The study of absorption, distribution, metabolism and excretion (ADME) of compounds within the body is of vital importance to health. PXR is a nuclear receptor which regulates the expression of a great many ADME genes and is responsible for the regulation of all parts of the ADME of many compounds. It is believed that the nuclear receptors, including PXR, activate genes on a ligand dependent basis, a mechanism which is probably regulated by the recruitment of a different battery of co-regulator proteins. The ligand dependent selection of genes by recruitment of alternate co-regulator complexes has not yet been directly demonstrated. The co-regulator proteins of PXR are not well characterised although co-regulator complexes of other receptors, including the oestrogen receptor, growth hormone receptor and retinoic acid receptor are comparatively well characterised. In order to demonstrate that different co-regulator complexes can be recruited by different ligands of PXR is was first necessary to identify the co-regulator complexes of PXR. An immunoprecipitation assay was developed and used in conjunction with an half GFP interaction assay as validation. The immunoprecipitated samples gave interesting data by mass spectrometry although it was not possible to validate these results using the half GFP interaction assay. This thesis contributes significantly to the effort to identify PXR co-regulator proteins although further investigation using alternate techniques may yield more information. Conclusive demonstration of a differential gene expression by different ligands came in the form of a gene array based on primary rat hepatocytes dosed with pregnenalone-16?-carbonitrile (PCN) and lithocholic acid (LCA). Appropriate dose ranges of these compounds were established in a separate, pilot, study and were within appropriate literature dose ranges. This gene array generated some 2000+ significantly altered gene expression profiles, as determined by statistical analysis of the data. Venn diagram analysis shows a clear difference between PCN and LCA with a number of commonly and individually expressed genes. A number of significantly altered genes were identified by principal components analysis as being important drivers of difference between the effects of PCN and LCA. Taqman QPCR analysis of these genes gave strong evidence that these two compounds are exerting different transcriptional effects.

Eukaryotic cells respond to extracellular stimuli by transmitting intracellular instructions via signalling pathways to coordinate appropriate responses. Mitogen-activated protein kinase (MAPK) pathways are often used to transmit these instructions to regulate gene expression, where Ternary Complex Factors (TCFs) are among their nuclear targets. Elk-1 is the founding member of the TCF family of transcription activators. The mechanism of function and regulation of Elk-1 has been extensively studied, therefore providing a paradigm for signal-induced transcription. The activity of Elk-1 is influenced by Post-Translational Modifications (PTMs), such as phosphorylation and sumoylation. Elk-1 ubiquitylation has also been reported in vitro, however little work has been done on this modification of Elk-1. This thesis sought to reveal the mechanism of regulation and function of Elk-1 ubiquitylation. Elk-1 was demonstrated to be both monoubiquitylated and polyubiquitylated in vitro and in cells. Using size exclusion chromatography and the dominant negative nedd8 conjugating E2 enzyme Ubc12, several features of the Elk-1 specific E3 ligases have been revealed in vitro. In cells, ternary complex formation was shown to be important for monoubiquitylation. Furthermore monoubiquitylated Elk-1 is diminished following ERK-mediated phosphorylation, hence activation, in response to mitogen stimulation. It was also demonstrated that an Elk-1 derivative that exhibits strong monoubiquitylation level also exhibits a reduced capability to transactivate gene expression at the Serum Responsive Element (SRE), indicating a negative role of monoubiquitylation on Elk-1 transcriptional ability.

RNA polymerase III (Pol III) is responsible for transcribing a relatively small but vital set of genes, including 5S rRNA and tRNAs. Pol III transcription has been shown to be upregulated in transformed and cancer cells, suggesting an important role in cell growth and proliferation. Its tight regulation is, therefore, fundamental for cell welfare, and a number of factors have been shown to be implicated in its control. These include the tumour suppressors p53 and Rb, as well as p107 and p130, and the basal transcription factor Dr1. The work in this thesis focused on the role of these repressors in regulating Pol III transcription in human cells.

