Thesis advisor: Gabor Marth / While a PhD student in Dr. Gabor Marth's laboratory, I have had primary responsibility for two projects focused on using RNA-Seq to measure differential gene expression. In the first project we used RNA-Seq to identify differentially expressed genes in four yeast species and I analyzed the findings in terms of the evolution of gene expression. In this experiment, gene expression was measured using two biological replicates of each species of yeast. While we had several interesting biological findings, during the analysis we dealt with several statistical issues that were caused by the experiment's low number of replicates. The cost of sequencing has decreased rapidly since this experiment's design and many of these statistical issues can now practically be avoided by sequencing a greater number of samples. However, there is little guidance in the literature as to how to intelligently design an RNA-Seq experiment in terms of the number of replicates that are required and how deeply each replicate must be sequenced. My second project, therefore, was to develop Scotty, a web-based program that allows users to perform power analysis for RNA-Seq experiments. The yeast project resulted in a highly accessed first author publication in BMC Genomics in 2011. I have structured my dissertation as follows: The first chapter, entitled General Issues in RNA-Seq, is intended to synthesize the themes and issues of RNA-Seq statistical analysis that were common to both papers. In this section, I have discussed the main findings from the two papers as they relate to analyzing RNA-Seq data. Like the Scotty application, this section is designed to be "used" by wet-lab biologists who have a limited background in statistics. While some background in statistics would be required to fully understand the following chapters, the essence of this background can be gained by reading this first chapter. The second and third chapters contain the two papers that resulted from the two RNA-Seq projects. Each chapter contains both the original manuscript and original supplementary methods and data section. Finally, I include brief summaries of my contributions to the two papers on which I was a middle author. The first was a functional analysis of the genomic regions affected by mobile element insertions as a part of Chip Stewart's paper with the 1000 Genome Consortium. This paper was published in Plos Genetics. The second was a cluster analysis of microarray gene expression in Toxoplasma gondii, which was included as part of Alexander Lorestani et al.'s paper, Targeted proteomic dissection of Toxoplasma cytoskeleton sub-compartments using MORN1. This paper is currently under review. The yeast project was a collaborative effort between Jesse Gray, Michael Springer, and Allen Costa at Harvard Medical School, Jeffery Chuang here at Boston College, and members of the Marth lab. Jesse Gray conceived of the project. While I wrote the draft for the manuscript, many people, particularly Gabor Marth, provided substantial guidance on the actual text. I conceived of and implemented Scotty and wrote its manuscript with only editorial assistance from my co-authors. I produced all figures for the two manuscripts. Chip Stewart provided extensive guidance and mentorship to me on all aspects of my statistical analyses for both projects. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A sinalização celular desencadeada na presença do hormônio andrógeno em células da próstata é dependente da ativação do receptor de andrógeno (AR), que é um fator de transcrição codificado pelo gene NR3C4. O AR quando não ligado ao hormônio andrógeno é encontrado no citoplasma, e a ligação do hormônio ao AR promove seu deslocamento para o núcleo. O AR se liga a motivos de DNA, promovendo a transcrição de determinados genes por meio de um complexo mecanismo de regulação ainda não totalmente conhecido. Neste trabalho exploramos a capacidade dos RNAs longos intergênicos não-codificadores (lincRNAs) se ligarem ao AR e contribuírem para a regulação transcricional de genes. Utilizamos a imunoprecipitação de RNA, seguida de sequenciamento em larga escala (RIP-Seq) para a identificação e quantificação de lincRNAs associados fisicamente ao AR na linhagem celular de próstata LNCaP. Em paralelo utilizamos dados de transcriptoma e de marcas epigenéticas para verificar se a interação física dos lincRNAs com o AR influenciaria a composição da cromatina e a expressão dos genes vizinhos desses ARA-lincRNAs (Androgen Receptor Associated LincRNAs). Como resultado deste estudo