The transcription elongation factor P-TEFb in 'Drosophila melanogaster' : characterization of a mutation of the Cdk9 kinase and of the different activities of two co-factors

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Thesis

Reference

The transcription elongation factor P-TEFb in 'Drosophila

melanogaster' : characterization of a mutation of the Cdk9 kinase and of the different activities of two co-factors

BASQUIN, Denis

Abstract

Transcription by RNA polymerase II is regulated, in part, by the positive transcription elongation factor b (P-TEFb), which promotes transition from abortive to productive elongation. The Drosophila P-TEFb complex is composed of the Cdk9 kinase and a cyclin partner, CyclinT or CyclinK. To investigate the physiological role of P-TEFb, we generated transgenic flies allowing the conditional expression of wild-type or mutant Cdk9, alone or together with CyclinT or CyclinK. We found that the two P-TEFb complexes have similar binding pattern on chromosomes and are recruited to transcriptionally active loci. By expressing a dominant-negative form of Cdk9 in specific tissues, we showed that P-TEFb function is required for endoreplication of larval tissues, for proper differentiation of imaginal discs and for oogenesis. We demonstrated that the two cyclin subunits have non-redundant activities in vivo, and that P-TEFb containing CyclinT, but not CyclinK, activates transcription when tethered to promoter.

BASQUIN, Denis. The transcription elongation factor P-TEFb in 'Drosophila melanogaster' : characterization of a mutation of the Cdk9 kinase and of the

different activities of two co-factors. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4080

URN : urn:nbn:ch:unige-19367

DOI : 10.13097/archive-ouverte/unige:1936

Available at:

http://archive-ouverte.unige.ch/unige:1936

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE FACULTÉ DESSCIENCES Département de zoologie et biologie animale Professeur P. Spierer

Docteur D. Pauli

The transcription elongation factor P-TEFb in Drosophila melanogaster : Characterization of a mutation of the Cdk9 kinase and of the different

activities of two co-factors.

THÈSE

Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologique

par

Denis Basquin de Beggingen (SH)

Thèse N° 4080

GENÈVE

Atelier de reproduction ReproMail Avril 2009

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Résumé

Une régulation appropriée de l’expression des gènes est essentielle lors du développement et de la différentiation des organismes complexes, lesquels acquièrent une grande variété de types cellulaires et de tissus spécialisés. La dérégulation du patron d’expression des gènes peut avoir des conséquences majeures sur la spécificité cellulaire et est responsable de nombreuses maladies. Bien que de nombreux processus cellulaires soient impliqués dans la régulation de l’expression génique, la transcription représente le mécanisme de contrôle le plus direct. Dans les cellules eucaryotes, la transcription des gènes codants pour les protéines est effectuée par l’ARN polymérase II (ARNPII). Le cycle de transcription est un processus complexe et séquentiel aboutissant à la production d’ARN pré-messagers, et comprend les étapes de pré-initiation, initiation, dégagement du promoteur, élongation et terminaison. Au delà de son rôle dans la synthèse d’ARNm, l’ARNPII régule également le remodelage de la chromatine et la maturation des ARNm. Par opposition aux ARN polymérases I et III, l’ARNPII se caractérise par l’existence de multiples copies d’un heptapeptide (YSPTSPS)n, présent en position C-terminale de la grande sous-unité Rpb1, et désigné CTD (C-terminal domain). Lors de la transcription, le CTD est abondamment phosphorylé à différentes positions et se comporte comme une plateforme d’intégration pour le recrutement de facteurs impliqués dans la synthèse d’ARN, la maturation des ARNm et la modification de la chromatine. La phosphorylation du CTD a lieu à différentes étapes du cycle de transcription et est exécutée par au moins trois kinases cycline-dépendant (Cdks) connues. Les sous-unités Cdk7/Cycline H du facteur général de transcription TFIIH et Cdk8/Cycline C de certains complexes Médiateurs modifient la sérine 5 du CTD à l’initiation de la transcription. En début d’élongation, l’activité kinase de Cdk9/Cycline T ou Cdk9/Cycline K est requise pour prévenir un arrêt prématuré de la transcription et stimuler l’élongation en phosphorylant la sérine 2 du CTD, ainsi que les facteurs DSIF et NELF.

