Genexpression in Eukaryoten beschreibt einen mehrstufigen Prozess, welcher auf Ebene der Transkription durch den positiven Transkriptionselongationsfaktor P-TEFb entscheidend reguliert wird. PTEFb bildet einen heterodimeren Komplex aus der Cyclin abhängigen Kinase 9 und deren Kofaktor Cyclin T1/2. Dieser Komplex aktiviert die Elongation der Transkription durch Phosphorylierung der negativen Elongationsfaktoren DSIF und NELF. Darüber hinaus phosphoryliert PTEFb Serin2 Reste in der C-terminalen Domäne von RNA PolII und stimuliert so die kotranskriptionelle Prozessierung der synthetisierten prä-mRNA. In Anpassung an unterschiedliche Wachstumsbedingungen wird die Aktivität dieses Faktors durch reversible Interaktion mit 7SK RNA und HEXIM Proteinen innerhalb eines katalytisch inaktiven Ribonukleoproteinpartikels (7SK RNP) streng kontrolliert. Dieses sensible Gleichgewicht zwischen P-TEFb auf der einen und dem 7SK RNP auf der anderen Seite bildet die Grundlage der Regulation der Transkriptionselongation. Trotz der hohen Abundanz von 7SK RNA in der Zelle, assoziiert in vivo jedoch nur ein relativ kleiner Teil hiervon mit P-TEFb, sodass die effektiv zur Verfügung stehende RNA-Menge für die Bildung des 7SK RNP vermutlich limitierend wirkt. Ziel der vorliegenden Arbeit war es daher neue 7SK RNA interagierende Faktoren zu identifizieren, welche die Interaktion von PTEFb mit dem 7SK RNP steuern und so die PolII abhängige Transkription regulieren. Anhand verschiedener chromatographischer Reinigungen konnte zunächst ein bislang uncharakterisiertes La ähnliches Protein (LARP7) mit einer spezifischen Affinität für Pyrimidinreiche RNAs isoliert werden. LARP7 bindet, wie durch immunbiochemische Analysen und RNA- Bindungsstudien gezeigt werden konnte, quantitativ an das hoch konservierte uridylreiche 3´- Ende von 7SK RNA. Diese Assoziation erfordert dessen La- und RRMDomänen und erhöht wesentlich die Stabilität der RNA. Darüber hinaus kofraktioniert LARP7 mit weiteren Faktoren des 7SK RNP, bindet direkt an HEXIM1 und P-TEFb und stellt somit ebenfalls eine integrale Komponente des 7SK RNP dar. Die gewonnenen Daten weisen außerdem erstmals darauf hin, dass P-TEFb durch einen vorgeformten trimeren Komplexes, bestehend aus HEXIM1, 7SK RNA und LARP7 inhibiert wird. Reportergenanalysen in TZMbl-Zellen, welche Luziferase unter der Kontrolle des streng P-TEFb abhängigen HIV-1-LTRPromotors exprimieren zeigten, dass diese Inhibition im Wesentlichen durch LARP7 vermittelt wird. So ließ sich nach Reduktion der LARP7 Expression mittels RNAi eine signifikante Steigerung der Transkription vom HIV-1-LTR-Promotor beobachten. Eine ähnliche Stimulation der Transkription von PolII konnte in LARP7 defizienten HeLa-Zellen durch quantitative Real-Time-PCR auch für eine Reihe zellulärer Gene nachgewiesen werden. Die Beobachtung, dass LARP7 die generelle PolII Transkription reprimiert, korreliert zudem mit einer bereits beschriebenen Tumorsupressorfunktion des LARP7 homologen mxc Proteins aus D. melanogaster. Somit beeinflusst LARP7 das zelluläre Gleichgewicht zwischen freiem und 7SK RNP-gebundenem P-TEFb und fungiert somit als negativer Regulator der PolII Transkription in vivo. / Eucaryotic gene expression is a multistep process, which is critically regulated on the level of RNA polII transcription by the positive transcription elongation factor P-TEFb. P-TEFb forms a heterodimeric complex, consisting of the cyclin-dependent kinase 9 and its cofactor cyclin T1/2. This complex stimulates transcriptional elongation as well as the cotranscriptional processing of the synthesized pre-mRNA by phosphorylation of negative elongation factors and the RNA polII Cterminal domain. To accommodate different growth conditions, P-TEFb activity is kept under tight control through its reversible interaction with 7SK RNA and HEXIM proteins in a catalytically inactive ribonucleoprotein particle (RNP). This sensitive balance between PTEFb on the one hand and the 7SK RNP on the other represents the basis of transcriptional regulation of elongation. Despite the high abundance of 7SK RNA in the cell, only a small part is associated with P-TEFb in vivo, suggesting that the actual