descrevemos pela primeira vez centenas de LincRNAs associados fisicamente ao receptor de andrógeno após o tratamento com o hormônio. Observamos que uma parte desses ARA-lincRNAs pode modificar in cis a expressão de genes codificadores de proteínas vizinhos e essa capacidade de regulação é reforçada pela presença de marcas epigenéticas que são características de reguladores in cis. Além disso, mostramos que as regiões da cromatina contendo domínios topologicamente associativos (TADs) que possuem ARA-lincRNAs apresentam fatores de transcrição, marcas epigenéticas e nível de transcrição gênica diferenciadas. Os resultados apresentados neste trabalho estendem a importância dos lincRNAs durante eventos regulatórios complexos e mostra pela primeira a atuação dessas moléculas em sinergia com um fator de transcrição modificando a cromatina e alterando a regulação gênica. / The cell signaling events triggered in the presence of the androgen hormone in prostate cells is dependent on the activation of the androgen receptor (AR), which is a transcription factor encoded by the NR3C4 gene. AR when not bound to the androgen hormone is found in the cytoplasm, and binding of the hormone to AR promotes its displacement to the nucleus. AR binds to DNA motifs, promoting the transcription of certain genes through a complex regulatory mechanism not yet fully undestood. In this work we explore the ability of long non-coding intergenic RNAs (lincRNAs) to bind to AR and contribute to the transcriptional regulation of genes. We used immunoprecipitation of RNA, followed by large-scale sequencing (RIP-Seq) for the identification and quantification of lincRNAs physically associated with AR in the LNCaP prostate cell line. In parallel we used transcriptome data and data on epigenetic marks to verify whether the physical interaction of lincRNAs with AR would influence the chromatin composition and the expression of genes neighboring these ARA-lincRNAs (Androgen Receptor Associated LincRNAs). As a result of this study we described for the first time in the literature hundreds of LincRNAs physically associated with the androgen receptor after treatment with the hormone. We have observed that part of these ARA-lincRNAs can modify in cis the expression of neighboring protein coding genes and this regulatory ability is enhanced by the presence of epigenetic marks that are characteristic of in cis regulators. In addition, we have shown that chromatin regions containing topologically associating domains (TADs) that possess ARA-lincRNAs have different transcription factors, epigenetic marks and gene transcription levels. The results presented in this work extend the importance of the lincRNAs during complex regulatory events and shows for the first time the performance of these molecules in synergy with a transcription factor modifying the chromatin and altering gene regulation.

Chez les eucaryotes, l’ADN est empaqueté dans des complexes protéiques d’histones nommés nucléosomes qui assurent sa conformation. Cet arrangement est hétérogène à travers le génome et peut être dynamiquement modifié. La régulation de l’architecture chromatinienne joue un rôle essentiel dans la stabilité des génomes ainsi que la dynamique transcriptionnelle. Certaines régions qualifiées d’ ‘’heterochromatine constitutive’’ sont toutefois connues pour être maintenues à l’état condensé. Régionalisées aux extrémités et centres des chromosomes, l’hétérochromatine constitutive participe des fonctions télomériques et centromériques.Spodoptera frugiperda (S.fru, Lépidoptère, Noctuelle) est un ravageur de culture endémique du continent américain, récemment invasif dans le continent africain. Comme tous les Lépidoptères, S.fru est une espèce holocentrique dont le centromère est réparti le long des chromosomes et non restreint en un point unique. Cette disposition interroge sur l’établissement, la distribution ainsi que la fonction conservée de l’HC puisque cette dernière est principalement décrite pour être majoritairement localisée dans de larges régions péricentriques. Comprendre l’architecture chromatinienne chez S.fru peut avoir un intérêt en lutte biologique mais également permettre d’approfondir les connaissances en épigénétique chez un organisme non-modèle.Dans le cadre de la thèse, nous nous sommes demandés si la diméthylation de la lysine 9 de l’histone 3 (H3K9me2), marqueur de l’hétérochromatine constitutive, possédait un rôle conservé chez S.fru. Pour ce faire, nous avons comparé des données de ChIP-seq d’H3K9me2 sur cellules et larves entières après avoir annoté les gènes et l’ensemble des