L’hétérodimère Cdk9/Cycline T ou Cdk9/Cycline K, nommé P-TEFb (positive transcription-elongation factor b), est un facteur d’élongation conservé au cours de l’évolution des organismes eucaryotes. Chez les mammifères, l’activité de P-TEFb est contrôlée par une grande variété de cofacteurs qui le recrutent aux promoteurs actifs. Inversement, P-TEFb peut être inhibé par interaction avec des régulateurs spécifiques. Une perturbation de l’activité enzymatique de Cdk9 peut avoir des conséquences sévères sur le fonctionnement cellulaire et est impliqué dans certaines maladies humaines. Bien que la compréhension des mécanismes

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par lesquels P-TEFb régule la transcription ait beaucoup progressé, le rôle physiologique de cette kinase reste largement inconnu. De récentes études laissent cependant apparaître un rôle important de P-TEFb dans divers processus biologiques tels que la croissance, la prolifération, la protection contre l’apoptose ou encore la différentiation cellulaire.

Dans la présente étude, nous avons examiné les fonctions biologiques de P-TEFb chez Drosophila melanogaster. Pour ce faire, nous avons opté pour une approche transgénique et avons généré des lignées de Drosophiles codant la forme sauvage de Cdk9 ainsi que des deux cyclines régulatrices connues, Cycline T et Cycline K. Ces transgènes nous ont permis de diriger une expression conditionnelle de chaque protéine dans une grande variété de tissus grâce au système UAS/GAL4. Nous avons débuté notre analyse en testant la fonctionnalité de chaque transgène in vivo et leur capacité de se substituer à la fonction des allèles endogènes.

Nous avons montré que Cdk9 s’associe de manière stable avec chacune des cyclines en culture de cellules, et que les deux complexes P-TEFb sont recrutés aux loci activement transcrits des chromosomes polytènes. Pour étudier le rôle physiologique de P-TEFb, nous avons également produit une forme dominante-négative de Cdk9 pour laquelle le site catalytique a été muté. Nous avons démontré qu’une expression de la protéine mutante dans un tissu donné interfère spécifiquement avec la fonction endogène de Cdk9, et affecte dramatiquement la phosphorylation de l’ARNPII à la sérine 2 sur les chromosomes. Par l’analyse des phénotypes associés à l’expression de la forme dominante-négative de Cdk9, nous avons constaté que P-TEFb est nécessaire pour la croissance larvaire, et que la perte partielle d’activité de la kinase dans les tissus larvaires affecte fortement le processus d’endoréplication. En revanche, l’expression de la protéine mutante ne semble pas altérer la prolifération des cellules dans les tissus embryonnaires ni dans les tissus imaginaux. Une expression spécifique de la protéine mutante dans les disques imaginaux interfère cependant avec la croissance et la différentiation terminale des structures adultes. Nous avons également examiné le rôle de P-TEFb dans la lignée germinale femelle. L’expression de la kinase dominante-négative dans ce tissu affecte différents mécanismes liés à l’ovogénèse et entraîne la stérilité. Nous avons notamment montré que P-TEFb est nécessaire pour la migration des cellules folliculaires autour des cystes germinaux et influence leur destin cellulaire. De plus, nous avons aussi constaté qu’une réduction de l’activité de Cdk9 peut engendrer une prolifération excessive des cellules germinales et affecter occasionnellement leur capacité à se différencier. Dans une dernière partie, nous avons recherché la signification fonctionnelle des deux cyclines sur l’activité de Cdk9. Nous avons montré dans un premier temps que les deux cyclines n’ont pas d’activité redondante lors du développement de la Drosophile. De plus

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nous avons constaté que l’activité de P-TEFb comprenant Cycline T ou Cycline K diffèrent au niveau transcriptionnel, et que seule la CyclineT est capable de stimuler l’expression d’un gène rapporteur lorsque le complexe est recruté à des sites d’activation en amont du promoteur. Finalement, l’examen de certains phénotypes associés à la co-expression de Cdk9 dominant-négatif avec Cycline T ou Cycline K suggère que les deux complexes P-TEFb ne sont pas actifs de la même manière in vivo, car la surexpression de Cycline T, mais pas Cycline K, tend à supprimer les effets dominant-négatifs de Cdk9 sur la croissance et la différentiation.

