Les propriétés centrales des cellules souches sont la pluripotence et la capacité d'auto-renouvellement. Les cellules souches hématopoïétiques (CSHs) sont dotées de ces caractéristiques qui leur permettent de générer toutes les cellules du compartiment hématopoïétique, tout en maintenant en parallèle leur compartiment. Nous menons des approches visant à amplifier ex vivo les CSHs en les activant par HOXB4 exogène (CSHs humaines) ou via la signalisation Notch/DLL-4 (CSHs murines). Or deux analyses transcriptomiques indépendantes de ces deux modes d'activation ont de manière étonnante convergé sur une augmentation de l'expression de deux gènes jamais identifiés auparavant comme étant impliqués dans le maintien des CSHs : Pumilio1 (Pum1) et Pumilio2 (Pum2). Pum1 et Pum2 sont des régulateurs post-transcriptionnels appartenant à la famille Pumilio-FBF (PUF) des protéines liant l'ARN. Bien qu'il ait été établi que le rôle princeps de ces protéines PUF est de soutenir la prolifération des cellules souches chez les Invertébrés, jusqu'à présent on ne sait rien du rôle de Pum1 et Pum2 dans les CSH humaines et murines.Pour toutes ces raisons, nous avons étudié le rôle et les mécanismes d'action de Pum1 et Pum2 dans les CSH murines et humaines en utilisant l'interférence ARN (ARNi). L'invalidation de Pum1 ou de Pum2 dans les CSHs murines conduit à une réduction de l'expansion et du potentiel clonogénique ex vivo, associée à une apoptose accrue et l'arrêt du cycle cellulaire en phase G0/G1. L'invalidation concomitante de Pum1 et Pum2 majore ces effets ce qui suggère un effet coopératif entre les deux protéines. L'expansion et le potentiel clonogénique des CSH invalidées pour Pum1 sont restaurés suite à l'expression forcée de Pum1 (insensible au shRNA utilisé), validant ainsi la spécificité de nos shRNAs. Par contre la surexpression de Pum1 dans les CSHs invalidées pour Pum2 ne restaure pas leurs fonctions, soulignant le rôle non redondant de chaque protéine. En outre, lorsque les CSHs invalidées pour Pum1 ou Pum2 sont inoculées à des souris irradiées létalement de suivre le potentiel hématopoïétique à long terme, seules quelques rares cellules de la moelle osseuse issues des CSH KD pour Pum1 ou Pum2 sont mises en évidence après 4 mois de reconstitution, contrairement aux CSH contrôles. Des résultats identiques ont été obtenus en invalidant Pum1 ou Pum2 dans les CSH humaines.En conclusion, nos résultats démontrent l'implication des facteurs Pumilio dans le maintien du potentiel souche, l'expansion et la survie des CSHs murines et humaines. L'identification des facteurs Pumilio et de leurs cibles comme nouveaux régulateurs des CSHs permettra d'envisager de nouveaux outils en vue de perspectives thérapeutiques. / The central properties of stem cells are the pluripotency and the capacity of self-renewal. Hematopoietic stem cells (HSCs) posses such common features that allows them to generate all the cells of the hematopoietic compartments, maintaining in the same time the HSC pool. We develop approaches focused on ex vivo HSC expansion through activation by exogenous HOXB4 (human HSCs) or Notch/Dll-4 ligand (murine HSCs). Two independent transcriptomic analyses surprisingly converged toward an increased expression of two genes never identified sofar as crucial for HSC functions: Pumilio1 (Pum1) and Pumilio2 (Pum2). Pum1 and Pum2 are posttranscriptional regulators belonging to the Pumilio-FBF (PUF) family of RNA-binding proteins. Although it was established that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells in Invertebrates, so far nothing is known about the role of Pum1 and Pum2 in human and murine HSCs.For these reasons, we have investigated the roles and mechanisms of action of Pum1 and Pum2 in murine and human HSCs through shRNA strategy. Pum1 and Pum2 knockdown (KD) in murine HSCs led to a decreased HSC expansion and clonogenic potential ex vivo, associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase. KD of both Pum1 and Pum2 enhanced these effects, suggesting a cooperative effect. Expansion and clonogenic potential of KD Pum1 HSCs were rescued by enforced expression of Pum1 (insensitive to our shRNA), thus validating the specificity of our shRNA. Enforced expression of Pum1 could not rescue the functions of Pum2 KD HSCs, highlighting the non-redundant role of these proteins. Furthermore, when Pum1 or Pum2 KD HSCs were inoculated into lethally irradiated mice to follow the long-term hematopoietic potential, only rare bone marrow cells derived from Pum1 and Pum2 KD HSCs were evidenced after 4 months, contrary to control HSCs. Identical results were obtained with human Pum1 or Pum2 KD HSCs.In conclusion, our results demonstrate the involvement of Pumilio factors in stemness maintenance, expansion and survival of murine and human HSCs. Identification of Pumilio factors and their targets as new regulators of HSCs expansion will allow consider them as new tools for therapeutic perspectives.

