La plus grosse sous-unité (Rpb1) de l'ARN polymérase II est caractérisée par une structure unique et flexible au niveau du domaine C-terminal (CTD) qui consiste en une répétition en tandem d'une séquence consensus heptapeptidique Y1-S2-P3-T4-S5-P6-S7. Le CTD est essentiel pour pour la viabilité cellulaire et est requis pour d'importantes activités associées avec la transcription par l'ARN Pol II, dont la régulation de la synthèse de l'ARN et des événements de maturation co-transcriptionnels (mise en place de la coiffe 5', épissage, terminaison en 3'... etc). Forts d'un système permettant l'expression conditionnelle de mutants du CTD, nous disséquons le CTD afin de comprendre l'implication de résidus/répétitions spécifiques sur la transcription dans des cellules mammifères. Nos résultats montrent que le CTD est crucial pour le contrôle de la transcription pervasive et indiquent une implication inédite des complexes Mediateur et Integrateur dans le processus de terminaison de la transcription. / The largest subunit (Rpb1) of RNA polymerase II has a unique and flexible structure at its C-terminal domain (CTD) that consists of tandem repeats with the consensus heptad sequence Y1-S2-P3-T4-S5-P6-S7. The CTD is essential for cellular viability and is required for important activities associated with RNA pol II transcription, including the regulation of RNA synthesis and co-transcriptional processing events (5’ capping, splicing, 3’ termination…etc). Using a conditional CTD mutant expression system, we dissect the importance of specific residues/repeats on transcription in mammalian cells. Our results indicate the importance of the CTD in the control of pervasive transcription and hints to novel roles of the Mediator and Integrator complexes in transcription termination processes.

<p>The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro and the identification of similar complexes from metazoans indicates that its function is conserved through evolution. Mediator subunit composition and structure is well characterized both by biochemical, genetic and biophysical methods. In contrast, little is known about the mechanisms by which Mediator operates and how the complex is regulated. The aim of my thesis was to elucidate how Mediator functions at the molecular level and to investigate functional interactions within Mediator. </p><p> It is possible to recruit RNA polymerase II to a target promoter and thus to activate transcription by fusing Mediator subunits to a DNA binding domain. In order to investigate functional interactions within Mediator, we made such fusion proteins where different Mediator subunits were fused to the DNA binding domain of lexA. The expression of a reporter gene containing binding sites for lexA was subsequently measured in both a wild type strain and in strains where genes encoding specific Mediator subunits had been disrupted. We found that lexA-Med2 and lexA-Gal11 are strong activators that function independently of all Mediator subunits tested. On the other hand, lexA-Srb10 is a weak activator that depends on Srb8 and Srb11 and lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. Both lexA-Med1 and lexA-Srb7 proteins showed a stable association with the Mediator subunits Med4 and Med8 in wild type cells and in all deletion strains tested, indicating that they were functionally incorporated into the Mediator complex. We also showed that both Med4 and Med8 exist in two forms that differed in electrophoretic mobility and that these forms differed in their ability to associate with Mediator immuno-purified from the LEXA-SRB7 and LEXA-MED1 strains. Dephosphorylation assays of purified Mediator indicated that the two mobility forms of Med4 corresponded to the phosphorylated and unphosphorylated forms of the Med4 protein respectively. </p><p> Some of the data presented in this study as well as previous genetic and biochemical data obtained in our lab suggested a functional link between the Med1, Med2, Srb10 and Srb11 proteins. We extended these findings by showing that the Srb10 kinase phosphorylates the Med2 protein at residue serine 208, both in vitro and in vivo. We also showed that a point mutation of the single phosphorylation site to an alanine or to an aspartic acid residue altered the gene