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Summary

The proper regulation of gene expression is essential during development and differentiation of complex organisms, in which functional specialization of cells and tissues is taking place. Disruption of the accurate expression pattern of the genes may have profound effects on cellular function and underlies many diseases. Although several cellular processes are involved in the regulation of gene expression, the most direct control occurs during transcription. In eucaryotes, transcription of protein-coding genes is performed by the RNA polymerase II (RNAPII). The transcription cycle is a multi-step process resulting in the production of full-length pre-mRNA transcript, and includes pre-initiation, initiation, promoter clearance, elongation and termination. In addition to its functions in RNA synthesis, RNAPII influences both chromatin remodeling and RNA processing. In contrast to the RNA polymerases I and III, RNAPII has a characteristic heptapeptide repeat (YSPTSPS)n in the C- terminus of its largest subunit Rpb1, referred to the CTD (C-terminal domain). During transcription, the CTD repeats became highly phosphorylated at several positions and act as an integrating platform for recruitment of factors involved in mRNA synthesis, mRNA processing and chromatin modification. CTD phosphorylation occurs at different stages of the transcription cycle and is mediated by at least three cyclin-dependant kinases (Cdk). The Cdk7/CyclinH subunits of the general transcription factor TFIIH and the Cdk8/CyclinC kinase of some Mediator complexes both target the serine 5 residue of the CTD repeats during transcription initiation. In early elongation, the Cdk9/CyclinT or Cdk9/CyclinK kinases are required to prevent RNAPII pausing and to trigger productive elongation by phosphorylating serine 2 of the CTD as well as the negative elongation factors DSIF and NELF.

The Cdk9/CyclinT or Cdk9/CyclinK heterodimers, so called P-TEFb (positive transcription-elongation factor b) complex, is an evolutionarily conserved factor among eucaryotes. In mammals, its activity is regulated by a wide variety of co-factors that recruit the kinase to active promoters. Conversely, P-TEFb can be inhibited through interaction with specific regulators. Disruption of Cdk9 activity may have profound effects on cellular function and is associated with certain human diseases. While many progress have been made towards the understanding of the mechanisms by which P-TEFb regulate transcription, little is known about the physiological role of this kinase. In recent years however, it became apparent that P-TEFb plays an important role in several biological processes, such as cell growth, proliferation, protection from apoptosis and differentiation.