Les bactéries sont en permanence confrontées à des changements d'environnements. La régulation transcriptionnelle joue alors un rôle majeur dans l'adaptation des bactéries. En particulier, la bactérie phytopathogène D. dadantii s'est récemment adaptée à l'hôte végétal. Elle produit en particulier des pectate lyases (Pel) qui dégradent la pectine, ciment des parois végétales, et jouent un rôle majeur dans le développement de la maladie. Les gènes pelD et pelE, malgré la forte divergence dans leur expression, sont issus d'un transfert horizontal suivi d'une duplication récente. La question de l'intégration de ces gènes avec les régulations préexistantes s'est alors posée.Dans un premier temps, les mécanismes moléculaires détaillés de la régulation de pelD ont été étudiés. Il a été montré que cette régulation s'appuie sur un promoteur divergent de forte affinité pour l'ARN polymérase mais de faible efficacité pour la transcription et sur un arrangement stratégique de quatre sites de fixation de répresseur FIS et deux sites de l'activateur CRP. Tous ces éléments interagissent entre eux pour produire une régulation fine de l'expression de pelD. L'origine de la divergence régulatrice entre les paralogues pelD et pelE a par la suite été explorée. De manière surprenante, la divergence entre ces deux gènes et leur sélection s'appuie presque exclusivement sur un décalage de la position du promoteur de pelE (« TSS turnover ») qui l'a transformé en initiateur de la dégradation de la pectine. Ce mécanisme très fréquent chez les eucaryotes pluricellulaires (homme, drosophile, souris…) n'avait jamais encore été décrit chez les bactéries.A travers l'étude des promoteurs pelD et pelE de D. dadantii, de nouveaux mécanismes renforçant l'importance de la régulation transcriptionnelle dans les processus adaptatifs ont ainsi été découverts / Bacteria face frequent environmental changes. Transcriptional regulation plays a major role in the adaptation to these changes. In particular, the phytopathogen bacteria Dickeya have recently adapted to vegetal hosts. They produce Pecate lyases (Pel), among others, to degrade pectin in plant cell walls, which is necessary for disease development. The pelD and pelE genes, despite the strong divergence in their expression, originate from a horizontal gene transfer followed by a recent duplication. This raises the question of their integration into the preexisting regulatory networks.Detailed molecular mechanisms of the transcriptional regulation of pelD were studied first. It was shown that this regulation relies on a high-affinity but low transcription efficiency divergent promoter and a strategic arrangement of four FIS repressor binding sites and two CRP activator binding sites. These elements interact together to fine-tune the expression of pelD. Next, the origin of the regulatory divergence between the paralogous genes pelD and pelE was explored. Surprisingly, their divergence and selection relies mostly on a TSS turnover which happened on the pelE regulatory region and transformed pelE into an initiator of pectin degradation. This widespread phenomenon in multicellular eukaryotes (human, fly, mouse…) had not yet been seen in bacteria. To conclude, through the study of D. dadantii pelD and pelE promoters, new mechanisms highlighting the relevance of transcriptional regulation in adaptation were discovered in this work

Dans son environnement, la bactérie Escherichia coli (E. coli) fait face à d’importantes fluctuations des ressources carbonées. Une capacité d’adaptation métabolique lui permet de coloniser ou de subsister en fonction des substrats disponibles. Cette adaptation est régie par un réseau complexe de régulations de l’expression génique. Le régulateur global post-transcriptionnel CSR (Carbon Storage Regulator) régule la stabilité et/ou l’initiation de la traduction d’ARNm par l’intermédiaire de la protéine CsrA. Ce système, essentiel en présence de glucose et est également supposé être impliqué dans la régulation d’une transition métabolique glycolyse vers gluconéogenèse. Le caractère essentiel de CSR est à ce jour inexploré, tout comme son implication dans la régulation d’une adaptation métabolique. Une approche de biologie intégrative menée pour différents mutants du système CSR a permis d’avancer pour la première fois, une explication de l’essentialité de CSR lors d’une croissance exponentielle sur glucose et de caractériser son