Listeria monocytogenes est un pathogène intracellulaire facultatif responsable d’une infection sévère d’origine alimentaire, la listériose. L’étude du processus d’infection cellulaire de cette bactérie a permis d’élucider divers mécanismes impliqués dans les interactions hôte-pathogène et dans le fonctionnement de la cellule eucaryote. En particulier, L. monocytogenes a été l’un des modèles pionniers dans la découverte du ciblage de la chromatine et de régulateurs nucléaires par des microbes. L’étude d’un facteur de virulence de L. monocytogenes, LntA, a permis l’identification d’un de ces régulateurs : BAHD1. En recrutant des protéines impliquées dans la formation de l’hétérochromatine, telles HDAC1/2 et HP1, BAHD1 stimule la formation d’une chromatine compacte à effet répressif. Lors d’une infection de cellules épithéliales par L. monocytogenes, BAHD1 réprime la réponse immunitaire stimulée par les interférons, une fonction inhibée par LntA. BAHD1 demeurant peu étudiée, mon doctorat a eu pour premier objectif de poursuivre la caractérisation de ce régulateur épigénétique. Par ailleurs, des données préliminaires suggéraient qu’un facteur de virulence de Listeria récemment découvert, InlP, avait la potentialité d’être, comme LntA, une nucléomoduline. Mon deuxième objectif a été d’explorer cette hypothèse.Les résultats de mon premier axe montrent que BAHD1 interagit avec MIER1 et que cette interaction est cruciale pour l’association de BAHD1 aux HDAC1/2. Nous reportons également que BAHD1 modifie la chromatine en changement la méthylation et l’acétylation des histones, ainsi que la méthylation de l’ADN, au niveau d’un gène cible, ESR1. Ces résultats nous permettent de proposer que BAHD1 forme, avec MIER1, un échafaudage assemblant un nouveau complexe de remodelage de la chromatine associé aux HDAC1/2 : le complexe BAHD1. Nous avons ensuite étudié un rôle de BAHD1 dans un organe ciblé par la Listeria, le cerveau. Nos résultats indiquent qu’une déficience totale en BAHD1 altère le transcriptome global de cet organe chez la souris. Les gènes majoritairement surexprimés sont impliqués dans des fonctions du système nerveux, le métabolisme et des troubles neurologiques. Les gènes majoritairement sous-exprimés sont impliqués dans des voies de l’immunité innée, dont des gènes de réponses aux interférons. Par ailleurs, une haplo-déficience en Bahd1 provoque des troubles comportementaux. En comparaison des souris Bahd1+/+, les souris Bahd1+/- souffrent d’une anxiété accrue et d’altérations du réflex de sursaut acoustique. Ces résultats suggèrent qu’une dérégulation de BAHD1, par des stimuli de l’environnement ou par des stimuli infectieux, pourrait avoir des effets neuro-pathologiques.Le second axe de ma thèse concernait l’étude des interactions d’InlP avec des protéines nucléaires de l’hôte, identifiées par un crible double-hybride. Nous montrons d’abord qu’InlP est une internaline atypique, avec des répétitions riches en leucine caractérisées par un motif LPX2. Nous identifions, ensuite, deux protéines nucléaires ciblées par InlP : le facteur d’épissage et suppresseur de tumeur RBM5 et le corépresseur RERE. Quand InlP est produite de façon ectopique dans les cellules humaines, elle se localise dans le noyau, où elle altère la formation de corps nucléaires enrichis en RERE. Dans des cellules sur-exprimant RBM5, InlP inhibe l’effet pro-apoptotique de RBM5 et stimule la formation de corps nucléaires denses associés à RBM5. Ces résultats suggèrent qu’InlP est une nucléomoduline agissant sur la l’assemblage et le désassemblage de compartiments de stockage de protéines cibles impliquées dans la synthèse et l’épissage d’ARNs de l’hôte.Ce travail ouvrent des perspectives dans la compréhension des interactions hôte-pathogène et dans une meilleure connaissance des mécanismes patho-épigénétiques, ainsi qu’en biologie cellulaire, dans la compréhension de la dynamique des organites nucléaires sans membrane. / Listeria monocytogenes is an optional intracellular pathogen responsible for a severe foodborne infection called listeriosis. The study of the cellular infection process of this bacterium has shed light on various mechanisms involved in host-pathogen interactions and in the functioning of the eukaryotic cell. In particular, L. monocytogenes has emerged as one of the pioneering models in the discovery of microbial targeting of chromatin and nuclear regulators. The study of a virulence factor of L. monocytogenes, LntA, allowed the identification of one of these regulators : BAHD1. By recruiting proteins involved in the formation of heterochromatin, such as HDAC1/2 and HP1, BAHD1 stimulates the formation of a compact chromatin with a repressive effect. When epithelial cells are infected with L. monocytogenes, BAHD1 suppresses the immune response stimulated by interferons, a function inhibited by LntA. Since BAHD1 is still under-researched, the first objective for my thesis was to further characterize this epigenetic regulator. In addition, preliminary data suggested that a recently discovered virulence factor of Listeria, InlP, had the potential to be, like LntA, a nucleomodulin. My second objective was to explore this hypothesis.The results of my first axis show that BAHD1 