expression of a specific set of genes. Taken together, these data indicate that posttranslational modification of Mediator subunits is a so far uncharacterized mechanism for regulation of gene expression. </p><p> In order to study the function of the Srb7 subunit of Mediator, we isolated a temperature sensitive strain where the amino acids 2 to 8 of srb7 were deleted. The Mediator subunits Nut2 and Med7 were isolated as high copy suppressor of srb7-∆(2-8) and we were also able to show that Srb7 interacted with Nut2 and Med7 both in a 2-hybrid system and in co-immuno precipitation experiments using recombinantly expressed proteins. Interestingly, a deletion of amino acids 2 to 8 of Srb7 abolishes its interaction with both Med7 and Nut2 in vitro. Med4 also interacted with Srb7 in the 2-hybrid system and surprisingly, the first eight amino acids of Srb7 were shown to be sufficient for this interaction.</p>

The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro and the identification of similar complexes from metazoans indicates that its function is conserved through evolution. Mediator subunit composition and structure is well characterized both by biochemical, genetic and biophysical methods. In contrast, little is known about the mechanisms by which Mediator operates and how the complex is regulated. The aim of my thesis was to elucidate how Mediator functions at the molecular level and to investigate functional interactions within Mediator. It is possible to recruit RNA polymerase II to a target promoter and thus to activate transcription by fusing Mediator subunits to a DNA binding domain. In order to investigate functional interactions within Mediator, we made such fusion proteins where different Mediator subunits were fused to the DNA binding domain of lexA. The expression of a reporter gene containing binding sites for lexA was subsequently measured in both a wild type strain and in strains where genes encoding specific Mediator subunits had been disrupted. We found that lexA-Med2 and lexA-Gal11 are strong activators that function independently of all Mediator subunits tested. On the other hand, lexA-Srb10 is a weak activator that depends on Srb8 and Srb11 and lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. Both lexA-Med1 and lexA-Srb7 proteins showed a stable association with the Mediator subunits Med4 and Med8 in wild type cells and in all deletion strains tested, indicating that they were functionally incorporated into the Mediator complex. We also showed that both Med4 and Med8 exist in two forms that differed in electrophoretic mobility and that these forms differed in their ability to associate with Mediator immuno-purified from the LEXA-SRB7 and LEXA-MED1 strains. Dephosphorylation assays of purified Mediator indicated that the two mobility forms of Med4 corresponded to the phosphorylated and unphosphorylated forms of the Med4 protein respectively. Some of the data presented in this study as well as previous genetic and biochemical data obtained in our lab suggested a functional link between the Med1, Med2, Srb10 and Srb11 proteins. We extended these findings by showing that the Srb10 kinase phosphorylates the Med2 protein at residue serine 208, both in vitro and in vivo. We also showed that a point mutation of the single phosphorylation site to an alanine or to an aspartic acid residue altered the gene expression of a specific set of genes. Taken together, these data indicate that posttranslational modification of Mediator subunits is a so far uncharacterized mechanism for regulation of gene expression. In order to study the function of the Srb7 subunit of Mediator, we isolated a temperature sensitive strain where the amino acids 2 to 8 of srb7 were deleted. The Mediator subunits Nut2 and Med7 were isolated as high copy suppressor of srb7-∆(2-8) and we were also able to show that Srb7 interacted with Nut2 and Med7 both in a 2-hybrid system and in co-immuno precipitation experiments using recombinantly expressed proteins. Interestingly, a deletion of amino acids 2 to 8 of Srb7 abolishes its interaction with both Med7 and Nut2 in vitro. Med4 also interacted with Srb7 in the 2-hybrid system and surprisingly, the first eight amino acids of Srb7 were shown to be sufficient for this interaction.