implication dans la régulation de la transition métabolique glucose-acétate. Des approches de transcriptomique et de stabilomique utilisées pour une souche sauvage au cours d’une adaptation métabolique ont mis en évidence l’importance des régulations de la stabilité des ARNm au cours de l’adaptation. En conclusion, ces travaux approfondissent grandement les connaissances concernant le système CSR et son implication dans la régulation du métabolisme d’E. coli. Ce système, indispensable à la régulation du métabolisme durant une phase de croissance sur glucose s’ajoute de façon indéniable au réseau déjà complexe de régulations du métabolisme d’E. coli / In its natural environment, Escherichia coli (E. coli) faces strong fluctuations of the nutrient availability. A complex gene regulatory network makes the bacterium able to switch between a state of growth in the presence of an appropriate carbon source and a non-growth state in its absence. Within this network, the global post-transcriptional regulator CSR (Carbon Storage Regulator) modifies mRNA stability and/or translation initiation by the CsrA protein. This system has been shown to be essential for cells to grow on glucose and is hypothesized to be involved in the regulation of metabolic transitions. However both observations remained unexplored so far. An integrative approach has been used to investigate for the first time the essentiality of CSR on glucose as well as its involvement in the regulation of the glucose-acetate transition. Molecular and phenotypic data for different mutants of the CSR system have been produced and integrated into mathematical models. Transcriptomic and Stabilomic approaches have been used eventually to characterize the importance of the control of mRNA stability during the metabolic adaptation. mRNA stability regulations appear to be of particular importance in gene expression regulation during metabolic adaptation. To conclude, this work shed a new light upon CSR’s involvement in the regulation of E. coli’s metabolism. CSR is definitely essential to regulate glycolysis and thus constitutes another regulator to be integrated into the already complex regulations network of E.coli’s metabolism

Le gène CFTR, impliqué lorsqu'il est muté dans la mucoviscidose, est finement régulé au niveau tissulaire (principalement exprimé dans les organes cibles de la mucoviscidose) et au cours du développement. Par exemple, dans les tissus pulmonaires, l'expression du gène CFTR est plus forte chez le fœtus que chez l'adulte (75:1), où seulement deux copies en moyenne par cellule sont détectées.L'objectif de ce travail était de déterminer les mécanismes moléculaires responsables de cette régulation. Nous avons identifié de nombreux motifs cis-régulateurs au niveau de la région promotrice et de la région 3'UTR. Nous avons également caractérisé des facteurs de transcription, dont certains présentent une spécificité tissulaire et développementale. C'est notamment le cas des protéines de la famille FOX, des régulateurs clés dans le développement du système reproducteur et pulmonaire. Cette étude a également permis d'identifier le rôle de certains microARNs dans la déstabilisation des transcrits CFTR. Finalement, nous proposons un rôle combiné de ces différents acteurs dans la régulation transcriptionnelle et post-transcriptionnelle du gène CFTR. L'identification des éléments répresseurs devrait fournir de nouvelles cibles thérapeutiques pour la mucoviscidose. / CFTR gene, involved in cystic fibrosis, displays a tightly regulated spatio-temporal pattern of expression (mainly expressed in taget tissues of cystic fibrosis). In lung, CFTR transcripts are abundant during fetal development compared to the adult stage (75:1), where only two copies per cell are detected. The aim of this work was to determine the molecular mechanisms involved in this regulation. We have identified several cis-regulatory motifs in the 5'UTR and the 3'UTR parts. We have characterized transcription factors with tissue- and temporal-specific activity. Members of FOX family are crucial regulators in reproductive duct and lung formation. We have also identified microRNAs in destabilizing CFTR transcripts. Finally, we propose a coupling role of trans-acting regulators in the transcriptional and post-transcriptional regulation of the CFTR gene. Characterizing the repressors would help to identify novel therapeutic tools in cystic fibrosis.