éléments répétés du génome, susceptibles d’être enrichis par cette marque. Parallèlement, des échantillons d’ARN-seq ont été étudiés afin de questionner le statut répressif de l’hétérochromatine constitutive. Nos résultats suggèrent un invariable maintien d’H3K9me2 dans les régions (sub)télomériques transcriptionnellement inactives ainsi qu’une forte association aux locus répétés d'ADN ribosomal (rDNA). Ces séquences ne constituent toutefois qu’une minorité des régions enrichies, le reste étant retrouvé dans des séquences répétées ainsi que dans le corps des gènes, indifféremment de leur état transcriptionnel. La persistante association d’H3K9me2 aux télomères et rDNA présagerait d’un maintien de la marque à proximité des centromères dont nous proposons un modèle d’établissement.La disposition de l’hétérochromatine constitutive questionne celle des régions euchromatiniennes, pauvres en nucléosomes, transcriptionnellement active et dynamiquement modifiées au cours du développement, du cycle cellulaire et des conditions environnementales. Afin de tester l’antagonisme de ces conformations, nous avons respectivement étudié la répartition des zones ouvertes et fermées du génome de la larve au stade L4 par approches de FAIRE-seq et de MAINE-seq. Ces structures ont été décrites dans la littérature pour être enrichies par de spécifiques modifications d’histones. Ainsi nous avons mis au point le protocole de native ChIP-seq d’H3K4me3 (marque active) et H3K9me2, H3K9me3, H3K27me3 (marques répressives). L’analyse en cours de l’ensemble de ces données de séquençages permettra d’avoir une vue intégrée de l’architecture chromatinienne au stade ravageur. / In eukaryotes, DNA is arranged in histones proteins complexes called nucleosomes that shape its conformation. This arrangement is heterogeneous across genomes and can be dynamically modified. Regulation of chromatin architecture plays an essential role in genome stability and transcription dynamics. Some regions named ‘’constitutive heterochromatin’’ are nonetheless known to remain highly condensed, regardless of conditions. Regionalized at extremities and chromosomes centers, constitutive heterochromatin contributes to telomeric and centromeric functions.Spodoptera frugiperda (S.fru, Lepidoptera, Noctuidae) is major crop pest in the Americas that recently invaded Africa. Like all Lepidopteran, S.fru is holocentric which means that its centromere is spread along chromosome and not restricted to a uniq point. This disposition question about establishment, distribution but also conserved function of constitutive heterochromatin since its usually and mainly localized in large pericentric regions.Deciphering chromatinian architecture in S.fru can be of interest in biological control but also allow to deepen epigenetic knowledge in a non-model organism.During my phD, we questionned the role of histone 3 lysine 9 demethylated (H3K9me2) in S.fru, a histone modification known in other yet described organisms to be a constitutive constitutive heterochromatinian hallmark.We compared H3K9me2 ChIP-seq data on cells and larvae after overall genomic functional annotation, potentially enriched for this mark. In parallel, RNA-seq samples were analyzed to question the putative repressive status of constitutive heterochromatin.Our results suggest an invariant retention of H3K9me2 in (sub)telomeric regions transcriptionally inactive but also a strong association of this mark in repeated ribosomal DNA locus (rDNA).These sequences constitutes nonetheless a minority of enriched regions since most of them regionalize in repeated sequences like transposons and tandem array but also gene bodies, independently of their transcriptional states.Persistent H3K9me2 association to telomeres and rDNA could predict of the conserved expression of this mark near centromeres. Based on literature and bioinformatics analysis, we proposed a model for S.fru holocentromeres.Constitutive heterochromatin questions euchromatin arrangement, described to be nucleosome poor, transcriptionally active and dynamically modified across development, cell cycle and environmental conditions. In order to test these structural antagonisms, we respectively studied open and closed genome conformations by FAIRE-seq and MAINE in larvae. These structures are reported to be associated to specific histones marks. We developed a native ChIP-seq protocol on H3K4me3 (active mark) and H3K9me2, H3K9me3, H3K27me3 (repressives marks). Overall analysis of these NGS data would help to picture an integrative view of chromatin architecture during larval pest stage.