interacts with MIER1 and that this interaction is crucial for the association of BAHD1 with HDAC1/2. We also report that BAHD1 modifies chromatin by changing histone methylation and acetylation, as well as DNA methylation, at a target gene, ESR1. These results allow us to propose that BAHD1 form, with MIER1, a scaffold assembling a new chromatin remodeling complex associated with HDAC1/2 : the BAHD1 complex. We then studied the role of BAHD1 in an organ targeted by Listeria, the brain. Our results indicate that a total deficiency in BAHD1 alters the overall transcriptome of this organ in mice. Most of the overexpressed genes are involved in nervous system functions, metabolism and neurological disorders. The predominantly downregulated genes are involved in innate immunity pathways, including interferon response genes. In addition, a haplodeficiency in Bahd1 causes behavioral problems. Compared to Bahd1+/+ mice, Bahd1+/- mice suffer from increased anxiety and changes in acoustic startle reflex. These results suggest that deregulation of BAHD1, through environmental or infectious stimuli, may have neuro-pathological effects.The second axis of my thesis focused on the study of InlP interactions with host nuclear proteins, identified by a double-hybrid screen. First, we show that InlP is an atypical internalin, with leucine-rich repeats characterized by an LPX2 motif. We then identify two nuclear proteins targeted by InlP: the splicing factor and tumor suppressor RBM5 and the corepressor RERE. When InlP is produced ectopically in human cells, it is localized in the nucleus, where it alters the formation of nuclear bodies enriched in RERE. In RBM5-overexpressing cells, InlP inhibits the pro-apoptotic effect of RBM5 and stimulates the formation of dense nuclear bodies associated with RBM5. These results suggest that InlP is a nucleomodulin acting on the assembly and disassembly of target protein storage compartments involved in the synthesis and splicing of host RNAs.This work opens perspectives in the understanding of host-pathogen interactions and in a better knowledge of patho-epigenetic mechanisms, as well as in cell biology and the understanding of membraneless nuclear organelles dynamics.

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New technologies have greatly shaped the scientific and medical landscape within the last years. The unprecedented expansion of data and information on RNA biology has led to the discovery of new RNA classes with unique functions and unexpected modifications. Today, the biggest challenge is to transfer the large number of findings in basic RNA biology into corresponding clinical RNA-based therapeutics. Lately, this research begins to yield positive outcomes. RNA drugs advance to the final phases of clinical trials or even receive FDA approval. Furthermore, the introduction of the RNA-guided gene-editing technology CRISPR and advances in the delivery ofmessenger RNAs have triggered a major progression in the field of RNA-therapeutics. Especially short interfering RNAs and antisense oligonucleotides are promising examples for novel categories of therapeutics. However, several issues need to be addressed including intracellular delivery, toxicity, and immune responses before utilizing RNAs in a clinical setting. In this review, we provide an overview on opportunities and challenges for clinical translation of RNA-based therapeutics, with an emphasis on advances in novel delivery technologies and abdominal aortic aneurysm disease where non-coding RNAs have been shown to play a crucial regulatory role.

During their lifetime, Black/African Americans have a higher likelihood of developing the diabetes mellitus metabolic disorder than other racial and ethnic groups in the United States. While research indicates that socioeconomic status, diet, and obesity factor into race disparities, the epigenetics field additionally identifies historical and contemporary racism as contributors to race disparities. This study is a qualitative analysis that examines a sample of health science research articles that use an epigenetics approach to understand diabetes among Black/African Americans. I analyzed the extent and mechanisms through which articles subtly reproduce dominant stereotypes of Black/African Americans and diabetes through representations of culture, diet, and sugar consumption, among other factors. Moreover, our analysis shows how these articles recreate new scripts that view biological differences as a product of historical and ongoing racism. The result of this analysis indicates three categories: 1) presentations of race as a social construction and racism as a cause of biological outcomes; 2) utilization of cultural perspectives that reify racial categories and point to social environments within households/neighborhoods and diet as a cause; 3) advocating for a multidisciplinary approach in medicine to foster collaborative change within minority communities. These results further emphasize the importance for sociologists and other research scholars to become more educated in the field of epigenetics. Furthermore, by becoming more educated on epigenetics, this can allow sociologists to further contribute to the field.