A fim de contornar a problemática imposta pela recalcitrância da parede celular à hidrólise, o processo de produção de etanol a partir de biomassa vegetal requer um passo denominado de pré-tratamento, que consiste em tornar a biomassa mais acessível à ação de glicosil hidrolases que atacam a parede celular. O melhor conhecimento de processos de degradação de parede operados pela própria planta tem o potencial de direcionar as pesquisas em bioernegia, indicando quais os mecanismos mais eficientes para desmontar o complexo de polímeros da parede celular. Cunhado pré-tratamento biológico, este consiste na hidrólise de polissacarídeos estruturais empregando mecanismos e elementos chave de processos de degradação de parede celular que ocorrem na própria planta. Um exemplo de evento com esta característica é a formação de aerênquima lisígeno, que consiste na abertura de espaços de gás em tecidos parenquimáticos. A formação do aerênquima em cana-de-açúcar se dá de forma modular, sendo o processo constituído por seis etapas: 1) percepção do sinal inicial; 2) separação celular: 3) expansão celular; 4) morte celular programada; 5) hidrólise de hemiceluloses e 6) hidrólise de celulose. O presente trabalho teve como principal foco estudar a regulação das duas etapas iniciais, visando obter conhecimentos que possibilitem para tornar a biomassa de cana de açúcar menos resistente à penetração de enzimas. O aerênquima ocorre na raiz de cana-de-açúcar por um processo constitutivo. Sua independência de um indutor externo foi corroborada mediante tratamento com nutrientes e inibidor da percepção por etileno (1-MCP) pela análise dos cinco centímetros mais apicais da raiz. O atraso na formação do aerênquima após tratamento com nutrientes levou à expressão diferencial de genes relacionados à degradação de parede celular, morte celular programada e sinalização por etileno. Por outro lado, o 1-MCP não afetou visivelmente a formação do aerênquima, porém alterou o balanço hormonal na raiz. O padrão transcricional das raízes tratadas com 1-MCP revelou aumento da expressão de genes relacionados à expansão celular e estresse oxidativo. Tais padrões são discutidos à luz da regulação hormonal da formação do aerênquima através do estabelecimento de um balanço hormonal definido entre auxina e etileno. Ambos os experimentos levaram à seleção de quatro genes candidatos: dois fatores de transcrição (ScRAV1 e ScERF1) e duas glicosil hidrolases (ScEPG1 e ScARA1), sequenciados e analisados quanto à diversidade hom(e)óloga, sequências promotoras e estrutura gênica em comparação a S. bicolor. A topologia das reconstruções filogenéticas e a distribuição diferencial de sítios para RAV e ERF nos promotores de ScEPG1 e ScARA1 não parece refletir padrões de expressão distintos, visto que os diferentes hom(e)ólogos demonstraram ser igualmente expressos. Em sistema heterólogo, o fator de transcrição RAV apresentou atividade repressora sobre o promotor de ScEPG1. Tal papel de RAV sugere regulação negativa sobre a degradação da lamela média e sobre o processo de perda de adesão e separação celular. A identificação de reguladores-chave da formação do aerênquima consiste em importante elo entre a sinalização hormonal e a degradação de parede celular, possibilitando a melhor compreensão dos mecanismos de controle da degradação endógena de parede celular. Os dados produzidos possibilitam a aplicação biotecnológica dos genes sequenciados, além de consistirem em significativo avanço no conhecimento sobre a fisiologia do processo estudado e na dinâmica da regulação da expressão gênica acoplada a aspectos filogenéticos das sequências alvo. / In order to circumvent the problems imposed by the cell wall recalcitrance to hydrolysis, the process underlying biofuel production requires a step called pretreatment, aimed at making biomass more accessible to the action of glycosil hydrolases that attack the cell wall. The increased knowledge of wall degradation processes operated by the plant itself has the potential to influence research in the bioernergy field, pointing out the most efficient mechanisms to disassemble the cell wall complex polymeric structure. The term coined biological pretreatment means taking advantage of key elements and mechanisms of cell wall degradation processes occurring in the plant itself in order to disassemble the entangled polysaccharide structure. One example of endogenous cell wall degradation process is the lysigenous aerenchyma formation, which consists in the opening of gas spaces in parenchyma tissue. The aerenchyma formation in sugarcane is thought to be a modular process that occurs in six steps: 1) target cells perception; 2) cell separation; 3) cell expansion; 4) programmed cell death; 