Le cancer du sein est la principale cause de mortalité par cancer chez la femme. Deux des facteurs de transcription de la famille Fos, Fra-1 et Fra-2, sont surexprimés dans les cancers du sein agressifs et contribuent au phénotype tumoral en favorisant entre autres, la prolifération, la motilité et l'invasivité. De façon surprenante, les mécanismes moléculaires via lesquels Fra-1 et Fra-2 (et plus généralement le complexe transcriptionnel AP-1 dont ils sont des constituants) gouvernent la transcription de leurs gènes cibles sont quasi-inconnus. Dans ce contexte, en combinant diverses approches (immunoprécipitation de chromatine, interférence à l'ARN…), j'ai étudié les mécanismes moléculaires par lesquels Fra-1 et Fra-2 contrôlent la transcription dérégulée du gène de l'urokinase ou uPA (sérine protéase cruciale dans la progression tumorale et l'établissement de métastases) qui est l'un des nouveaux marqueurs utilisés en clinique pour la mise en place des choix thérapeutiques. Mes travaux montrent de façon originale que (i) Fra-1 et Fra-2 agissent de façon non redondante et coopèrent pour réguler l'expression d'uPA via leur fixation sur un enhancer AP-1 localisé à -1,9 kb du site d'initiation de la transcription (TSS), (ii) Fra-2 est nécessaire au recrutement de RNA Pol II au niveau de l'enhancer, tandis que Fra-1 stimule le passage de RNA Pol II de sa forme initiatrice à sa forme élongatrice et (iii) que la polymérase recrutée à l'enhancer rejoint le TSS par un mécanisme de « tracking », très rarement décrit dans la littérature, en produisant de petits ARNs non codants, bidirectionnels et instables. / Breast cancer is the most frequent malignant disease among women. Two transcription factors, Fra-1 and Fra-2, belonging to the Fos family members, are overexpressed in aggressive breast cancers and contribute to the tumorigenic phenotype by favoring proliferation, motility and invasion. Surprisingly, the molecular mechanisms governed by Fra-1 and Fra-2 (and more generally by the AP-1 transcriptional complex, which they are components of) for the transcription of their target genes are still largely unknown. In this context, by combining different approaches (chromatin immunoprecipitation, RNA interference…), I studied the molecular mechanisms orchestrated by Fra-1 and Fra-2 for the expression of the urokinase (or uPA) gene (encoding a serine protease crucial for tumor progression and metastasis), which is one of the new diagnostic markers now taken into consideration for deciding therapeutic strategies. Interestingly, my results show that (i) Fra-1 and Fra-2 have non redundant functions and cooperate for the transcriptional regulation of uPA through their binding to AP-1 enhancer located 1.9 kb upstream of the transcriptional start site (TSS), (ii) Fra-2 is required for the recruitment of RNA Pol II on this enhancer while Fra-1 allows the conversion of RNA Pol II initiating form into its elongating form and (iii) enhancer-recruited RNA Pol II reaches the TSS by a tracking mechanism, mechanism very rarely described in the literature, during which it synthetizes small, unstable bidirectional, non coding RNAs.

<p>Cellular processes involved in the conversion of the genetic information from DNA into a protein are often regulated by reversible phosphorylation reactions. By modulating the phosphorylated status of key proteins their activity can either be enhanced or repressed. In this thesis I have studied the significance of phosphorylation in the regulation of transcription and splicing using human adenovirus as a model system.</p><p>The results show that the activity of the cellular SR family of splicing enhancer or repressor proteins are reduced in adenovirus infected nuclear extracts by a virus-induced hypophosphorylation. The viral E4-ORF4 was shown to induce SR protein dephosphorylation by recruiting the cellular protein phosphatase PP2A. The E4-ORF4/PP2A complex was shown to relieve the SR protein-mediated repression of late virus-specific splicing and further activate alternative splicing in transiently transfected cells. Collectively, these results showed that alternative splicing, like many other biological processes, is regulated by reversible protein phosphorylation.</p><p>Similarly, the cellular p32 protein was shown to cause hypophosphorylation of the SR protein ASF/SF2 resulting in a reduced RNA binding capacity of ASF/SF2. This change in ASF/SF2 RNA binding also had a drastic effect on the function of ASF/SF2 as a regulatory protein affecting splice site choice. The cellular p32 protein and the viral E4-ORF4 protein both target the same cellular splicing factor, ASF/SF2. However, they regulate splicing by different mechanisms. E4-ORF4 recruits a phosphatase to dephosphorylate ASF/SF2, while p32 sequester ASF/SF2 in an inactive complex.