Objective: Obesity is driven by modifiable lifestyle factors whose effects may be mediated by epigenetics. Therefore, we investigated lifestyle effects on blood DNA methylation in participants of the LIFE-Adult study, a well-characterised population-based cohort from Germany. Research design and methods: Lifestyle scores (LS) based on diet, physical activity, smoking and alcohol intake were calculated in 4107 participants of the LIFE-Adult study. Fifty subjects with an extremely healthy lifestyle and 50 with an extremely unhealthy lifestyle (5th and 95th percentiles LS) were selected for genome-wide DNA methylation analysis in blood samples employing Illumina Infinium⃝R Methylation EPIC BeadChip system technology. Results: Differences in DNA methylation patterns between body mass index groups (<25 vs. >30 kg/m2) were rather marginal compared to inter-lifestyle dif- ferences (0 vs. 145 differentially methylated positions [DMPs]), which identified 4682 differentially methylated regions (DMRs; false discovery rate [FDR <5%) annotated to 4426 unique genes. A DMR annotated to the glutamine-fructose-6- phosphate transaminase 2 (GFPT2) locus showed the strongest hypomethylation (∼6.9%), and one annotated to glutamate rich 1 (ERICH1) showed the strongest hypermethylation (∼5.4%) in healthy compared to unhealthy lifestyle individu- als. Intersection analysis showed that diet, physical activity, smoking and alcohol intake equally contributed to the observed differences, which affected, among others, pathways related to glutamatergic synapses (adj. p < .01) and axon guid- ance (adj. p < .05). We showed that methylation age correlates with chronological age and waist-to-hip ratio with lower DNA methylation age (DNAmAge) acceler- ation distances in participants with healthy lifestyles. Finally, two identified top DMPs for the alanyl aminopeptidase (ANPEP) locus also showed the strongest expression quantitative trait methylation in blood. Conclusions: DNA methylation patterns help discriminate individuals with a healthy versus unhealthy lifestyle, which may mask subtle methylation differ- ences derived from obesity.

Epigenetic regulation of gene expression occurs at a number of levels including covalent DNA and histone modifications, nucleosome positioning and ATP-dependent chromatin remodelling as well as higher order chromatin folding and 3D genome organisation. ATP-dependent chromatin remodelling complexes modulate nucleosome structure, positioning and chromatin de-compaction and are involved in gene activation and repression. SWI/SNF ATP-dependent chromatin remodelling complexes contain either BRG1 or BRM as the core ATPase together with other common and variable subunits. BRG1 is required for terminal epidermal differentiation in mice and humans and for hair follicle stem cell activation during mouse hair follicle regeneration and cutaneous wound healing. However, the role of SWI/SNF complexes in human hair growth and wound healing remain unknown. Here it is demonstrated that genes encoding SWI/SNF complex subunits are expressed in human hair follicles. It also highlights that siRNA mediated suppression of SWI/SNF complexes in hair follicle culture has no effect on hair growth, or anagen-catagen transition in the short term, but a significant increase in proliferation of the outer root sheath keratinocytes was seen. The data also documents the expression of several SWI/SNF subunits in human epidermis and that siRNA mediated SMARCA4 gene suppression in primary human keratinocyte monolayers defined the requirements of BRG1 for wound closure through control of cell migration, but not proliferation. In summary, this data revealed a diverse SWI/SNF complex subunit composition in human epidermis and hair follicle, and an essential role of the core complex ATPase BRG1 in keratinocyte migration during wound closure and re-epithelisation.