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In the present study, we have investigated the biological functions of P-TEFb in Drosophila melanogaster. To this end, we generated transgenic flies encoding the wild-type Cdk9 kinase as well as the two known Cdk9-regulatory subunits CyclinT and CyclinK. These genetic tools allowed us to direct expression of the proteins in a wide variety of tissues using the UAS/GAL4 system. We started our analysis by demonstrating that the transgenes are functional in vivo and able to substitute for the endogenous function of the genes. By immunochemistry, we showed that Cdk9 can form stable heterodimers with one or the other cyclin in tissue culture cells, and that both complexes are recruited to transcriptionally active genes on chromosomes. To get insight into the physiological role of P-TEFb, we generated a dominant-negative form of Cdk9 by mutation of the catalytic site. We showed that a transgenic expression of Cdk9 dominant-negative in selective tissues specifically alter P- TEFb function and dramatically affects serine 2 phosphorylation of the RNAPII CTD on chromosomes. By analysing the phenotypes associated with Cdk9 dominant-negative, we found that P-TEFb function is required for normal larval growth, and that impairing Cdk9 activity strongly affects endoreplication of the larval tissues. Interestingly, we found that P- TEFb function is not involved in cell proliferation within the embryonic tissues neither in imaginal discs. In the imaginal tissues however, disruption of Cdk9 activity eventually results in decrease size of the adult appendages and interferes with the terminal differentiation program. We also investigated the requirement for P-TEFb in the female germline. The expression of Cdk9 dominant-negative in that tissue causes multiple oogenesis defects resulting in female sterility. We notably demonstrated that P-TEFb function is required for the proper migration of the follicle cells around the germline cysts and influence their cellular fate. Moreover, we also showed that impairing Cdk9 activity results in overproliferation of the germ cells and occasionally interfere with their ability to differentiate as germline cysts. In the last part of this study, we attempted to determine the functional significance of the two cyclin subunits on P-TEFb activity. We started this analysis by demonstrating that both cyclins have non-redundant activity in developing Drosophila. In addition, we showed that P-TEFb containing CyclinT or CyclinK do not mediate similar effects in reporter transcription assays, and that CyclinT but not CyclinK can stimulate transcription when tethered to sites upstream of the promoter. Finally, the examination of certain phenotypes associated with the co- expression of Cdk9 dominant-negative with CyclinT or CyclinK suggests that the complexes are activated differently in Drosophila tissues, and that CyclinT overexpression, but not CyclinK, tend to suppressed the dominant-negative effects of Cdk9 mutation on cell growth and differentiation.

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Introduction

1. Transcription cycle by RNA polymerase II.

The production of pre-mRNA by the RNAPII is a stepwise process including pre- initiation, initiation, promoter clearance, elongation and termination. Each step is regulated by specific sets of transcription factors that recruit the polymerase to promoters, stimulate its catalytic activity and/or ensure the co-transcriptional processing (capping and splicing) of the pre-mRNA during elongation. The final step of the transcription cycle involve the 3’end processing of the transcripts such as the cleavage of the mRNA, its polyadenylation and its export to the cytoplasm, which are regulated through interaction of factors with the transcription machinery. Change of the chromatin architecture is also necessary for efficient transcription, and is promoted by association of chromatin modifiers with the elongating polymerase. All these processes, pre-mRNA synthesis, mRNA processing and chromatin remodeling are tightly coupled during transcription, and are coordinated through modifications of the RNAPII CTD.

1.1. Assembly of the pre-initiation complex (PIC) and transcription initiation.

The transcription cycle starts with the assembly of the pre-initiation (PIC) complex at the core promoter of protein-coding genes. Five general transcription factors (GTFs) assist RNAPII positioning to the promoter and are necessary for accurate transcription initiation.

These factors include TFIIB, TFIID, TFIIE, TFIIF and TFIIH (reviewed by Orphanides et al., 1996 ; Bushnell et al., 1996 ; Roeder, 1996). The assembly of the PIC to chromatin is modulated by specific cis-acting elements within the promoter. The primary step of PIC assembly involve the binding of TFIID, a protein complex composed of the TATA-binding protein (TBP) and several TBP-associated factors (TAFs) (reviewed by Tora, 2002 ; Muller and Tora, 2004). TFIID recognized several DNA motifs present in most core promoters such as the TATA box through the TBP subunit, the initiator (Inr) through TAF1 and TAF2 (Kaufmann and Smale 1994 ; Purnell et al. 1994 ; Verrijzer et al. 1994 ; Burke and Kadonaga 1996 ; Chalkley and Verrijzer 1999), and the downstream promoter element (DPE) through TAF6 and TAF9 (Burke and Kadonaga 1997 ; Chen and Manley 2003) (Fig. 1A). In addition, the recruitment of TFIID to active promoter is influenced by specific nucleosome marks, as it

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was recently shown that TAF3 directly interacts with histone H3 trimethylated at lysine 4 (H3K4me3) while TAF1 associates with acetylated nucleosomes (Vermeulen et al., 2007 ; Van Ingen et al., 2008) (Fig. 1B).