5) hemicellulose hydrolysis and 6) cellulose hydrolysis. This work has as the main objective to study the regulation of the two initial steps, aiming at acquiring knowledge that would afford to turn biomass less resistant to enzyme penetration. The aerenchyma develops within the roots of sugarcane as a constitutive process. Its independence from an external inducer was corroborated in this work by subjecting sugarcane plants to treatment with nutrients and one inhibitor of ethylene perception (1-MCP) when the five more apical centimeters of the root were analyzed. After treatment with nutrients, the delayed aerenchyma formation led to differential expression of cell wall degradation-, programmed cell death- and ethylene signalling-related genes. Under visual inspection, 1-MCP did not show effect on aerenchyma development. Instead, it changed the hormone balance mainly in the two most apical root segments. The transcriptional pattern of 1-MCP treated roots revealed increased expression of cell expansion- and oxidative stress-related genes. Such patterns are discussed in the light of the hormone regulation of the aerenchyma development through the establishment of a balance between auxin and ethylene within root segments. Both experiments led to the selection of four two candidate genes, two transcription factors (ScRAV1 and ScERF1) and two glycosil hydrolases (ScEPG1 and ScARA1), that were sequenced and analyzed regarding homEURologous diversity, promoter sequences and gene structure compared to S. bicolor. The topology of the phylogenetic reconstructions and the differential distribution of sites for RAV and ERF within ScEPG1 and ScARA1 promoters did not reflect distinct expression patterns. Different hom(e)ologous of each target gene were equally expressed. In an heterologous system, RAV transcription factor interacted with ScEPG1 promoter, reducing the activity encoded by the reporter gene. This suggests the repressive role of RAV on pectin degradation within the middle lamella, leading to reduced cell adhesion and cell separation, possibly regulated by this glycosyl hydrolase. The identification of key regulators of the aerenchyma formation is an important link between hormone signaling and the wall degradation within the sugarcane roots. It enables a better understanding of control mechanisms underlying wall modifications when performed by the plant itself. Altogether, the data produced in this work allows biotechnological application of sequenced genes. Moreover, the produced data unveil key aspects regarding physiologycal aspects of aerenchyma development and highlights features related to the dynamics of gene expression in sugarcane coupled to the phylogenetic aspects of the four target sequences.

Les cellules souches embryonnaires (cellules ES) constituent un excellent système modèle pour étudier les mécanismes épigénétiques contrôlant la transcription du génome mammifère. Un nombre important de membres de la famille des facteurs de remodelage de chromatine ATP-dépendants ont une fonction essentielle pour l’auto-renouvellement des cellules ES, ou au cours de la différentiation. On pense que ces facteurs exercent ces rôles essentiels en régulant l’accessibilité de la chromatine au niveau des éléments régulateurs de la transcription, en modulant la stabilité et le positionnement des nucléosome.Dans ce projet, nous avons conduit une étude génomique à grande échelle du rôle d’une dizaine des remodeleurs (Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Ep400, Brg1, Smarca3, Smarcad1, Smarca5, ATRX et Chd1l) dans les cellules ES. Une double stratégie expérimentale a été utilisée : Des expériences d’immunoprécipitation de la chromatine suivi par un séquençage à haute-débit (ChIP-seq) sur des cellules ES étiquetées pour les différents remodeleurs, pour étudier leur distribution sur le génome, et un approche transcriptomique sur des cellules déplétées de chaque remodeleur par traitement avec des vecteurs shRNA (knockdown). Nous avons établi les profils de liaison des remodeleurs sur des éléments régulateurs (promoteurs, enhancers et sites CTCF) sur le génome, et montré que ces facteurs occupent toutes les catégories d’éléments régulateurs du génome. La corrélation entre les données ChIP-seq et les données transcriptomiques nous a permis d’analyser le rôle des remodeleurs dans les réseaux de transcription essentiels des cellules ES. Nous avons notamment démontré l’importance particulière de certains remodeleurs comme Brg1, Chd4, Ep400 et Smarcad1 dans la régulation de la transcription chez les cellules ES. / The characteristics of embryonic stem cells (ES cells) make them one of the best models to study the epigenetic regulation exerted by different