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<p dir="ltr">Epigenetics involves the study of mechanisms that influence gene expression. These mechanisms are heritable and dynamic, and despite altering gene transcriptional activity, they do not change the underlying DNA sequence. While epigenetic mechanisms govern gene expression throughout the lifetime of an organism, the dynamic nature and precision of the transcriptional control afforded by processes such as histone modifications and chromatin architecture remodeling are exemplified in early mammalian development. Perhaps unsurprisingly, perturbations to the epigenetic status of a cell can alter its function, and widespread epigenome disruptions due to changes in the developmental environment can compromise the growth, and even viability, of an embryo or fetus. By studying epigenetic mechanisms and the patterns they impart, we can better understand not only how developmental progression is regulated during embryonic development and beyond, but also what the consequences of aberrant epigenetic disturbances may be to developing organisms.</p><p><br></p><p dir="ltr">Many gaps remain in our knowledge concerning epigenetic mechanisms in domestic livestock species, particularly regarding early development. Pigs represent a compelling model organism for study in this area, as they are increasingly being used as a biomedical model for human-oriented research due to their physiological similarities to humans, and they remain a staple meat species in many parts of the world. Chapter 2 investigates the presence and transcriptional dynamics of the SWI/SNF chromatin remodeling complex GBAF in porcine trophectoderm (PTr2) and fetal fibroblast (PFF) cells. These cell lines represent two discrete developmental stages during early swine development, with the PTr2 cells originally obtained from the trophectoderm of a gestation day 12 elongating porcine conceptus, and the fetal fibroblast cells were collected from a fetus on day 40 of gestation. Using immunocytochemistry and Western blotting techniques, GBAF was identified in both cell lines. Further, co-immunoprecipitation of GBAF constituent subunits and other BAF family subcomplex subunits revealed a previously undescribed interaction between the GBAF subunit GLTSCR1 and the BAF subunit BAF170, the latter of which has not been shown to be present in human and mouse GBAF. This may suggest a species-specific GBAF composition in swine. Analysis of RNA-seq data from porcine embryos, PTr2 cells, and PFF cells showed that while transcription of GBAF-specific subunits BRD9 and GLTSCR1 was detectable, expression levels were lower compared to other BAF family subunits. Taken together these results suggest that, while GBAF is detectable in swine early development contexts, it may have a comparatively minor contribution as an epigenetic mechanism during the represented developmental timepoints.</p><p><br></p><p dir="ltr">Details in the literature about the epigenetic landscape and the resulting chromatin state during porcine early development are also limited at present. Chapter 3 involves the global epigenetic profiling of the histone marks H3K4me3, H3K27ac, and H3K27me3 and the SWI/SNF central ATPase BRG1 in PTr2 and PFF cells using CUT&RUN. The enrichment patterns observed for these features were consistent with known patterns described in the literature. H3K4me3 was primarily enriched in gene promoter regions, and H3K27ac showed enrichment in both promoter regions and distal intergenic regions, some of which are likely active enhancers. H3K27me3 showed broad genomic localization and was detected at genes known to be transcriptionally inactive in these cell types, as well as in distal intergenic regions. BRG1 showed some co-enrichment with H3K4me3 and H3K27ac in promoter regions, as well as several instances of H3K27ac co-enrichment at intergenic sites. The sequencing files were used to build a chromatin state prediction model for 10 states in each cell line, ranging from TSS to repressed genomic regions. Additionally, the transcriptomes of PTr2 and PFF cells were compared to those of human cells taken from comparable gestational time points to determine if these swine cell lines could potentially serve as translational <i>in vitro</i> models. PTr2 cells and human trophectoderm (TE) cells were relatively dissimilar in their cell-type specific gene identities (~24% overlap) and corresponding transcriptional levels, but the porcine and human fibroblast cells shared around 50% of the same cell type-specific genes, and expression levels were broadly similar among them. Altogether, these findings provide foundational epigenetic landscape information for PTr2 and PFF cells and potential insights regarding similarities and differences in cell identity between human and pig trophectoderm and fetal fibroblast cells.</p><p><br></p><p dir="ltr">The placenta is a transient organ that provides essential support to the developing fetus in the form of nutrient and gas exchange. Despite its significance in facilitating fetal development, our understanding of how the placenta is affected by its environment is greatly limited, and only a handful of studies exploring the placental epigenome in swine exist to date. To address these gaps, and building upon the epigenetic profiling methods developed in Chapter 3, Chapter 4 investigated whether, and to what extent, the placental epigenome changes in response to fetal endocrine perturbations. Placental tissue was collected at day 86 of gestation from untreated pregnant gilts and pregnant gilts treated for 21 days with methimazole (MMI) to induce fetal hypothyroidism. CUT&RUN was used to evaluate the enrichment of H3K4me3, H3K27ac, and BRG1 in placental tissue derived from n=6 male and female fetuses in each treatment group (n=12 samples per group). Differential enrichment of all three epigenetic features was seen in placental tissues obtained from MMI-treated fetuses, and, notably, existing sex-specific differences in placental epigenetic features were exacerbated by MMI-induced fetal hypothyroidism. This may suggest that the porcine placenta may be impacted by fetal endocrine status during late gestation. Together, these findings show that sex-specific differences in placental chromatin state exist and that a fetal hypothyroid state is sufficient to perturb the placental epigenome, ultimately providing novel insights into the intricate interplay between fetal endocrine status and regulation of the placental epigenome.</p>