The anchoring of TFIID to promoter is a prerequisite for TFIIB recruitment, which interacts with TBP and assists TFIIB binding to the TFIIB-recognition element (BRE) upstream to the TATA box (Nikolov et al., 1995 ; Lagrange et al., 1998) (Fig. 1A). The bending of the promoter, induced by TFIID and TFIIB recruitment, is necessary for subsequent positioning of RNAPII at the transcription start site. Prior to its recruitment, free RNAPII can associate with TFIIF resulting in conformational changes of the polymerase (Sopta et al., 1985 ; Chung et al., 2003 ; Rani et al., 2004). Biochemical and structural studies have shown that TFIIB interacts with TFIIF-prebound RNAPII, and both TFIIB and TFIIF collaborate for accurate positioning of the polymerase at the transcription initiation site (Gnatt et al., 2001 ; Bushnell et al., 2004 ; Chen and Hahn, 2004). TFIIE is next recruited to stabilize the complex through interactions with TFIIB, TFIIF and RNAPII (Orphanides et al., 1996 ; Roeder, 1996 ; Ohkuma, 1997) (Fig. 1C). Finally, TFIIE recruits TFIIH to the PIC and assists TFIIH in the formation of an open complex between RNAPII and the promoter through its DNA-helicase activity (Goodrich and Tijan, 1994 ; Holstege et al., 1996 ; Kim et al., 2000 ; Douziech et al., 2000 ; Okuda et al., 2008). Once the open complex is established, transcription initiation occurs upon addition of the two initiating nucleoside triphosphates (NTPs) dictated by the DNA sequence and the formation of the first phosphodiester bound.

The supply of all NTPs and ATP is then necessary to allow RNAPII to clear the promoter and to prevent transcriptional arrest in early elongation (Dvir et al., 1996).

1.2. Promoter clearance and early transcription elongation.

At the transition between transcription initiation and elongation, RNAPII passes through a stage known as promoter clearance or promoter escape (reviewed by Dvir, 2002).

During this stage, the PIC is partially disassembled. Some factors such as TBP remain associated to the promoter following transcription initiation and serve as a scaffold for the assembly of the next initiation complex at the promoter (Van Dyke et al., 1989 ; Jiang and Gralla, 1993 ; Zawel et al., 1995 ; Yudkovsky et al., 2000). Other GTFs including TFIIB and TFIIE are quickly dissociated from the PIC to allow RNAPII to leave the promoter, and TFIIB can subsequently re-associate with TBP at the promoter. TFIIH was shown to be associated with the early elongation complex that have synthesized short transcripts of about

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Figure 1. Pre-initiation complex (PIC) assembly. (A, left) Location and consensus sequence of the TATA box, Initiator (Inr), Downstream promoter element (DPE) and TFIIB recognition motif (BRE). The drawing on the right recapitulate the binding of TFIID to the DNA elements and its interactions with H3K4me3 (red stars) and acetylated nucleosomes (yellow circles) (Vermeulen et al., 2007). (B) Crystal structure (right) and schematic model (left) of RNAPII indicating the position of all its subunits (Rpb1 to Rpb12) (Hirose and Ohkuma, 2007). (C) TFIIF binding induces conformational changes of the clamp region (red arrow) and the Rpb4- Rpb7 subdomain (black arrow) of RNAPII. (D) Binding of TFIID (TBP) and TFIIB to the promoter is a prerequisite for RNAPII recruitment at the transcription initiation site. TFIIE is next recruited to stabilize the complex and to close the RNAPII structure through interaction with Rpb5 (red arrow).

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20-30 nucleotides in length, and would be required to prevent premature arrest (Goodrich and Tjian 1994 ; Dvir et al., 1997a ; Kumar et al., 1998 ; Spangler et al., 2001). Among all GTFs of the PIC, only TFIIF has been detected within the transcription elongation complex (TEC) (Price et al., 1989 ; Bengal et al., 1991 ; Tan et al., 1995 ; Zawel et al., 1995 ; Lei et al., 1999).