amount of RNA available limits the formation of the 7SK RNP. Hence, the objective of the present study was to identify novel 7SK RNA interacting factors, which direct the interaction of P-TEFb with the 7SK RNP, thereby regulating polII dependent transcription. At first, using different chromatographic purification strategies, an as yet uncharacterized La related protein (LARP7) with an affinity to pyrimidine- rich RNAs was isolated. Immunobiochemical analysis and RNA binding studies revealed, that LARP7 quantitatively associates with the highly conserved 3´-UUU-OH terminus of 7SK RNA. This binding requires its La- and RRM-domain and dramatically increases RNA stability. Moreover, LARP7 co-fractionates with additional factors of the 7SK RNP, binds directly to HEXIM1 and P-TEFb and therefore likewise constitutes an integral component of the 7SK RNP. Data presented here indicate that P-TEFb is inhibited upon association with a trimeric complex consisting of HEXIM1, 7SK RNA and LARP7. Furthermore, reporter gene analysis in TZMbl cells - cells expressing luciferase under the control of the strictly P-TEFb dependent HIV-1-LTR promoter - demonstrated this inhibition to be mainly mediated by LARP7. Thus, reduction of LARP7 expression by RNA-interference led to a significant stimulation of transcription in TZMbl cells. In addition, quantitative real time pcr revealed a similar effect on transcription for a series of cellular genes in LARP7 deficient HeLa cells. Moreover, the observation, that LARP7 represses polII transcription in general correlates well with a known function of the d. melanogaster LARP7 homologue mxc as a tumor suppressor. Thus, LARP7 affects the cellular P-TEFb/7SK RNP equilibrium und serves as a negative regulator of polII transcription in vivo.

La latence post-intégrative du VIH-1 génère des réservoirs qui empêchent l'éradication du virus avec les thérapies actuelles. La compréhension des mécanismes moléculaires de la latence servirait à l'identification de nouvelles cibles thérapeutiques et le développement de molécules de type LRA (Latency Reversing Agent) permettant la guérison fonctionnelle des patients et un arrêt des traitements. Nous avons démontré que la combinaison de deux types différents de LRAs à base de composés libérant P-TEFb (JQ1, I-BET, I-BET151 et HMBA) ou des agonistes de PKC (prostratine, bryostatine-1 et Ing-B) conduit à une robuste activation synergique de la production du VIH-1 dans divers modèles cellulaires de latence post-intégrative et dans des réservoirs de lymphocytes T CD4+ primaires. Le répresseur transcriptionnel CTIP2 recrute des complexes multienzymatiques pour favoriser l'établissement et la persistance de la latence du VIH-1 dans les cellules microgliales, le principal réservoir viral du système nerveux central. De plus, CTIP2 s’associe au complexe 7SK snRNP pour inhiber P-TEFb, un facteur d’élongation indispensable à l’expression du VIH-1 et à la réactivation des provirus latents. Des expériences d’immunoprécipitations de CTIP2, couplées à la spectrométrie de masse, nous ont permis d’identifier près de 900 partenaires protéiques de CTIP2. Parmi ces nouveaux partenaires, nous avons identifié la SUMO E3 ligase KAP1. Nous montrons que KAP1 réprime les phases précoce et tardive Tat dépendante de la transcription du VIH-1. KAP1 induit une dégradation de Tat, qui est sensible aux modulations de la voie SUMO. En effet, la sumoylation favorise l'association de Tat avec KAP1, de même que sa dégradation. Globalement, nos résultats suggèrent que KAP1 contribue à l'établissement et à la persistance des réservoirs latents du VIH-1. Cibler les voies SUMO constituerait un nouveau champ d'investigation dans le cadre du développement de nouvelles classes de LRAs. / The HIV-1 post-integration latency generates reservoirs that prevent the eradication of the virus with the current therapies. The understanding of the molecular mechanisms of this latency enable the identification of new therapeutic targets and the development of LRA (Latency Reversing Agent) for functional cure and treatments interruption. We have demonstrated that the combination of two different LRAs based on P-TEFb releasing