</p><p>Further, we demonstrated that overexpression of p32 during a lytic infection suppressed transcription from the adenovirus major late transcription unit. p32 induced a selective repression of CAAT-box containing promoters indicating the involvement of the transcription factor CBF/NF-Y in this regulation. A further analysis showed that p32 caused a hyperphosphorylation of the CTD of RNA Pol II, which resulted in a significant reduction in the processivity of Pol II during the elongation phase of transcription.</p><p>In summary, we have shown that E4-ORF4 regulates the activity of splicing regulatory SR proteins, and that p32 regulates the activity of the SR protein ASF/SF2 in splicing and Pol II processivity during transcription elongation. Mechanistically, both E4-ORF4 and p32 appears to function by regulating the phosphorylated status of key cellular proteins involved in these processes.</p>

Cellular processes involved in the conversion of the genetic information from DNA into a protein are often regulated by reversible phosphorylation reactions. By modulating the phosphorylated status of key proteins their activity can either be enhanced or repressed. In this thesis I have studied the significance of phosphorylation in the regulation of transcription and splicing using human adenovirus as a model system. The results show that the activity of the cellular SR family of splicing enhancer or repressor proteins are reduced in adenovirus infected nuclear extracts by a virus-induced hypophosphorylation. The viral E4-ORF4 was shown to induce SR protein dephosphorylation by recruiting the cellular protein phosphatase PP2A. The E4-ORF4/PP2A complex was shown to relieve the SR protein-mediated repression of late virus-specific splicing and further activate alternative splicing in transiently transfected cells. Collectively, these results showed that alternative splicing, like many other biological processes, is regulated by reversible protein phosphorylation. Similarly, the cellular p32 protein was shown to cause hypophosphorylation of the SR protein ASF/SF2 resulting in a reduced RNA binding capacity of ASF/SF2. This change in ASF/SF2 RNA binding also had a drastic effect on the function of ASF/SF2 as a regulatory protein affecting splice site choice. The cellular p32 protein and the viral E4-ORF4 protein both target the same cellular splicing factor, ASF/SF2. However, they regulate splicing by different mechanisms. E4-ORF4 recruits a phosphatase to dephosphorylate ASF/SF2, while p32 sequester ASF/SF2 in an inactive complex. Further, we demonstrated that overexpression of p32 during a lytic infection suppressed transcription from the adenovirus major late transcription unit. p32 induced a selective repression of CAAT-box containing promoters indicating the involvement of the transcription factor CBF/NF-Y in this regulation. A further analysis showed that p32 caused a hyperphosphorylation of the CTD of RNA Pol II, which resulted in a significant reduction in the processivity of Pol II during the elongation phase of transcription. In summary, we have shown that E4-ORF4 regulates the activity of splicing regulatory SR proteins, and that p32 regulates the activity of the SR protein ASF/SF2 in splicing and Pol II processivity during transcription elongation. Mechanistically, both E4-ORF4 and p32 appears to function by regulating the phosphorylated status of key cellular proteins involved in these processes.

Les protéines Hox sont des facteurs de transcription à homéodomaine, dont les propriétés de liaison à l’ADN contrastent avec leur spécificité fonctionnelle in vivo. Ils interagissent avec les cofacteurs PBC (Extradenticle (Exd) chez la drosophile) formant des complexes multimériques dont la spécificité fonctionnelle est accrue. Cette interaction repose sur le motif l’hexapeptide (HX), conservé dans la plupart des protéines Hox. Récemment, nous avons identifié le domaine UbdA (UA), spécifique aux protéines Hox de classe centrale Ultrabithorax (Ubx) et AbdominalA (Abd-A), comme un nouveau motif d'interaction avec la protéine Exd. Des analyses in vivo de la contribution de l’HX et de UbdA dans l’activité des protéines Ubx et Abd-A ont indiqué que les protéines Ubx et Abd-A partagent des fonctions (Exd dépendantes et indépendantes), qui ne sont pas médiées