Although an arbitrary boundary has been placed between promoter clearance and early elongation, no clear distinction between these two stages can be done in many experimental systems. Following disassembly of the PIC, the earliest phase of transcription elongation is mark by the instability of the complex which tends to release the nascent transcript even in presence of all NTPs (Luse and Jacob, 1987 ; Jacob et al., 1991 ; Jacob et al., 1994 ; Jiang et al., 1995 ; Dvir et al., 1996 ; Dvir et al., 1997b). The frequency of abortive transcription events is dependent on the length of nascent RNAs. During transcription initiation RNAPII tends to slip laterally, a phenomenon that is greatly reduced when the RNA reaches a size of about 9 nucleotides in length and is no longer observed after synthesis of a 23 nucleotides RNA (Kireeva et al., 2000 ; Pal and Luse 2002 ; Pal and Luse 2003). Thus, the stability of the elongation complex appears dependent on the formation of a RNA-DNA hybrid within the transcription bubble, which cannot be established prior to the synthesis of a 8-9 nucleotides RNA (Kireeva et al., 2000 ; Gnatt et al., 2001 ; Pal and Luse 2003).

1.3. Transcription elongation.

After promoter clearance, transcription elongation occurs in a discontinuous fashion and the TEC has to overcome several blocks that are intrinsic to the RNAPII catalytic activity and the chromatinized DNA template. Particularly, transcriptional pausing occurs shortly after promoter clearance and maintains the polymerase in latent period before resuming productive elongation. This promoter-proximal pausing has been demonstrated for all three eukaryotic RNA polymerases as well as viral and prokaryotic RNA polymerases, and its role in the control of gene expression has been discussed in several recent reviews (Landick, 2006 ; Lis, 2007 ; Margaritis and Holstege, 2008 ; Gilmour, 2009). In contrast to transcriptional arrest, pausing is a self-reversible process which is consider as a natural mode of transcription regulation. Genome-wide studies of RNAPII distribution and associated epigenetic marks in human (Kim et al., 2005 ; Guenther et al., 2007) and Drosophila cells (Muse et al., 2007 ; Zeitlinger et al., 2007) revealed that a large proportion of silent protein-coding genes (10 to more than 20%) exhibits the hallmarks of transcription initiation without completing elongation. Therefore, the control of gene expression is not only regulated by modification of

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the chromatin and PIC assembly at the promoter, but also during the elongation phase through transcriptional quiescence. The release of pausing RNAPII into a productive elongation state is a highly regulated process that requires the concert action of a variety of factors involved in transcription, RNA processing and chromatin remodeling. The integrated recruitment of these factors to the TEC is governed by phosphorylation of the C-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII.

1.3.1. Phosphorylation cycle of the RNAPII C-terminal domain.

The CTD of RNAPII contains multiples repeats of the heptapeptide sequence (YSPTSPS)n. The number of repeats is variable from one species to another and tends to increase with the organism complexity so that 26 repeats in yeast, 32 in Caenorhabditis elegans, 45 in Drosophila and 52 in human have been enumerated. The functional relevance of the number of CTD repeats was first demonstrated by Nonet et al. (1987), who showed that deletion of the yeast CTD from 26 repeats to less than 10 caused severe lethality. The existence of distinct phosphorylation state of the CTD was primarily described by Dahmus laboratory. The production of specific antibodies recognizing either hypo- and hyperphosphorylated CTD led to the demonstration that RNAPII assembled into the PIC is unphosphorylated, while the elongating polymerase became extensively phosphorylated (Christmann and Dahmus, 1981 ; Laybourn and Dahmus, 1989 ; Lu et al., 1991). The two residues serine 2 (Ser-2) and serine 5 (Ser-5) have been shown to be the major phosphorylation sites of the CTD, which carries on average one phosphate per repeat (Corden et al., 1985 ; Zhang and Corden, 1991a ; Payne and Dahmus, 1993). Subsequently, the distribution pattern of these two modifications across the transcription unit has been explored by chromatin immunoprecipitation (ChIP) in yeast and Drosophila using specific antibodies (O’Brien et al., 1994 ; Komarnitsky et al., 2000 ; Gomes et al., 2006). These studies revealed that RNAPII is primarily phosphorylated at Ser-5 in the promoter-proximal region, while Ser- 2 phosphorylation increases toward the 3’ end of the genes. More recently, it has been shown that the third serine residue of the CTD in position 7 (Ser-7) is also phosphorylated in vivo during transcription of a range of protein-coding genes and snRNA genes (Egloff et al., 2007 ; Chapman et al., 2007). By ChIP analysis using a specific antibody, these authors showed that Ser-7 phosphorylation peaks toward the 3’ end of genes, in a pattern similar to that of Ser-2 phosphorylation. In addition to the serine residues, phosphorylation of the tyrosine (Tyr-1) and threonine (Thr-4) also occurs in vivo to minimal extents (Zhang and Corden, 1991b ;