Espécies muito próximas da família Calliphoridae apresentam hábitos alimentares muito distintos, como alimentação em tecido de um hospedeiro vivo (parasitismo obrigatório) e alimentação em matéria orgânica em decomposição (necro-saprofagia). As origens evolu-tivas do hábito de parasitismo nesta família ainda são desconhecidas. No entanto, o que a torna ideal para o estudo da evolução do hábito alimentar é o aparecimento do parasitis-mo obrigatório em pelo menos três ocasiões independentes em sua história. Neste traba-lho, foram utilizados métodos para entender a evolução do parasitismo obrigatório assim como os genes envolvidos em três diferentes hábitos alimentares. O primeiro passo foi a inferência do hábito ancestral de Calliphoridae. Com o mapeamento de caracteres na filo-genia da família, o hábito ancestral mais provável seria a necro-saprofagia e o parasitismo obrigatório teria surgido posteriormente (independentemente do parasitismo facultati-vo). Ensaios de escolha com fêmeas permitiram a classificação precisa das espécies quan-to ao hábito alimentar. Ensaios com as larvas mostraram que tanto espécies necro-saprófagas como parasitas facultativas se alimentam tanto de carne em decomposição como carne fresca. Por outro lado, a espécie parasita obrigatória Co. hominivorax, apresentou um comportamento aversivo pela carne em decomposição e se desenvolveu somente na carne fresca. Esses resultados permitiram a formulação da hipótese de que o parasitismo tenha surgido a partir de uma mudança da atração das fêmeas pelos substra-tos para oviposição, seguida da especialização da larva parasita. A busca dos genes envol-vidos nos diferentes hábitos foi realizada por meio da análise de expressão gênica dife-rencial em dados de RNA-seq gerados para seis califorídeos. No total, foram encontrados 230 potenciais genes candidatos para investigação futura. Além disso, o padrão geral observado indicou que variações tanto nas regiões regulatórias como codificadoras, sofrem a ação predominante de seleção purificadora / Closely related species of the Calliphoridae family have contrasting feeding habits, such as feeding on living tissues of a host (obligate parasitism) and feeding on decaying organ-ic matter (necro-saprophagy). The evolutionary origins of parasitism in Calliphoridae are still unknown. However, what makes this family ideal for the study of the evolution of feeding habits is the appearance of obligate parasitism in at least three independent oc-casions. In this study, we used methods to understand the evolution of parasitism, as well as the genes involved in in three different feeding habits. First, we inferred the ancestral habit of Calliphoridae. By using stochastic character mapping along the phylogeny of the family, the most likely ancestral habit was revealed as necro-saprophagy. Obligate para-sitism could have evolved later (with an independent evolution of the facultative parasit-ism). Two-choice essays with females allowed the precise classification of the species regarding their feeding habits. Essays with larvae showed that both necro-saprophagous and facultative parasites feed on decaying flesh and fresh meat. On the other hand, the obligate parasite, Co. hominivorax, showed an aversive behavior to decaying meat and able to develop only in fresh meat. These results led to the formulation of the hy-pothesis that parasitism arose from a shift in the attraction of the female attraction to new oviposition sites, followed by the specialization of the parasitic larvae. The search for the genes involved in the different feeding habits was performed through an analysis of differential gene expression using RNA-seq data generated for six califorids. Within a da-taset containing more than 1000 candidate genes, 230 genes potential candidate genes were found for future research. In addition, the general pattern observed indicated that both regulatory and coding regions have predominantly undergone the action of purifying selection

Neste estudo foi implementado um pipeline bioinformático para processar e analisar dados de RNA-Seq total e fita-específico gerados em nosso laboratório a partir de amostras pareadas de tumor e tecido adjacente não tumoral de 14 pacientes com o objetivo de catalogar com alta-resolução a composição e alterações no transcritoma no PDAC incluindo genes codificadores e não codificadores de proteína. / In the present work, we applied a bioinformatic pipeline to process and analyse data from total RNA-seq strand-oriented generated in our laboratory from matched samples of tumor and non-tumor adjacent pancreatic tissue from 14 patients with the goal of generate a high resolution catalog of the composition and the alterations in the transcriptome of PDAC, including protein coding and non coding genes.