par une utilisation identique des motifs protéiques HX et UA.L’objectif de ces travaux est d’analyser les mécanismes moléculaires qui sous-tendent une utilisation ciblée/sélective des motifs protéiques HX et UA de Ubx et de Abd-A en absence du cofacteur connu Exd. Pour cela, des lignées cellulaires S2 DRSC exprimant les protéines Ubx sauvages et mutantes sur les motifs HX et UA, ont été générées et analysées par des expériences de ChIP-Seq. Nos données comparées à celles obtenues précédemment dans l’équipe pour la protéine Abd-A posent les bases permettant d’appréhender la contribution fonctionnelle et l’utilisation sélective des motifs protéiques HX et UA, au-delà de leurs fonctions dans la médiation de l'interaction avec le cofacteur Exd. / Hox proteins are homeodomain-containing transcription factors, whose poor DNA-binding properties contrast with their functional specificity in vivo. They interact with PBC cofactors (Extradenticle (Exd) in Drosophila), forming multimeric complexes with increased functional specificity. This interaction involve a conserved motif called the hexapeptide (HX), found in most Hox proteins. Recently, we the UbdA domain (UA), specific to the central class Hox proteins Ultrabithorax (Ubx) and Abdominal-A (Abd-A), as a novel interaction motif with the Exd protein. In vivo analysis of the HX and UA contributions to Ubx and Abd-A protein activity indicated Ubx and Abd-A shared functions (Exd dependent or independent) do not necessarily rely on a similar use of the HX or UA protein motifs. The aim of this work was to investigate the molecular mechanisms underlying the targeted/selective use of the HX and UA protein motifs in Ubx and Abd-A in the absence of the usual Hox Exd cofactor. For this, S2 DSRC cell lines stably expressing the Ubx protein, as well as HX or UA variants have been generated and analysed by ChiP-Seq experiments. Our data, compared to those previously obtained for Abd-A in the laboratory, set bases for apprehending the functional contribution and selective use of the HX and UA protein motifs, outside their established function in mediating interaction with the Exd cofactor.

RNA polymerase II (Pol II) is the enzyme responsible for the synthesis of all mRNAs in eukaryotic cells. As the central component of the eukaryotic transcription machinery, Pol II is the final target of transcription regulatory pathways. While the role for different Pol II associated proteins, co-activators and general transcription factors (GTFs) in regulation of transcription in response to different stimuli is well studied, a similar role for some subunits of the core Pol II is only now being recognized. The studies reported in this thesis address the role of the fourth largest subunit of Pol II, Rpb4, in transcription and stress response using Saccharomyces cerevisiae as the model system. Rpb4 is closely associated with another smaller subunit, Rpb7 and forms a dissociable complex (Edwards et al. 1991). The rpb4 null mutant is viable but is unable to survive at extreme temperatures (>34ºC and <12ºC) (Woychik and Young, 1989). This mutant has also been shown to be defective in activated transcription and unable to respond adequately to several stress conditions (Pillai et al. 2001; Sampath and Sadhale, 2005). In spite of wealth of available information, the exact role of Rpb4 in transcription process remains poorly understood. In the present work, we have used genetic, molecular and biochemical approaches to understand the role of Rpb4 as described in three different parts below: I. Role of Rpb4 in various pathways related to Transcription Elongation The genome-wide recruitment study of RNA pol II in presence and absence of Rpb4 has indicated role of Rpb4 in transcription elongation (Verma-Gaur et al. 2008). However, a recent proteomics based report has argued against it (Mosley et al. 2013). To address this conflict and understand Rpb4 functions, we monitored recruitment of RNA pol II on a few individual long genes in wild type and rpb4∆ cells. It was observed that RNA pol II recruitment on genes with longer coding regions is not significantly affected in rpb4∆ as compared to wild type thus ruling out role of Rpb4 in transcription elongation of these genes. However, our genetic interaction studies have shown a strong interaction (synthetic lethality) between RPB4 and the PAF1 and SPT4 genes, the products of which code for well-known transcription elongation factors. The studies based on Rpb4 overexpression in mutants for elongation factors, 6-Azauracil sensitivity of cells, effect