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Baskaran et al., 1993 ; Baskaran et al., 1997). However, the functional significance of these modifications during transcription has not yet been elucidated. Beside phosphorylation of the heptapeptide repeats, others post-translational modifications of the CTD have been reported.

These include isomerization of the proline residues (Pro-3 and Pro-6) and glycosylation of the serines and threonine residues (reviewed by Egloff and Murphy, 2008). Pro-3 and Pro-6 can modulate the structure and accessibility of the CTD to kinases and phosphatases through conformational changes in cis or trans (Morris et al., 1999). Notably, the phosphorylation- specific peptidyl-prolyl cis/trans isomerase (PPIase) Pin1 has been shown to regulates RNAPII transcription by affecting the phosphorylation status of the CTD through inhibition of the Fcp1 phosphatase (Verdecia et al., 2000 ; Xu et al., 2003 ; Xu and Manley, 2007).

Glycosylation of the CTD repeats at the serines and threonine residues has also been reported in vivo (Kelly et al., 1993). Interestingly, glycosylation and phosphorylation appeared to be mutually exclusive, thus it is possible that glycosylation of RNAPII regulates transcriptional events prior to promoter clearance and CTD phosphorylation.

The potential modifications of the CTD repeats at every position enable a wide range of signalling combinations (Fig. 2A). Particularly, phosphorylation of the serine residues has been shown to direct the sequential recruitment of multiple factors involved in elongation and mRNA maturation. The phosphorylation cycle of the CTD is regulated by specific kinases and phosphatases, some of which have been well characterized during the last decade.

1.3.2. Transcription regulation by CTD kinases and phosphatases.

As mention above, the CTD of RNAPII is subjected to cycles of phosphorylation and dephosphorylation as the TEC progress through the elongation phase. Although all CTD kinases have not been yet identified, the enzymes responsible for Ser-5 and Ser-2 phosphorylation became well documented. These enzymes are all members of the evolutionarily conserved cyclin-dependent kinase (Cdk) family, whose substrate specificity is modulated by association with a regulatory cyclin subunit (reviewed by Loyer et al., 2005).

By contrast to other Cdks dedicated to cell cycle progression, the abundance of the Cdks involved in transcription does not fluctuate during the cell cycle (Tassan et al., 1994 ; Grana et al., 1994 ; Rickert et al., 1996 ; Garriga et al., 2003). The regulation of their activities is nevertheless ensured by different mechanisms including post-translational modification of either subunits and/or interaction with specific inhibitory factors.

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Figure 2. CTD phosphorylation by Cdk kinases. (A) The CTD code. All possible serine phosphorylation and proline isomerization combinations of the CTD heptapeptide are shown.

The proline residues can adopt a cis or trans (tra) conformation catalyzed by specific enzymes such as Pin1. Distinct combinations of serine phosphorylation and possibly proline isomerization occur to regulate specific stages of the transcription cycle. Tyrosine (Y1) and threonine (T4) phosphorylation also occurs in vivo but their function in transcription regulation has not been determined (Zhang and Corden., 1991 ; Baskaran et al., 1993). Mammalian CTD can be glycosylated at the threonine and all serine residues, which might counteract CTD phosphorylation (Kelly et al., 1993). Multiple combinations of differentially glycosylated heptapepetides are also possible (reviewed by Egloff and Murphy, 2008). (B) Schematic drawings of the mammalian TFIIH, Mediator and P-TEFb complexes respectively. The subunits involved in their kinase activities are shown in orange.