AN ABSTRACT OF THE DISSERTATION OF SOLOMON G. HAILU, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry, presented on August 22, 2017, at Southern Illinois University, School of Medicine. TITLE: DISTINCT GENOME WIDE FUNCTIONS OF CHROMATIN REMODELERS IN NUCLEOSOME ORGANIZATION AND TRANSCRIPTION REGULATION MAJOR PROFESSOR: Dr. Blaine Bartholomew Chromatin remodelers are conserved from yeast to humans and are the gatekeepers of chromatin. They regulate transcription by occluding or exposing DNA regulatory elements globally. They are crucial for DNA processes such as DNA replication, repair and recombination. In addition, they are critical in developmental processes and differentiation. Chromatin remodelers are categorized into several families based on their conserved ATPase domain, an essential component required for their DNA translocation ability. In this study, we investigated the role yeast ISWI and SWI/SNF family of chromatin remodelers play on nucleosome rearrangement and transcription regulation by targeted mutagenesis of domains in accessory subunits and at the C-terminus of the catalytic subunit. All members of the ISWI family (ISW1a, ISW1b, ISW2) share a conserved C-terminal HAND, SANT and SLIDE domains, which are important for sensing linker DNA. We find an auto-regulation of ISWI complexes by the SLIDE domain, independent of the histone H4 Nterminal tail. Our protein-protein chemical crosslinking and mass spectrometry (CX-MS) analysis indicate that the SLIDE domain regulates the ATPase core through N terminal domains of the accessory subunit Itc1. Moreover, we show that the accessory subunits of ISWI modulate the ATPase activity and specificity of ISWI complexes. The DNA sensing ability of the SLIDE domain is required for the in vivo nucleosome spacing and transcription regulation by ISWI. We find that while ISW2 primarily regulates transcription at the 5’ end of genes, ISW1a is important in transcription elongation by rearranging nucleosomes starting at the +2 nucleosome and through the rest of the body of genes towards the 3’ end. ISW1b on the other hand rearrange nucleosomes in the gene body to facilitate suppression of cryptic transcription. For the first time, we show the potential division of labor between ISW1a and ISW1b during transcription elongation. On the other hand, SWI/SNF chromatin remodelers are essential epigenetic factors that are frequently mutated in cancer and neurological disorders. They harbor a C-terminal SnAC and AT hook domains that positively regulate their DNA dependent ATPase activity and nucleosome mobilizing capabilities. By deleting the AT hook motifs, we have identified the role of SWI/SNF in organizing the -1 and +1 nucleosomes at transcription start sites flanking the nucleosome free region (NFR). Our RNA-seq analysis shows SWI/SNF positively regulates the bi-directional transcription of non-coding RNA (ncRNA) which are activated when the AT hook motifs are deleted. Moreover, AT hooks regulate such activities of SWI/SNF through direct protein-protein interactions with the ATPase core as evidenced by our chemical crosslinking and mass spectrometry (CX-MS) analysis.