compounds (JQ1, I-BET, I- BET151 and HMBA) or PKC agonists (prostratin, bryostatin-1 and Ing-B) leads to robust synergistic activation of HIV-1 production in various cellular models of post-integration latency and in primary CD4+ T cells reservoirs. The transcriptional repressor CTIP2 recruits multienzymatic complexes to promote the establishment and persistence of HIV-1 latency in microglial cells, the main viral reservoir of the central nervous system. Furthermore, CTIP2 binds to the 7SK snRNP complex to inhibit P-TEFb, an elongation factor essential for HIV-1 expression and reactivation of latent proviruses. Immunoprecipitation experiments of CTIP2 coupled to mass spectrometry allowed us to identify almost 900 proteins partners of CTIP2. Among these new partners, we have identified the E3 SUMO ligase KAP1. We found that KAP1 contributes to HIV-1 gene silencing by repressing the initiation and the Tat–dependent steps of the viral gene transcription. KAP1 induces Tat degradation via a SUMO-sensitive pathway. Indeed, favoring the sumoylation promotes Tat association with KAP1 and the resulted Tat degradation. Altogether, our results suggest that KAP1 contributes to the establishment and the persistence of the latently infected HIV-1 reservoirs. Moreover, these results suggest that targeting the SUMO pathways may be a new field of investigation to develop new classes of LRAs for cure strategies.

In humans, protein-coding genes and most small nuclear (sn)RNA genes are transcribed by RNA polymerase II (pol II).The carboxy-terminal domain (CTD) of the largest subunit of pol II possesses multiple heptapetide repeats of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Phosphorylation of Ser2, Ser5 and Ser7 mediates the recruitment of transcription and RNA processing factors during the transcription cycle. There are notable differences between snRNA genes and protein-coding genes in terms of mechanisms controlling their expression. Pol II does not appear to make the transition to long-range productive elongation during transcription of snRNA genes, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. Initiation of transcription of most human genes transcribed by pol II requires the formation of a preinitiation complex (PIC) comprising TFIIA, B, D, E, F and H and pol II. The general transcription factor, TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors (TAFs). Differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. It has already been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5, has been shown to be associated with pol II-transcribed snRNA genes, the full complement of TAFs associated with these genes remained unclear. Here I show, using a ChIP and siRNA-mediated knockdown approach, that the TBP/TAF complex on snRNA genes differs from that on protein-coding genes. Interestingly, the largest TAF, TAF1 and the core TAFs, TAF10 and TAF4 are not detected on snRNA genes. I propose that this snRNA gene-specific TAF subset plays a key role in gene-type-specific control of expression. In addition, in order to further understand the molecular mechanism underlying the differences between expression of protein-coding genes and snRNA genes, I have investigated the role of RNA pol II-associated protein 2 (RPAP2) in transcription of snRNA genes. Here I show that RPAP2 recognizes the phospho-Ser7 mark on the pol II CTD, siRNA mediated knockdown of RPAP2 causes defects in snRNA gene expression and that RPAP2 is a CTD Ser5 phosphatase. I also present my studies of the mechanism of inhibition of phospho-Ser2 by herpes simplex virus-1 (HSV-1) protein ICP22. Phosphorylation of Ser2 by the positive transcription elongation factor (P-TEFb) is associated with productive transcriptional elongation. However, P-TEFb is not required for elongation of transcription of snRNA genes, but functions only to activate 3' box-directed RNA processing. In addition, there are conflicting data as to whether Cdk9 is acting as a Ser2 kinase during transcription of pol II-transcribed snRNA genes. As ICP22 is thought to inhibit P-TEFb, this protein could provide an alternative means to study P-TEFb function in expression of snRNA genes.

