<p>Precise regulation of the complex process of gene expression is essential for all aspects of life, and a large degree of this precision is mediated at the posttranscriptional level. The global and individual mechanisms by which posttranscriptional control is coordinated to maintain or alter levels of gene expression as necessary are not fully understood. Identification of the mRNA target sets of individual RNA binding proteins (RBPs) and characterization of the mechanisms by which RBPs regulate the expression of individual mRNAs provide some insight into the global structure of the posttranscriptional environment. However, few studies have integrated these findings into a global model of posttranscriptional control. We have explored the structure and function of the posttranscriptional regulatory system through a combination of global modeling approaches, global studies of mRNA translation and decay, and mechanistic studies of the function of individual RBPs, specifically HuR and Pum1. </p><p>By combining RBP-mRNA association data and transcription factor (TF) target data from separate global studies in yeast, we developed an integrated model of gene expression regulation. Evaluation of this model indicates that posttranscriptional regulation may be responsible for substantially greater contributions to the overall gene expression program than transcriptional regulation. Further, we identified a self-regulatory feature of the posttranscriptional network that suggests a `regulators of regulators' structure may be a defining feature of the posttranscriptional control of gene expression.</p><p>Additionally, we explored the mechanisms and functional consequences of the dynamic association between an RBP, HuR, and its target RNAs through a combination of modeling and experimental approaches, including polysome profile analysis and global measurement of RNA stability. Our model indicates that changes in total mRNA abundance are insufficient to fully explain the dynamics of association between HuR and its targets, suggesting a role for competition and cooperation with other RBPs. Our findings also indicate that HuR may play a role in inhibition of translation in a dynamic immunological system (T cell activation). </p><p>Finally, we performed a mechanistic analysis of the function of the Pum1 RBP and characterized the role of this protein in the translational regulation of several important target mRNAs through the use of luciferase reporter assays. We also provided the first in vivo evidence of a role for specific regions of the Pum1 protein in the mediation of gene expression. However, we were unable to verify previous in vitro reports of a role for Pum1 in the control of translation elongation of verified in vivo mRNA targets, suggesting that Pum1's regulatory function may be context dependent.</p><p>Ultimately, the approaches and findings in this study will provide a framework for the development of a global integrated model of posttranscriptional control. Through the iterative development of models and experimentation, hypotheses can be generated and, tested in the laboratory, and the results of these experiments will then further improve the development of the models. An integrated approach of this type will be necessary to fully understand the highly complex and interconnected nature of the gene expression regulatory system.</p> / Dissertation

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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.

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.