Les spermaphytes majoritairement représentés par les angiospermes ou plantes à fleur, ont le trait fondamental de pouvoir se multiplier par la formation de graine. Les graines représentent un caractère essentiel pour la survie des plantes, leur dissémination et un usage important pour l’industrie, notamment agro-alimentaire. Lors de la formation de la graine, des transitions cruciales ont lieu, en particulier pour le développement, l’établissement des réserves, la maturation, l’acquisition de la tolérance à la dessiccation et la dormance. Ces étapes sont régulées par des facteurs de transcription et notamment les LAFL (LEC1, ABI3, FUS3, LEC2). Les LAFL sont composés de deux familles, les facteurs de transcription à domaine B3 (ABI3, FUS3, LEC2) et les NF-Y (LEC1), qui sont impliqués dans la régulation génique dans différentes étapes du développement de la graine. Des études suggèrent la possibilité de la formation d’un complexe composé de ABI3, LEC1 et LEC2 sur le promoteur de l’OLEOSINE1 permettant d’activer plus fortement la transcription du gène. Néanmoins la caractérisation des protéines, leur action, régulation et leur capacité à interagir entre elles et former un complexe, restent encore méconnues. Dans un premier temps, je me suis attachée à caractériser la formation du complexe protéique composé d’ABI3, LEC1 et LEC2, plus particulièrement, l’interaction entre LEC1 et LEC2. La localisation subcellulaire des deux protéines par étiquetage in planta, ou grâce à des anticorps spécifiques, leur interaction dans un système double hybride et la mesure de l’effet de LEC1 sur LEC2 par thermophorèse ont permis de montrer que LEC2 et LEC1 peuvent former un dimère sur le promoteur de l’OLEOSINE1 et que cette association change l’affinité de LEC2 pour son ADN cible. Dans une deuxième approche, j’ai cherché à identifier des partenaires avec (données bibliographiques) et sans (approche de TAP-tag) a priori pour compléter l’étude. / The spermaphytes predominantly represented by angiosperms or flowering plants, have the fundamental ability to disseminate through seeds. Seeds are essential for the survival of plants, their dissemination and important resource for industry, especially food industry. During seed formation, critical transitions occur, in particular for development, establishment of reserves, maturation, acquisition of desiccation tolerance and dormancy. These steps are regulated by transcription factors and especially LAFL (LEC1, ABI3, FUS3, LEC2). LAFLS are composed of two families, B3 domain containing transcription factors (ABI3, FUS3, and LEC2) and NF-Y (LEC1) that are involved in gene regulation in different stages of seed development. Studies have suggested the possibility that a complex composed of ABI3, LEC1 and LEC2 is formed on the promoter of OLEOSINE1, allowing activating the transcription of the gene. Nevertheless, the characterization of proteins, their action, regulation and their capacity to interact with each another and form a complex, are still unknown. In a first step, I focused on characterizing the formation of the protein complex composed of ABI3, LEC1 and LEC2, more particularly on the interaction between LEC1 and LEC2. The subcellular localization of the two proteins were followed by labelling in planta, or by means of specific antibodies, their interaction tested in a double hybrid system and the measurement of the effect of LEC1 on LEC2 validated by thermophoresis. All the results demonstrated that LEC2 and LEC1 can form a dimer on the promoter of OLEOSINE1 and that this association change the affinity of LEC2 for its target DNA. In a second approach, I sought to identify partners with (bibliographic data) and without (TAP tagging approach) a priori to complete the study.

Few transcription factors (TFs) have been studied in the context of an integrative analysis incorporating genomic datasets from diverse genome regulatory mechanisms. Such an analysis allows the testing of specific regulatory associations in an unbiased and comprehensive manner. The promoter binding TF complex NF-Y regulates a diverse set of constitutive, inducible, developmental, oncogenic and tissue-specific genes. Using cancer models, ChIP-Seq, shRNA, and genomics, I have undertaken a genome-wide study of NF-Y. NF-Y binds to not only promoters but also extensively to enhancers, select classes of repetitive elements, inactive chromatin domains and insulators. NF-Y is a “pioneer”-like factor able to access its motif within closed, transcriptionally inactive chromatin domains. NF-Y pervasively associates with FOS, usually in the absence of JUN and the AP-1 motif, and with a group of growth controlling oncogenic TFs. I also show that NF-Y asymmetrically binds to its motif and stereo-aligns with specific TFs and their motifs. My results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather functions at a more diverse set of genomic elements. The dynamics of TF occupancy, cis-regulatory element (CRE) usage and their linkage to gene expression during a differentiation process, from a genome-wide perspective, is poorly understood and is critical to the understanding of fundamental aspects of development and disease. I utilize a model of inflammation-mediated oncogenic transformation, siRNA, ChIP-Seq, FAIRE-Seq, and microarrays to study the genomic aspects of transformation driven by Src-mediated activation of the inflammatory TF STAT3. I show that CRE usage is static, even in the presence of induced STAT3 activity, and large-scale transcriptional and phenotypic changes. STAT3 induced occupancy is tightly associated with FOS, pre-existing CREs, and does not create CREs de novo. I also highlight a putative role of TSC22D3 in inhibiting an epigenetic switch and in STAT3 and AP-1 factors driving the embryonic-like and bone-like phenotypes of breast cancer. The research presented here suggests that phenotypic alterations occurring during disease are not driven by large-scale perturbations of CRE usage. Overall, this dissertation provides an invaluable resource of genome-scale datasets within cancer models that will assist in future endeavors of scientific discovery.

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