In humans, RNA polymerase II is the sole source of messenger RNAs that are ultimately translated into proteins and its transcriptional activity is highly regulated. Mechanisms have evolved to control which, when, and to what degree genes are transcribed. Because most cells have the same genome, control of transcription is essential in maintaining cellular identity. Misregulation of Pol II transcription is a hallmark of both cancer and retroviral infection. This research investigates the regulation of Pol II transcription and related co-transcriptional mRNA capping. Chromatin immunoprecipitation experiments were used to characterize the composition of nucleosomes and Pol II, DSIF and NELF occupancies at bidirectional promoters and enhancers. In collaboration with Alberto Bosque and Vicente Planelles, sequencing experiments were performed in a primary T cell model of HIV latency and a role for sequence-specific recruitment of STAT5 was established in HIV reactivation. In contrast, analysis of Myc binding in vitro and in cells demonstrated that transcription machinery played a major role in recruiting Myc to genomic sites. A precise method was also developed to detect polymerase-associated nascent transcripts in nuclei. The roles of Cdk7, a subunit of TFIIH that phosphorylates Pol II during initiation, were characterized by treatment of nuclear extracts and cells with THZ1, a recently developed covalent inhibitor with anti-cancer properties. Inhibition of Cdk7 was demonstrated to cause defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. Capping of nascent RNAs was found to be spatially and temporally regulated in part by a previously undescribed THZ1-sensitive factor present in nuclear extract. THZ1 impacted pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to misloading of DSIF. In vitro and sequencing methods were used to describe an extremely rapid and global transcriptional response to hydrogen peroxide. During periods of oxidative stress, termination was likely inhibited and Pol II accumulated at promoters and enhancers after as few as two minutes, and clearance of these polymerases required P-TEFb. In the presence of flavopiridol, a potent P-TEFb inhibitor, non-productive elongation was observed and a potential role for P-TEFb in termination was proposed.

Regulation of transcription elongation by P-TEFb is critical for proper gene expression and cell survival. The cell possesses large quantities of P-TEFb, but the vast majority of it is locked away and inactive in the 7SK snRNP. Since the discovery of the 7SK snRNP, research has been conducted to determine how P-TEFb is released from this complex. The goal of the research presented in this thesis is to better understand how the 7SK snRNP regulates P-TEFb and ultimately, gene expression. This work documents the discovery and characterization of the 7SK stability protein LARP7. LARP7 is is associated with 7SK regardless of the presence of P-TEFb and HEXIM1. Stabilization of 7SK is essential for maintenance of the RNP because loss of LARP7 results in an increase in free P-TEFb and a significant reduction in the amount of 7SK. These results indicate that stabilization of the 7SK snRNP by LARP7 is important for regulating P-TEFb homeostasis. Although P-TEFb was first characterized from Drosophila lysates, the conservation of the 7SK snRNP and the mechanisms regulating P-TEFb inhibition have not been described. Here, the Drosophila melanogaster homologues of LARP7 and 7SK are characterized. These studies show that the system of P-TEFb regulation is similar in flies and this makes Drosophila an attractive model system for studying P-TEFb regulation through embryonic and larval development. Finally, factors and modifications involved in releasing P-TEFb directly are explored. An assay was developed for discovering proteins that can bind to and release P-TEFb from the 7SK snRNP. Use of this assay showed that post-translational modification of the components of the 7SK snRNP do not cause P-TEFb release directly. However, HIV Tat and the C-terminal P-TEFb binding region of the bromodomain containing protein, Brd4, are capable of extracting P-TEFb directly. Most importantly, the release of P-TEFb is followed by a conformational change in 7SK RNA that prevents the continued binding of HEXIM1 to the complex. P-TEFb release from the 7SK snRNP is the result of direct extraction of P-TEFb by viral or cellular proteins, and not post-translational modifications or a competition between HEXIM1 and hnRNP proteins for 7SK binding.