Cid1 is a cytoplasmic member of a novel class of regulatory poly(A) polymerases discovered recently in yeast, worms and vertebrates. Previous genetic studies in the fission yeast, Schizosaccharomyces pombe, suggested a role for Cid1 in the checkpoint response to replication stress, but it was not known how a poly(A) polymerase might contribute to this response. Further investigations into the mode of action of Cid1 were therefore undertaken in this study. Cid1 is likely to target specific RNAs for polyadenylation; potential RNA substrates were identified using the complementary methods of microarray hybridisation and whole proteome analysis using two-dimensional liquid chromatography. These experiments revealed that Cid1 does not affect RNAs during normal, unperturbed growth but instead alters the expression of specific subsets of genes during replication stress. Many RNAs affected by Cid1 in these circumstances were cell-cycle dependent and telomeric transcripts, including those encoding histones and a novel RecQ helicase, Rqh2. As Cid1 lacks an RNA recognition motif, it is unlikely to bind selectively to RNA targets on its own. Cid1-interacting proteins were identified using yeast two-hybrid and tandem affinity purification methods. From these studies, novel members of a Cid1 complex have been discovered including: a previously uncharacterised metallo-beta-lactamase, RNA-binding proteins, ribosomal proteins and a telomere-binding protein. Together, these approaches are leading to a model for the role of cytoplasmic polyadenylation by Cid1 in checkpoint control.

Transcription factor E2F plays in important role in growth control by co-ordinating early cell cycle events. In addition, certain E2F family members, including E2F-1, are endowed with apoptotic activity. E2F-1 is regulated during cell cycle progression and inducible by cellular stress, such as DNA damage. Within the DNA damage signalling pathway, checkpoint kinases act as effectors of the damage response through phosphorylating key substrates involved in growth control. Here, I report that checkpoint kinase Chk2 regulates E2F-1 activity in response to etoposide. A Chk2 kinase phosphorylation site resides in E2F-1, and undergoes physiological phosphorylation in response to DNA damage. Phosphorylation of E2F-1 by Chk2 leads to protein stabilization, increased half-life and transcriptional activation, and phosphorylated E2F-1 resides in discrete nuclear structures. A dominant-negative derivative of Chk2 blocks the induction of E2F-1, and prevents E2F-1-dependent apoptosis. Moreover, E2F-1 fails to be induced by etoposide in tumour cells that carry mutant chk2. Chk2 therefore phosphorylates and activates E2F-1 in the cellular response to DNA damage, and the damage induction of E2F-1 leads to apoptosis. The results suggest a role for E2F-1 in response to stress, perhaps in checkpoint control, and provide a plausible mechanistic and physiological explanation for the tumour suppressor activity of E2F-1.

