maternal weight trajectories, and particularly those of the histone acetylation pathway. Overall, this thesis demonstrated that genetics, obesogenic environment stimuli and maternal programming impact epigenetic marks at genomic locations relevant in the pathogenesis of obesity.

• Argonaute proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Argonaute proteins, but little is know about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes. • Here we investigated the evolutionary dynamics of an Argonaute-binding platform in NRPE1, the largest subunit of RNA Polymerase V. We compared NRPE1 sequences from more than 50 species, including dense sampling of two plant lineages. • This study demonstrates that the Argonaute-binding platform of NRPE1 retains Ago-hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is due to relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million years. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism. • The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.