actors in order to control the transcription of the mammalian genome. Members of the Snf2 family of ATP-dependent chromatin remodeling factors were shown to be of specific importance for ES cell self-renewal and during differentiation. These factors are believed to play essential roles in modifying the chromatin landscape through their capacity to position nucleosomes and determine their occupancy throughout the genome, making the chromatin more or less accessible to DNA binding factors.In this project, a genome-wide analysis of the function of a number of ATP-dependent chromatin remodelers (Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1, Ep400, ATRX, Smarca3, Smarca5, Smarcad1 and Alc1) in mouse embryonic stem (ES) cells was conducted. This was done using a double experimental strategy. First, a ChIP-seq (Chromatin Immunoprecipitation followed by deep sequencing) strategy was done on ES cells tagged for each factor in the goal of revealing the genomic binding profiles of the remodeling factors. Second, loss-of-function studies followed by transcriptome analysis in ES cells were performed in order to understand the functional role of remodelers. Data from both studies were correlated to acquire a better understanding of the role of remodelers in the transcriptional network of ES cells. Specific binding profiles of remodelers on promoters, enhancers and CTCF binding sites were revealed by our study. Transcriptomic data analysis of the deregulated genes upon remodeler factor knockdown, revealed the essential role of Chd4, Ep400, Smarcad1 and Brg1 in the control of transcription of ES cell genes. Altogether, our data highlight how the distinct chromatin remodeling factors cooperate to control the ES cell state.

Transcriptional regulation refers to the molecular systems that control the concentration of mRNA species within the cell. Variation in these controlling systems is not only responsible for many diseases, but also contributes to the vast phenotypic diversity in the biological world. There are powerful experimental approaches to probe these regulatory systems, and the focus of my doctoral research has been to develop and apply effective computational methods that exploit these rich data sets more completely. First, I present a method for mapping genetic regulators of gene expression (expression quantitative trait loci, or eQTL) using Random Forests. This approach allows for flexible modeling and feature selection, and results in eQTL that are more biologically supportable than those mapped with competing methods. Next, I present a method that finds interactions between genes that in turn regulate the expression of other genes. This is accomplished by finding recurring decision motifs in the forest structure that represent dependencies between genetic loci. Third, I present a method to use distributional differences in eQTL data to establish the regulatory roles of genes relative to other disease-associated genes. Using this method, we found that genes that are master regulators of other disease genes are more likely to be consistently associated with the disease in genetic association studies. Finally, I present a novel application of Random Forests to determine the mode of regulation of toxin-perturbed genes, using time-resolved gene expression. The results demonstrate a novel approach to supervised weighted clustering of gene expression data.

1 Abstract Diabetes mellitus is characterized by the dysfunction and reduction of insulin-producing cells, resulting in hyperglycemia, which in long term harms the organism. For future therapy, it is crucial to understand the function of various factors participating in the differentiation and maturation of endocrine pancreatic cells. The aim of this study was to unravel the functional role of ISL1 during the development of the pancreas. ISL1 is expressed in all endocrine cells of the islets of Langerhansbut its function remains unclear, especially during early pancreatogenesis. As the global deletion of this gene is embryonically lethal, we used the tissue specific deletion of Isl1 in Neurod1 possitive cells using the Cre-loxP system. In this work we studied the effect of this deletion on the structure of islets of Langerhans, the formation of endocrine cell types and relative expression of genes during early pancreatic development. A defective achitecture of islets together with postnatal absence of α-cells was found in the Isl1 deletion mutant. Also, the expression of genes important for the specification of α-cell lineage and their subsequent function was decreased. The secondary outcome was the optimalization of a protocol for effective sorting of endocrine cells using fluorescent flow cytometry, which...