Developmental processes are carefully controlled at the level of transcription to ensure that the fertilized egg develops into an adult organism. The mechanisms that controls transcription of protein-coding genes ultimately ensure that the Pol II machine synthesizes mRNA from the correct set of genes in every cell type. Transcriptional control involves Pol II recruitment as well as transcriptional elongation. Recent genome-wide studies shows that recruitment of Pol II is often followed by an intermediate step where Pol II is halted in a promoter-proximal paused configuration. The release of Pol II from promoter-proximal pausing is thus an additional and commonly occurring mechanism in metazoan gene regulation. The serine kinase P-TEFb is part of the Super Elongation Complex that regulates the release of paused Pol II into productive elongation. However, little is known about the role of P-TEFb mediated gene expression in development. We have investigated the function of P-TEFb in early Drosophila embryogenesis and find that P-TEFb and other Super Elongation Complex subunits are critical for activation of the most early expressed genes. We demonstrate an unexpected function for Super Elongation Complex in activation of genes with non-paused Pol II. Furthermore, the Super Elongation Complex shares phenotypes with subunits of the Mediator complex to control the activation of essential developmental genes. This raises the possibility that the Super Elongation Complex has an unappreciated role in the recruitment of Pol II to promoters. The unique chromatin landscape of each cell type is comprised of post-translational chromatin modifications such as histone methylations and acetylations. To study the function of histone modifications during development, we depleted the histone demethylase KDM4A in Drosophila to evaluate the role of KDM4A and histone H3 lysine 36 trimethylation (H3K36me3) in gene regulation. We find that KDM4A has a male-specific function and regulates gene expression both by catalytic-dependent and independent mechanisms. Furthermore, we used histone replacement to investigate the direct role of H3K14 acetylation in a multicellular organism. We show that H3K14 acetylation is essential for development, but is not cell lethal, suggesting that H3K14 acetylation has a critical role in developmental gene regulation. This work expands our knowledge of the mechanisms that precisely controls gene regulation and transcription, and in addition highlights the complexity of metazoan development. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript.</p>

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Grâce à un grand nombre d’études biochimiques, génétiques et structurales effectuées dans les dernières années, des avancements considérables ont été réalisés et une nouvelle vision du processus par lequel la machinerie transcriptionnelle de l’ARN polymérase II (Pol II) décode l’information génétique a émergé. De nouveaux indices ont été apportés sur la diversité des mécanismes de régulation de la transcription, ainsi que sur le rôle des facteurs généraux de transcription (GTFs) dans cette diversification. Les travaux présentés dans cette thèse amènent de nouvelles connaissances sur le rôle des GTFs humains dans la régulation des différentes étapes de la transcription. Dans la première partie de la thèse, nous avons analysé la fonction de la Pol II et des GTFs humains, en examinant de façon systématique leur localisation génomique. Les patrons obtenus par immunoprécipitation de la chromatine (ChIP) des versions de GTFs portant une étiquette TAP (Tandem-Affinity Purification) indiquent de nouvelles fonctions in vivo pour certains composants de