Sex differences in mammalian gene expression result from differences in genotypic sex as well as in hormonal regulators between males and females. In rat, mouse and human liver, ~1000 genes are expressed in a sex-dependent manner, and contribute to sex differences in metabolism of drugs, steroids and lipids, and in liver and cardiovascular disease risk. In rats and mice, sex-biased liver gene expression is primarily dictated by the sexually dimorphic pattern of pituitary growth hormone (GH) release and its STAT5-dependent transcriptional activities. Studies presented in this thesis include the following. (1) A computational approach based on DNA sequence and phylogenetic conservation was developed and used to identify novel functional STAT5 binding sites - both consensus and non-consensus STAT5 sequences - near prototypic GH-responsive genes. (2) Global gene expression analysis of livers from pituitary-ablated male and female mice identified four major classes of sex-biased genes differing in their profiles of GH dependence. (3) Sex-differences in DNase-hypersensitive sites (DHS, corresponding to open chromatin regions) were identified genome-wide in mouse liver. These sex-differential DHSs were enriched for association with sex-biased genes, but a majority was distant from sex-biased genes. Furthermore, many were responsive to GH treatment, demonstrating that GH-mediated regulation involves chromatin remodeling. Analysis of sequence motifs enriched at sex-biased DHSs implicated STAT5 and novel transcription factors such as PBX1 and TAL1 in sex-biased gene regulation. (4) Genome-wide mapping of histone modifications revealed distinct mechanisms of sex-biased gene regulation in male and female liver: sex-dependent K27me3-mediated repression is an important mechanism of repression of female-biased, but not of male-biased, genes, and a sex-dependent K4me1 distribution, suggesting nucleosome repositioning by pioneer factors, is observed at male-biased, but not female-biased, regulatory sites. STAT5-mediated activation was most strongly associated with sex-biased chromatin modifications, while BCL6-mediated repression primarily occurs in association with sex-independent chromatin modifications, both at binding sites and at target genes. The relationships between sex-dependent chromatin accessibility, chromatin modifications and transcription-factor binding uncovered by these studies help elucidate the molecular mechanisms governing sex-differential gene expression, and underscore the utility of functional genomic and epigenetic studies as tools for elucidating transcriptional regulation in complex mammalian systems.

La voie Wnt/β-caténine est impliquée dans la prolifération et le contrôle du destin cellulaire de nombreux tissus à la fois au cours du développement et pour le maintien de l’homéostasie chez l’adulte. L’équipe a montré qu’une activation aberrante de la β-caténine conduisait au développement de carcinomes hépatocellulaires (CHC), alors que son activation physiologique dans une souspopulation d'hépatocytes du foie adulte (les hépatocytes éricentraux) permet la mise en place et le maintien du zonage métabolique. Ce zonage hépatique peut être considéré comme une différenciation terminale des hépatocytes, et permet au foie d'être pleinement fonctionnel. L’objet de ma thèse a été d’identifier les déterminants moléculaires expliquant la diversité d’action de la β-caténine dans le foie. J'ai tiré profit de modèles génétiques développés au laboratoire d'activation ou d'inactivation inductibles de la voie β-caténine dans le foie. A partir d'hépatocytes isolés de ces modèles, j'ai recherché par ChIp-seq (immunoprécipitation de chromatine couplée à un séquençage haut-débit) les sites de fixation sur l’ADN de la β-caténine et de Tcf4, que j'ai identifié comme principal médiateur de l’activité nucléaire de la β-caténine dans le foie. Ces données, couplées à des études d’accessibilité de la chromatine et de transcriptome ont permis d’identifier la structure de la chromatine et Hnf4a comme déterminants majeurs permettant à la beta-caténine de contrôlerpositivement et négativement des programmes génétiques spécifiquement hépatiques, qui luipermettent d'assurer le zonage métabolique du foie. En effet, Hnf4a a un rôle antagoniste de la β-caténine, contrôlant positivement la transcription des gènes réprimés par la β-caténine. Ce contrôles'exerce grâce à un dialogue d'Hnf4a avec Tcf4 et la β-caténine, qui s'opère majoritairement via des interactions entre ces protéines. Nous proposons ainsi que la β-caténine coopère