<p>  </p> <p>Plant complex traits are controlled by multi-layer of dynamic and complicated gene networks regulated at different levels. To better inform crop breeding to promote desired traits, a comprehensive and fundamental understanding of their genetic basis is much needed. With the rapid developments of <em>omics</em> planforms and next generation sequencing technology, we now have large-scale data from genome, epigenome, transcriptome, metabolome, and others for the crop plants. Integration of those multiple <em>omics</em> data together with computational approaches led to the establishment of a novel science known as system biology. Research described in this thesis used system biology approaches to dissect complex crop traits such as disease response of tomato (Chapter2 and Chapter3) and the heterosis of nitrogen use efficiency of maize (Chapter4).</p> <p>Plant disease response is an elaborate, multilayered complex trait involving several lines of defense signaling. In the past decades, progress in molecular analyses of plant immune system has revealed key elements of a complex response network in Arabidopsis, a model species. Histone modifications, a type of epigenetic regulation, have emerged as key modulators that regulate defense responses, while our understanding of the role of histone-modifying enzymes in this process is still in its infancy. Here, we described the immune function of two histone methyltransferases SDG33 and SDG34 in tomato. We found the single mutants in <em>sdg33</em> and <em>sdg34</em> showed increased susceptibility to hemibiotrophic bacterial pathogen <em>Pseudomonas syringae</em> whereas the double mutant <em>sdg33sdg34</em> is comparable to wild type. Using RNA-seq and histone ChIP-seq approaches, we investigated the possible underlying mechanisms and found that the expression of a set of immune-related genes is misregulated by <em>P. syringae</em> only in the single mutants but not in the double mutant. Integrating with epigenomic data, we found that the misexpression of those SDG33/SDG34 dependent immune-response genes was associated with altered histone methylation status in the single mutant. Intriguingly, the double mutant also showed altered histone methylation but unaffected gene expression, suggesting a compensating regulatory mechanism at play. The function of SDG33 and SDG34 in immune response seems to be specific for the pathogen, as the double mutants exhibited enhanced resistance the single mutants showed no altered responses when treated with necrotrophic fungal pathogen <em>Botrytis cinerea</em>. Network analysis found the most regulatory gene by <em>B. cinerea</em> in a SDG33/SDG34 dependent manner have been implicated in biotic stress response such as <em>ERF4, TOPLESS, PUB23 </em>and<em> RCD1</em>. Comparing the immune response of double mutant against <em>P. syringae</em> and <em>B. cinerea</em>, we found that the disease related genes are only mis-regulated in the interaction of <em>B. cinerea</em> treatment not in the <em>P. syringae</em> treatment, which could be the reason of enhanced resistance to <em>B. cinerea</em> but not for <em>P. syringae</em> in the double mutants. In summary, we found the histone methyltransferases SDG33 and SDG34 has different functions in the immune response against <em>P. syringae</em> and <em>B. cinerea</em>, which might be direct or indirect relevant to the histone methylation level of the expression of downstream immune related gene.</p> <p>In addition to biotic stress, another complex trait studied in this thesis is the heterosis of nitrogen use efficiency (NUE) in Maize. NUE is another complex trait associated with multiple physiological processes including N sensing, uptake, assimilation, transport, and storage. Heterosis refers to a phenomenon where the progeny generated by crossing two different cultivars of the same species exhibit superior fitness than the inbred parents. Even though, heterosis has been exploited to improve complex traits including NUE, the underlying molecular mechanisms is not completely understood. Here, we analyzed N-responsive transcriptomes and physiological traits of a panel of six maize hybrids and their corresponding inbreds grown in the field at two different N levels. We observed diverse levels of trait heterosis that are dependent on the N conditions and organ types. We discovered dramatic pattern shift of beyond-parental-range gene expression in hybrids in response to varying N levels. We identified through integrative analyses a set of genes whose expression heterosis are quantitatively correlated to trait heterosis. These genes are involved in response to stimulus, photosynthesis, and N metabolism, and likely mediate the heterosis phenotype of N-use and growth traits in maize. In summary, our integrated analysis provided insights into the mechanistic basis of the heterosis of NUE. </p> <p>Together, applying systems and functional genomics approaches to investigate important agricultural traits could lead to a comprehensive understanding of plant complex traits to inform future engineering and breeding for better crops.</p>

