RNA polymerase II-mediated transcription of HIV-1 genes depends on positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We show that P-TEFb interacts with H1 and that H1 phosphorylation in cell culture correlates with P-TEFb activity. Importantly, P-TEFb also directs H1 phosphorylation during Tat transactivation and wild type HIV-1 infection. Our results also show that P-TEFb phosphorylates histone H1.1 at a specific C-terminal site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb disrupts Tat transactivation as well as transcription of the c-fos and hsp70 genes in HeLa cells. P-TEFb phosphorylation of H1 also plays a role in the expression of muscle differentiation marker genes in the skeletal myoblast cell line C2C12. Additionally, ChIP experiments demonstrate that H1 dissociates from the HIV-1 LTR in MAGI cells, stress-activated genes in HeLa cells, and muscle differentiation marker genes in C2C12 cells under active P-TEFb conditions. Our results overall suggest a new role for P-TEFb in both cellular and HIV-1 transcription through chromatin.

Gene expression of human immunodeficiency virus type-1 (HIV-1), which causes Acquired Immunodeficiency Syndrome (AIDS), is regulated at the transcriptional level, where negative factors can block elongation that is overcome by HIV Tat protein and P-TEFb. P-TEFb, a positive elongation transcription factor with two subunits, CDK9 and Cyclin T1 (CycT1), catalyzes Tat-dependent phosphorylation of Ser-5 in the Pol II C-terminal domain (CTD), allowing production of longer mRNAs. Ser-5 phosphorylation enables the CTD to recruit mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. This dissertation demonstrates that stable binding of Mce1 and cap methyltransferase to template-engaged Pol II depends on CTD phosphorylation, but not on nascent RNA. Capping and methylation doesn't occur until nascent pre-mRNA become 19-22 nucleotides long. A second and novel pathway for recruiting and activating Mce1 involved direct physical interaction between the CTD, Tat and Mce1. Tat stimulated the guanylyltransferase and triphosphatase activities of Mce1, thereby enhancing the otherwise low efficiency of cotranscriptional capping of HIV mRNA. These findings imply that multiple mechanisms exist for coupling transcription elongation and mRNA processing at a checkpoint critical to HIV gene expression. To elucidate P-TEFb's function in human (HeLa) cells, RNA interference (RNAi) was used to degrade mRNA for hCycT1 or CDK9. Down-regulation of P-TEFb expression by RNAi can be achieved without causing major toxic or lethal effects and can control Tat transactivation and HIV replication in host cells. High-density oligonucleotide arrays were used to determine the effect of P-TEFb knockdown on global gene expression. Of 44,928 human genes analyzed, 25 were down-regulated and known or likely to be involved in cell proliferation and differentiation. These results provide new insight into P-TEFb function, its potent role in early embryonic development and strong evidence that P-TEFb is a new target for developing AIDS and cancer therapies. To fulfill the promise of RNAi for treating infectious and human genetic diseases, structural and functional mechanisms underlying RNAi in human cells were studied. The status of the 5' hydroxyl terminus of the antisense strand of short interfering RNA (siRNA) duplexes determined RNAi activity, while a 3' terminus block was tolerated in vivo. A perfect A-form helix in siRNA was not required for RNAi, but was required for antisense-target RNA duplexes. Strikingly, crosslinking siRNA duplexes with psoralen did not completely block RNAi, indicating that complete unwinding of the siRNA helix is not necessary for RNAi in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.

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.