Chez les bactéries sporulantes du genre Bacillus, des mécanismes importants tels que la sporulation et la virulence sont régulés par des systèmes de communication cellulaire qui impliquent des peptides de signalisation et des régulateurs de la famille RNPP (Rap, NprR, PlcR, PrgX). L'objectif de mon travail de thèse a été de déterminer le rôle du régulateur NprR chez les bactéries du groupe B. cereus. Ce travail se divise en trois parties complémentaires. La première partie a consisté à montrer que NprR est impliqué dans un système de communication cellulaire. Nous avons montré que NprR est un régulateur transcriptionnel de début de phase stationnaire qui est dépendant du peptide de signalisation NprX. Associé à NprX, NprR active la transcription du gène nprA qui code pour une protéase extracellulaire. Nous avons démontré que le peptide NprX est sécrété, maturé puis réimporté dans la cellule bactérienne par deux systèmes d'oligopeptide perméase (Opp et Npp). Une fois dans la cellule, la forme mature de NprX (vraisemblablement l'heptapeptide SKPDIVG) se lie à NprR et permet la transcription du gène nprA. Nous avons ensuite cherché à déterminer la fonction de ce régulateur au cours du cycle infectieux de B. thuringiensis (Bt) chez l'insecte. Nous avons montré que NprR est actif après la mort de l'insecte et permet aux bactéries de survivre, sous forme de cellules végétatives, dans les cadavres. Une analyse transcriptomique indique que NprR régule l'expression d'au moins 41 gènes qui codent notamment pour des enzymes dégradatives et un locus de gènes impliqués dans la production d'un peptide synthétisé de façon non ribosomique (la kurstakine). Nous avons démontré que les gènes codant pour les enzymes dégradatives s'expriment spécifiquement après la mort de l'hôte et que les produits de ces gènes sont essentiels pour hydrolyser différents substrats (protéines, lipides, chitine), ce qui suggère que Bt a un mode de vie nécrotrophe dans le cadavre. La kurstakine est essentielle pour la survie de Bt pendant son développement nécrotrophe et nous avons montré que cette molécule est nécessaire pour le swarming et la formation de biofilm. Par ailleurs, un mutant du gène nprR ne se développe pas et ne sporule pas efficacement dans le cadavre. L'ensemble de nos résultats indiquent que le necrotrophisme est un mode de vie hautement régulé, qui est essentiel dans le cycle infectieux de Bt car il contribue à la transmission horizontale de ce micro-organisme. Enfin, nous avons étudié la régulation de l'expression des gènes nprR et nprX. Nous avons montré que les gènes nprR-nprX sont co-transcrits à partir d'un promoteur dépendant de sigma-A (PA) situé en amont du gène nprR. La transcription à partir de ce promoteur débute lors de l'entrée en phase stationnaire et est contrôlée par deux régulateurs transcriptionnels: CodY et PlcR. Le répresseur CodY pourrait se lier à l'ADN en amont du promoteur PA et réprimer la transcription des gènes nprR-nprX pendant la phase exponentielle de croissance. Au début de la phase stationnaire, le contrôle négatif de CodY est levé et PlcR active la transcription de nprR-nprX en se liant à une boîte PlcR située en amont de PA. Nos résultats indiquent que nprX est également transcrit indépendamment de nprR à partir de deux promoteurs, PH et PE, respectivement dépendant de sigma-H et sigma-E. Les deux promoteurs permettent d'assurer la transcription de nprX en phase stationnaire tardive alors que la transcription à partir du promoteur PA est achevée. Cette étude met en évidence le role clé des régulateurs CodY, PlcR and Spo0A dans la régulation de l'expression des gènes nprR-nprX. / In sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signaling peptides and regulators of the RNPP family. In this work, we investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. This work can be divided into three complementary parts.The first part consisted to demonstrate that NprR is involved in a quorum-sensing system. We showed that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene (nprA) during the first stage of the sporulation process. We demonstrated that the NprX peptide is secreted, processed and then reimported within the bacterial cell by two oligopeptide permease systems (Opp and Npp). Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. The second part was to explore the function of NprR during the infectious cycle of B. thuringiensis (Bt). We showed that NprR is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes encoding degradative enzymes or involved in the synthesis of a non-ribosomal peptide named kurstakin. The degradative enzymes include chitinases, proteases and lipases. The corresponding genes are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We showed that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. Altogether, our results show that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to horizontal transmission. Finally, the last part of my PhD consisted to study the regulation of nprR and nprX expression. We showed that the nprR-nprX genes are cotranscribed from a sigma A-dependent promoter (PA) located upstream from nprR. The transcription from PA starts at the onset of the stationary phase and is controlled by two transcriptional regulators: CodY and PlcR. The nutritional repressor CodY binds a DNA target site upstream from PA and represses nprR-nprX transcription during the exponential growth phase. At the onset of the stationary phase, the negative control of CodY is relieved and PlcR activates nprR-nprX transcription by binding a PlcR box located upstream from PA. We showed that nprX is also transcribed independently of the nprR transcription from two promoters, PH and PE, dependent on sigma-H and sigma-E, respectively. Both promoters ensure nprX transcription during late stationary phase and sporulation while transcription from PA is complete. This study highlights the key role played by CodY, PlcR and Spo0A in nprR-nprX transcription.