avec le facteur de transcription Hnf4a, connu pour contrôler les gènes du métabolisme hépatocytaire, pour assurer la différenciation terminale hépatique. J'ai également recherché les partenaires protéiques nucléaires de la β-caténine par coimmunoprécipiatation couplée à de la spectrométrie de masse. Plusieurs protéines jouant un rôle dans l'épissage des ARN ont pu être identifiées de cette manière. La quantification exon par exon des transcrits séquencés par RNA-Seq m'ont également permis d'identifier une possible régulation directede l’épissage des gènes SLC39A14 et NDRG2 par la β-caténine. Enfin, nous nous sommes attachés à comprendre comment la β-caténine jouait un rôle prolifératif suite à son activation aberrante dans le foie d'une part (par l'utilisation d'un modèle transgénique prénéoplasique) et lors de la régénération hépatique d'autre part. Nous avons pu démontré que ce rôle prolifératif était complexe, seulement partiellement autocrine, mettant en jeu la Cycline D1 et le facteur de croissance Tgfa, que nous avons définis comme étant des cibles directes de la β-caténine dans le foie. Ces derniers résultats évoquent également un dialogue fonctionnel entre la voie β-caténine et la cascade de signalisation Tgfa-Egf/Egfr/Erk pour assurer la prolifération hépatocytaire. / The Wnt/β-catenin pathway is involved in proliferation and cell fate control and regulatesdevelopmental stages as well as adult tissue homeostasis. Our team has previously shown that aberrantB-catenin signaling induced Hepatocellular Carcinoma (HCC) development, whereas physiologicalactivation in a subpopulation of adult liver hepatocytes (pericentral hepatocytes) patterns liverzonation. This hepatic zonation can be considered as hepatocytes terminal differenciation.My thesis aimed to identify key molecular determinants allowing diversity upon β-catenin activation.I took advantage of genetically engineered mice models of activation or inactivation of β-catenin,developped in the laboratory. Using hepatocytes obtained from these models I identified β-catenin andTcf4, that I previously identifed as the main effector of nuclear β-catenin in liver, binding sites byChIp-seq (Chromatin Immunoprecipitation followed by high-throughput sequencing). Couplingbinding site localization to transcriptomic and chromatin accessibility studies allowed to identifychromatin structure and tissue specific transcription factor HNF4A as key modulators of β-cateninsignaling in the liver and therefore modulators of metabolic zonation. Indeed, β-catenin negativelyregulates Hnf4a target genes. This negative control exerts essentially via protein interactions. Wepropose that β-catenin and Hnf4a cooperates to ensure terminal hepatic differenciation.I also searched for nuclear β-catenin partners by co-immunoprecipitation followed by massspectrometry. This approach revealed many splicing factors as beta-catenin partners. RNA seqanalysis revealed a possible direct regulation of splicing of SL39A14 and NDRG2 genes.Finally we tried to understand the proliferative role of β-catenin during liver regeneration. Wedemonstrated a complex and partially autocrine role of B-catenin. We defined CyclinD1 and thegrowth factor Tgfa as β-catenin direct targets in the liver. These latest results also imply a functionnaldialog between the β-catenin pathway and Tgfa-Egf/Egfr/Erk signalisation cascade to allowhepatocytes proliferation.

Herein we tested the repeatability of RAD-seq phylogeographic construction by creating a second, independent phylogeography of the pitcher-plant mosquito, Wyeomyia smithii. We sampled 25 populations drawn from different localities nearby previous collection sites and used these new data to construct a second, independent phylogeography to test the reproducibility of phylogenetic patterns. Our previous phylogeography was based on 3,741 phylogenetically informative markers from 21 populations and rooted with mitochondrial COI. The present phylogeography was based on 16,858 informative markers and rooted with RAD-seq. We found correspondence between clades at the extremes of W. smithii's distribution; however, there were several discrepancies between the trees, including the refugium that gave rise to all post-glacial populations. We observed that combining all 46 populations resolved these discrepancies and, equally importantly, that extensive taxon sampling in areas of historical importance is more valuable than increasing the number of informative sites in establishing an accurate, robust phylogeography. This thesis includes unpublished co-authored material.