Pre-mRNA splicing is an important level in posttranscriptional gene regulation that is essential for accurate protein synthesis and generating protein diversity. The abundance of cryptic splice sites and long intronic DNA sequences makes their splicing a complex one. The identification of correct exons and introns needs additional information in the form of splicing regulatory elements (SREs) along with canonical splice signals. The interplay among these SREs and the trans factors (which bind to SREs) gives the identity to introns and exons which in turn leads to precise pre-mRNA splicing. Previous studies from our laboratory showed, that when expressed in mammalian cells from an expression vector, PDGF-B ORF was re-spliced at 4/5 exon junction with the downstream SV40 splice acceptor site in the vector. However, deletion of the 66-nt PDGF-B 3’ UTR region resulted in about 25% reduction in re-splicing. Sequence analysis of this region revealed presence of binding sites for splicing factors ASF/SF2 and SRp55, and an AU-rich element (ARE), mutation each of which affected re-splicing partially. In mammals, AREs are commonly found in the 3’UTR of mRNAs encoding proteins involved in diverse functions and are involved in selective mRNA degradation. Several ARE binding proteins are crucial for ARE’s function. Since mutation of the single ARE in the 3’UTR region altered the re-splicing efficiency, the role of AU-rich elements and ARE-binding proteins (AU-BPs) in modulation of splicing was investigated using siRNAs against AU-BPs, BRF1, hnRNPD, HuR, GAPDH and TTP. Down regulation of expression of these factors indeed affected the level of re-spliced product. We have studied the interactions between the full-length splicing factors (U1-70K and U2AF35) and the AU-BPs (BRF1, hnRNPD and HuR) as well as among the AU-BPs using three different assay methods: Yeast-two hybrid, co-immunoprecipitation and pull down assays. Our study has revealed that the BRF1 interacts with U1-70K and U2AF35 as well as the other AU-BPs hnRNPD and HuR but with different affinities. We have also analyzed the ability of AU-BPs to interact with SR proteins SRp20 and 9G8. We did find strong interaction of BRF1 with SRp20 and 9G8. Generation of a large number of nested deletion mutants of all the proteins allowed us to identify the interaction regions on the surface of BRF1, U1-70K, hnRNPD, U2AF35 and HuR. The results of Y2H analyses were further confirmed by pull down assay using purified interacting regions. It was found that a single region from aa 181-254 in BRF1 interacts with multiple partners i.e., splicing factors and the AU-BP hnRNPD. However, the RNA-binding zinc-finger domain from residue 120-181 independently interacts with HuR. Further, the multiple protein interacting region (MPIR) (aa 181-254) in BRF1 exhibits different affinities towards its interacting partners with that for U1-70K and hnRNPD being stronger than that for U2AF35 and HuR. This observation suggests that BRF1 activity can be modulated by interaction with different partners at different sites. U1-70K interacted only with BRF1 among the proteins tested in this study and this interaction appears to be RNA independent .This could have implications in splice site selection and RNA stability since BRF1 has been shown to promote RNA degradation. While the Arg/Glu-rich C-terminal region in U1-70K is sufficient for its interaction with BRF1, U2AF35 requires both the zinc-finger 2 and the arg/Gly/Ser-rich C-terminal regions for its association with BRF1. hnRNPD also interacts with multiple partners that include BRF1, HuR and U2AF35 using the N-terminal region that harbors a Ala-rich domain. The interaction of hnRNPD with HuR is RNA dependent while with BRF1 and U2AF35, it is RNA independentt. Further, its interaction with all the partners is equally strong. This suggests that hnRNPD could exert differential influence depending on the context of its interaction and abundance of the interacting partner. HuR, primarily known as an mRNA stabilizing factor, interacts with both BRF1 and hnRNPD with equal affinity involving the hinge region, the interaction with the former being RNA-independent and the later being RNA-dependent. This differential RNA-dependent and independent interactions with the two AU-BPs using a single interacting domain suggests a balancing act of HuR on the activities of BRF1 and hnRNPD. These interactions can further be differentially modulated by posttranslational modifications on one or all of the interacting partners depending on the physiological status of the cell. We have also analyzed the multiple protein complexes formed in absence of cellular RNA. Though we are unable to see direct protein-protein interaction between HuR and U1-70K in Yeast two hybrid analysis, we could detect the presence of U1-70K in HuR immunoprecipitate. It appears that U1-70K associates with HuR via BRF. We also detected the presence of HuR in U1-70K complexes which could be due to its association with BRF1. We are unable to find hnRNPD and U2AF35 in these complexes indicating that they may have been excluded. In anti-U2AF35 immunoprecipitates, we detected the presence of U1-70K as well as hnRNPD but no HuR. This may be due to RNase treatment as hnRNPD and HuR interactions are RNA dependent. Our findings that AU-rich elements in conjunction with AU-BPs function as intronic splicing modulators or enhancers, reveal hitherto unidentified new players in the poorly understood complex mechanisms that mediate alternative splicing. The possibility of dynamic nature of the interactions among splicing factors and AU-BPs mediated by post-translational modifications provide a basis for rapid cellular responses to changing environmental cues through generation of differentially spliced mRNAs and corresponding protein products that differ in their stability and hence their relative abundance. Our results also unfold enormous possibilities for future investigations on interactions among the many splicing factors and AU-BPs, and in understanding these complex interactions in modulation of pre-mRNA splicing, mRNA translation and degradation. The finding of coupling of AU-BPs to splicing machinery could further lead to better understanding of the mechanism of AU-BP-mediated targeting of mRNAs to processing bodies and ultimate degradation of the mRNAs.