of Dst1 overexpression in rpb4∆ cells and mitotic recombination rate in rpb4∆ cells have indicated functional interactions of Rpb4 with many of the transcription elongation factors. II. Studies on Genetic and Functional Interactions of Rpb4 with SAGA Complex in Promoter- Specific Transcription Initiation To carry out transcription, RNA pol II depends on several general transcription factors, mediators, activators, co-activators and chromatin remodeling complexes. In the present study, we explored the genetic and functional relationships between Rpb4 and the SAGA complex of transcription machinery, to gain some insight on the role of Rpb4 during transcription. Our chromatin immunoprecipitation data suggest that RNA pol II does not associate with promoters of heat shock genes during transcription activation of these heat stress induced genes in absence of Rpb4. SAGA coactivator complex is required for RNA pol II recruitment and transcription activation of these genes (Zanton and Pugh, 2004). However, recruitment of the SAGA complex at promoters of these heat shock genes was not affected in rpb4∆ cells after heat stress. Our genetic interaction analysis between RPB4 and components of SAGA complex (spt20∆) showed synthetic lethality indicating that fully functional Rpb4 and SAGA complex are required for cellular functions in the absence of heat stress and the simultaneous deletion of factors in the two complexes leads to cell death. III. Role of Rpb4 in phosphorylation cycles of Rpb1-CTD The C-Terminal Domain (CTD) of Rpb1 protein of RNA pol II undergoes several rounds of phosphorylation cycles at Ser-2 and Ser-5 residues on its heptad repeats during transcription. These phosphorylation marks are to be erased before the start of next round of transcription. Using protein pull down assay, we observed that hyperphosphorylated form of Rpb1 is reduced in rpb4∆ as compared to that seen in wild type cells among the free RNA pol II molecules. The level of Rpb2 protein was unaffected in both wild type and rpb4∆. These preliminary data hints at role of Rpb4 in the regulation of Rpb1 phosphorylation.

El gen humano 11&#61538;-HSD2 es un modelo para investigar la contribución de los efectos de los receptores de esteroides en células de cáncer de mama. El análisis del promotor mostró que la región distal está implicada en la mayor parte de la activación dependiente de hormona. En respuesta a hormona, STAT5A se recluta a la región distal y PR a las regiones distal y proximal del promotor. El reclutamiento de PR se debe a dos mecanismos diferentes, la unión directa de PR a la región proximal, y la implicación vía JAK/STAT en el reclutamiento a la región distal. La inducción del gen 11&#61538;-HSD2 por hormonas disminuye parcialmente por inhibidores de MAPK y PI3K/Akt y totalmente por inhibidores de JAK/STAT. Así, los efectos citoplasmáticos del PR están implicados en la inducción del gen progesterona. La forma activa de la ARN-polimerasa II es reclutada por la inducción con hormonas a la región distal del promotor 11&#61538;-HSD2 y la región distal tiene respuesta a hormonas por sí misma, indicando que la inducción del gen por hormonas empieza antes del sitio de inicio de transcripción descrito previamente. / The human 11&#61538;-HSD2 gene is a model to investigate the contribution of steroid hormone receptors effects on a progesterone responsive promoter in breast cancer cells. Deletion analysis of the 11&#61538;-HSD2 promoter showed that the distal region is involved in most of the hormone-dependent activation. ChIP showed hormone-dependent STAT5A-recruitment to the distal region and PR-recruitment to the distal and proximal promoter regions. Results suggest two different mechanisms of hormone-induced PR-recruitment, since cells stably expressing PR containing a mutated DNA-binding domain have affected hormone-dependent PR-recruitment to proximal promoter, and JAK/STAT pathway inhibition blocks PR-recruitment to distal promoter. Hormone-stimulated 11&#61538;-HSD2 gene-expression was partially decreased by MAPK and PI3K/AKT pathway inhibitors and totally blocked by JAK/STAT pathways inhibitors, indicating that cytoplasmic PR effects involvement in progestin-induced 11&#61538;-HSD2 expression. Importantly, upon hormone induction active RNA-polymerase II is recruited from the 11&#61538;-HSD2 distal promoter region and the distal minimal promoter has hormone-responsiveness by itself, suggesting that progesterone-dependent 11&#61538;-HSD2 expression starts upstream the previously characterized transcription start site.