Tfap2a gehört zur Familie der AP-2-Transkriptionsfaktoren. Heterozygote Mutationen von TFAP2A im Menschen führen zum Branchio-Okulo-Fazialen-Syndrom (BOFS) und sind mit Nierenanomalien assoziiert. Molekulare Mechanismen, die zu diesen BOFS-assoziierten Nierenanomalien führen, sind noch unbekannt. In diesem Projekt wurde die Expression von Mitgliedern der AP-2-Familie in neugeborenen und erwachsenen Wildtyp-Mäusen analysiert. Tfap2a wurde in der Ureterknospe und der distalen Region des S-förmigen Körpers in den Nieren neugeborener Mäuse exprimiert. Die Expression blieb in ausgereiften distalen Tubuli und Sammelrohren erhalten. Tfap2b, ein zweites Mitglied der AP-2-Familie, das in der Niere exprimiert wird und mit Zystenbildung assoziiert ist, wurde im aufsteigenden Ast der Henleschen Schleife sowie in den distalen Tubuli und dem in der Nierenrinde liegenden Sammelrohr exprimiert. Um die Rolle von Tfap2a in der Niere zu untersuchen, wurden Mäuse mit einer sammelrohrspezifischen Deletion von Tfap2a (Tfap2a-KO) erzeugt. Phänotypische und morphologische Analysen ergaben, dass Tfap2a-KO-Mäuse mäßig reduzierte Nierengewichte und eine fortschreitende Dilatation der äußeren medullären Sammelrohre aufwiesen. Einzelkern- und RNA-Sequenzierung der Nieren adulter Mäuse zeigte eine deregulierte Expression von Genen, die mit der Organisation von Aktinfilamenten, Zelladhäsion, Wnt-Signalen und anderen Signalwegen der Nierenentwicklung in Verbindung stehen. In einem isolierten Modell von kultivierten Sammelrohrzellen mit einer Deletion von Tfap2a waren ähnliche Signalwege dereguliert. Insgesamt deutet diese Studie darauf hin, dass Tfap2a für die Differenzierung des Sammelrohrepithels und die Regulierung des Durchmessers des Tubuluslumens erforderlich ist. Dies ermöglicht Einblicke in die molekularen Grundlagen der beim BOFS beobachteten Nierenfehlbildungen. / The transcriptional regulator Tfap2a is part of the AP-2 transcription factor family. Heterozygous mutations of TFAP2A in humans lead to branchio-oculo-facial syndrome (BOFS) and are associated with renal anomalies. Molecular mechanisms leading to BOFS-associated renal anomalies are still unknown. In this project, expression patterns of AP-2 family members were analyzed in newborn and adult wildtype mice. Tfap2a was expressed in the ureteric bud and distal region of the S-shaped body in kidneys of newborn mice. Expression was maintained in mature distal tubules and collecting ducts. Tfap2b, a second AP-2 family member expressed in the kidney and associated with cyst formation, was found in the ascending limb and showed overlapping expression with Tfap2a in distal tubules and the cortical collecting duct. To investigate the role of Tfap2a in the kidney, mice with a collecting duct-specific deletion of Tfap2a (Tfap2a-KO) were generated by crossing mice carrying a Cre-recombinase under the Hoxb7 promotor and mice with floxed Tfap2a alleles. Phenotypic and morphological analyses revealed that Tfap2a-KO mice displayed moderately reduced kidney weights and a progressive dilation of outer medullary collecting ducts. Single-nucleus and bulk RNA sequencing of kidneys of three months old Tfap2a-KO mice and littermate controls indicated deregulated expression of genes associated with actin filament organization, cell adhesion, Wnt signaling, and other kidney developmental pathways. Genes deregulated in Tfap2a-deficient mice included several genes previously implicated in the development of congenital anomalies of the kidney and urinary tract. In an isolated model of cultured collecting duct cells carrying a Tfap2a knockout similar pathways were deregulated. Taking together, this study indicates that Tfap2a is required for collecting duct epithelium differentiation and tubular lumen diameter regulation, providing insights into the molecular basis of renal defects observed in BOFS.