Les facteurs environnementaux, tels que les pesticides, peuvent induire des changements phénotypiques dans une variété d'organisme incluant les mammifères. Nous avons étudié chez la souris les effets d'un pesticide largement utilisé, l'atrazine (ATZ), sur la méiose, une étape clé du processus de spermatogenèse. L'utilisation des méthodes de puces à ADN (Gene-Chip) et de séquençage de chromatine immunoprécipité (ChIP-seq) nous a permis de mettre en évidence l'effet de l'ATZ sur une variété de fonctions cellulaires, incluant l'activité GTPase, la fonction mitochondriale et le métabolisme des hormones stéroïdes. De plus, les souris traitées présentent un enrichissement des marques d'histone H3K4me3 au niveau des régions de forte recombinaison (sites de cassures double brin) de gènes très long et une réduction de ces mêmes marques au niveau des régions pseudo-autosomal du chromosome X. Nos données démontrent que l'exposition à l'ATZ interfère avec le déroulement normal de la méiose, ceci affectant la production des spermatozoïdes. Nous avons trouvé que les marques H3K4me3, chez la souris mâle, sont largement affectées par l'ATZ grâce à l'utilisation de technique de séquençage du génome entier. La reprogrammation embryonnaire nécessite l'action coordonnée d'un grand nombre de gène et de facteurs épigénétiques afin de permettre la transition de cellules somatique en cellules germinales. Les modifications épigénétiques imposées pendant la transition des cellules somatiques en cellules germinales et affectées par des expositions nocives, peuvent être héritées et transmises aux générations suivantes via les gamètes. Dans cette étude, nous avons examiné l'héritage des histones modifié aux générations suivantes. Nous avons exposés des femelles gestantes CD1 non consanguines à l'ATZ et les mâles issus de ces femelles ont été croisés pendant trois générations avec des femelles non traitées. Nous avons démontré ici que l'exposition à l'ATZ réduit le nombre de spermatozoïdes sans affecter la morphologie cellulaire ou la proportion des différents types cellulaires constituant l'épithélium séminifère chez les individus issus de la 3ème génération après traitement. Beaucoup de gènes associés avec la réparation de l'ADN, la reproduction et les fonctions mitochondriales sont dérégulés chez les mâles issus de la 3ème génération après traitement. De façon importante, l'exposition à l'ATZ change dramatiquement l'initiation de la transcription, l'épissage et la polyadénylation alternative des ARN. Nous avons aussi observé chez les mâles F3 issus de souris traitées à l'ATZ une altération de la localisation des marques H3K4me3 dans le promoteur de gène associé à la régulation de processus métaboliques cellulaires, à la régulation de la transcription et à la mitose. Les changements de localisation des marques H3K4me3 chez les mâles F3 issus de souris traitées à l'ATZ correspondent à des changements de la localisation de ces marques au niveau de gènes impliqués dans la différenciation des cellules de type souche de la génération F1.Nos données suggèrent que l'héritage transgénérationnel est permis grâce à de multiples voies et repose sur le statut épigénétique de gènes impliqués dans la différenciation des cellules de type souches tels que Pou5f1 et Sox2, l'action des facteurs de transcription et la rétention d'histones dans le sperme. / Environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice. We demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome. Our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.We found that the H3K4me3 marks in male mice are broadly affected by the widely used herbicide atrazine with genome wide ChIP-sequencing. Embryonic reprogramming requires the coordinated action of many genes and epigenetic factors to perform somatic to germline transition. The epigenetic modifications imposed during somatic to germline transition and affected by harmful exposure can be inherited and transferred to subsequent generations via the gametes. In this study, we examine the inheritance of altered histone modifications by subsequent generations. We exposed pregnant outbred CD1 female mice to the widely used herbicide atrazine (ATZ), and the male progeny were crossed for three generations with untreated females. We demonstrate here that exposure to ATZ reduces the number of spermatozoa without changing the cell morphology or types in testis tissue in the third generation after treatment. Many genes associated with DNA repair, reproduction and mitochondrial function became dysregulated in the third generation (F3) of males after treatment. Importantly, exposure to ATZ dramatically changes the transcription initiation, splicing and alternative polyadenylation of RNA. We also observed altered occupancy of H3K4me3 markers in the F3 generation of ATZ-derived males in gene promoters associated with the regulation of cellular metabolic processes, transcriptional regulation and mitosis. The changes in H3K4me3 occupancy in F3 ATZ-derived males correspond to changes in the H3K4me3 occupancy of stem cell differentiation genes in the F1 generation. Our data suggest that transgenerational inheritance is accomplished through multiple pathways and relies on the epigenetic state of stem cell differentiation genes such as Pou5f1 and Sox2, transcription factor action and sperm histone retention.