cette machinerie et pour des éléments structuraux de la Pol II. Nos résultats suggèrent que TFIIF et l’hétérodimère Rpb4–Rpb7 ont une fonction spécifique pendant l’étape d’élongation transcriptionnelle in vivo. De plus, notre étude amène une première image globale de la fonction des GTFs pendant la réaction transcriptionnelle dans des cellules mammifères vivantes. Deuxièmement, nous avons identifié une nouvelle fonction de TFIIS dans la régulation de CDK9, la sous-unité kinase du facteur P-TEFb (Positive Transcription Elongation Factor b). Nous avons identifié deux nouveaux partenaires d’interaction pour TFIIS, soit CDK9 et la E3 ubiquitine ligase UBR5. Nous montrons que UBR5 catalyse l’ubiquitination de CDK9 in vitro. De plus, la polyubiquitination de CDK9 dans des cellules humaines est dépendante de UBR5 et TFIIS. Nous montrons aussi que UBR5, CDK9 and TFIIS co-localisent le long du gène  fibrinogen (FBG) et que la surexpression de TFIIS augmente les niveaux d’occupation par CDK9 de régions spécifiques de ce gène, de façon dépendante de UBR5. Nous proposons que TFIIS a une nouvelle fonction dans la transition entre les étapes d’initiation et d’élongation transcriptionnelle, en régulant la stabilité des complexes CDK9-Pol II pendant les étapes précoces de la transcription. / Biochemical, genetic and structural studies made over the last years bring a new view on the RNA polymerase II (Pol II) machinery and the process by which it decodes the genetic information. They provided new insights into the diversity of the transcriptional regulation mechanisms, and on the role played by the general transcription factors (GTFs). The studies presented in this thesis provide new evidence on the role of human GTFs in the regulation of different stages of transcription. In the first part of the thesis, we investigated the function of the human Pol II and GTFs in living cells, by systematically analyzing their genomic location. The location profiles obtained by chromatin immunoprecipitation (ChIP) of TAP (tandem-affinity purification) tagged versions of these factors indicate new in vivo functions for several components of this machinery, and for structural elements of the Pol II. These results suggest that TFIIF and the heterodimer Rpb4–Rpb7 have a specific function during the elongation stage in vivo. Additionally, our study offers for the first time a general picture of GTFs function during the Pol II transcription reaction in live mammalian cells, and provides a framework to uncover new regulatory hubs. Secondly, we report on the identification of a new function of the factor TFIIS in the regulation of CDK9, the kinase subunit of the Positive Transcription Elongation Factor b (P-TEFb). We identify two interaction partners for TFIIS, namely CDK9 and the E3 ubiquitin ligase UBR5. We show that UBR5 catalyzes the ubiquitination of CDK9 in vitro. Moreover, the polyubiquitination of CDK9 in human cells is dependent upon both UBR5 and TFIIS, and does not signal its degradation. We also show that UBR5, CDK9 and TFIIS co-localize along specific regions of the  fibrinogen (FBG) gene, and that the overexpression of TFIIS increases the occupancy of CDK9 along this gene in a UBR5 dependant manner. We propose a new function of TFIIS in the transition between initiation and elongation stages, by regulating the stability of the early CDK9-Pol II transcribing complexes. Key words: chromatin immunoprecipitation, general transcription factors, tandem-affinity purification, RNA polymerase II, Rpb4–Rpb7 heterodimer, transcription factor IIF (TFIIF), transcription factor IIS (TFIIS), UBR5 ubiquitin ligase, Positive Transcription Elongation Factor b (P-TEFb), CDK9 ubiquitination.

Grâce à un grand nombre d’études biochimiques, génétiques et structurales effectuées dans les dernières années, des avancements considérables ont été réalisés et une nouvelle vision du processus par lequel la machinerie transcriptionnelle de l’ARN polymérase II (Pol II) décode l’information génétique a émergé. De nouveaux indices ont été apportés sur la diversité des mécanismes de régulation de la transcription, ainsi que sur le rôle des facteurs généraux de transcription (GTFs) dans cette diversification. Les travaux présentés dans cette thèse amènent de nouvelles connaissances sur le rôle des GTFs humains dans la régulation des différentes étapes de la transcription. Dans la première partie de la thèse, nous avons analysé la fonction de la Pol II et des GTFs humains, en examinant de façon systématique leur localisation génomique. Les patrons obtenus par immunoprécipitation de la chromatine (ChIP) des versions de GTFs portant une étiquette TAP (Tandem-Affinity Purification) indiquent de nouvelles fonctions in vivo pour certains composants de cette machinerie et pour des éléments structuraux de la Pol II. Nos résultats suggèrent que TFIIF et l’hétérodimère Rpb4–Rpb7 ont une fonction spécifique pendant l’étape d’élongation transcriptionnelle in vivo. De plus, notre étude amène une première image globale de la fonction des GTFs pendant la réaction transcriptionnelle dans des cellules mammifères vivantes. Deuxièmement, nous avons identifié une nouvelle fonction de TFIIS dans la régulation de CDK9, la sous-unité kinase du facteur P-TEFb (Positive Transcription Elongation Factor b). Nous avons identifié deux nouveaux partenaires d’interaction pour TFIIS, soit CDK9 et la E3 ubiquitine ligase UBR5. Nous montrons que UBR5 catalyse l’ubiquitination de CDK9 in vitro. De plus, la polyubiquitination de CDK9 dans des cellules humaines est dépendante de UBR5 et TFIIS. Nous montrons aussi que UBR5, CDK9 and TFIIS co-localisent le long du gène  fibrinogen (FBG) et que la surexpression de TFIIS augmente les niveaux d’occupation par CDK9 de régions spécifiques de ce gène, de façon dépendante de UBR5. Nous proposons que TFIIS a une nouvelle fonction dans la transition entre les étapes d’initiation et d’élongation transcriptionnelle, en régulant la stabilité des complexes CDK9-Pol II pendant les étapes précoces de la transcription. / Biochemical, genetic and structural studies made over the last years bring a new view on the RNA polymerase II (Pol II) machinery and the process by which it decodes the genetic information. They provided new insights into the diversity of the transcriptional regulation mechanisms, and on the role played by the general transcription factors (GTFs). The studies presented in this thesis provide new evidence on the role of human GTFs in the regulation of different stages of transcription. In the first part of the thesis, we investigated the function of the human Pol II and GTFs in living cells, by systematically analyzing their genomic location. The location profiles obtained by chromatin immunoprecipitation (ChIP) of TAP (tandem-affinity purification) tagged versions of these factors indicate new in vivo functions for several components of this machinery, and for structural elements of the Pol II. These results suggest that TFIIF and the heterodimer Rpb4–Rpb7 have a specific function during the elongation stage in vivo. Additionally, our study offers for the first time a general picture of GTFs function during the Pol II transcription reaction in live mammalian cells, and provides a framework to uncover new regulatory hubs. Secondly, we report on the identification of a new function of the factor TFIIS in the regulation of CDK9, the kinase subunit of the Positive Transcription Elongation Factor b (P-TEFb). We identify two interaction partners for TFIIS, namely CDK9 and the E3 ubiquitin ligase UBR5. We show that UBR5 catalyzes the ubiquitination of CDK9 in vitro. Moreover, the polyubiquitination of CDK9 in human cells is dependent upon both UBR5 and TFIIS, and does not signal its degradation. We also show that UBR5, CDK9 and TFIIS co-localize along specific regions of the  fibrinogen (FBG) gene, and that the overexpression of TFIIS increases the occupancy of CDK9 along this gene in a UBR5 dependant manner. We propose a new function of TFIIS in the transition between initiation and elongation stages, by regulating the stability of the early CDK9-Pol II transcribing complexes. Key words: chromatin immunoprecipitation, general transcription factors, tandem-affinity purification, RNA polymerase II, Rpb4–Rpb7 heterodimer, transcription factor IIF (TFIIF), transcription factor IIS (TFIIS), UBR5 ubiquitin ligase, Positive Transcription Elongation Factor b (P-TEFb), CDK9 ubiquitination.