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1.3.2.1. Regulation of CTD kinases.

At the promoter region, the RNAPII CTD becomes phosphorylated at Ser-5 by two distinct Cdk/cyclin pairs : Cdk7/CyclinH and Cdk8/CyclinC. Cdk7/CyclinH associates with the MAT1 (ménage à trois-1) protein to form the Cdk-activating kinase (CAK), which phosphorylates and activates other Cdks involved in cell cycle regulation (reviewed by Harper and Elledge, 1998 ; Kaldis, 1999). The CAK kinase is also part of the TFIIH factor and is responsible for Ser-5 phosphorylation during transcription initiation (Akoulitchev et al., 1995

; Valay et al., 1995). TFIIH consists of the holoenzyme IIH, which contains six subunits involved in its ATP-dependent helicase activity, and the CAK complex (Fig. 2B). The CTD kinase activity of TFIIH required both the presence of MAT1 and phosphorylation of Cdk7 at its activating residue threonine 170 (Thr-170) or serine 164 (Ser-164) (Fisher et al., 1995 ; Devault et al., 1995 ; Martinez et al., 1997 ; Larochelle et al., 2001). In addition, phosphorylation of CyclinH by the casein kinase 2 (CK2) results in full activation of the TFIIH kinase activity towards the CTD (Schneider et al., 2002). Conversely, a decrease in the phosphorylation level of Cdk7 at Ser-164 reduces the kinase activity of the complex (Akoulitchev and Reinberg, 1998 ; Long et al., 1998). The phosphorylation of CyclinH by Cdk7 itself or by a subset of Mediator complexes might also occurs in vivo to regulate the CTD kinase of TFIIH (Akoulitchev et al., 2000 ; Lolli et al., 2004). Earlier genome-wide studies for Cdk7 requirement in gene expression indicated that the majority of RNAPII transcripts are affected by Cdk7 mutation in both yeast and Drosophila (Holstege et al., 1998 ; Lee and Lis, 1998 ; McNeil et al., 1998 ; Schwartz et al., 2003). More recent analysis in yeast has restricted the set of genes severely affected by inactivation of the TFIIH-associated kinase to around 5% of all transcripts (Lee et al., 2005). This data suggests that the loss of Ser-5 phosphorylation mediated by Cdk7 could be partially compensated by other CTD kinases including Cdk9 (Zhou et al., 2000 ; Ramanathan et al., 2001 ; Ni et al., 2004).

The Cdk8/CyclinC pair associates with Med12 and Med13 to form the kinase module of some Mediator complexes, which phosphorylates the CTD in a manner similar to that of TFIIH (reviewed by Bourbon et al., 2004 ; Casamassimi and Napoli, 2007). The Mediators are known to function in transcriptional events prior to elongation such as PIC assembly, and dissociates from the TEC during transcription initiation. The kinase activity of these complexes is thought to be mainly implicated in repressive functions, although both Kin28 and Serb10, the orthologues of Cdk7 and Cdk8 in yeast, have overlapping roles in promoting transcription (Liu et al., 2004). The Mediators are large complexes of more than 20 subunits

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organized in three distinct structural sub-modules termed “head”, “middle” and “tail” (Dotson et al., 2000 ; Kang et al., 2001) (Fig. 2B). The head and middle modules mediate interactions of the complexes with the CTD of RNAPII and several GTFs including TFIID (TBP) and TFIIF, while the tail interacts with gene-specific regulators. The Mediator complexes are involved in both positive and negative regulation of transcription. Mediators can stimulate transcription through interactions with the initiation complex, activators bound at regulatory elements and multiple factors required for PIC assembly at the promoter (Malik and Roeder, 2000 ; Cantin et al., 2003 ; Wu et al., 2003a ; Johnson and Carey, 2003 ; Liu et al., 2004 ; Baek et al., 2006 ; Black et al., 2006). Conversely, Mediators complexes containing Med12, Med13, Cdk8 and CyclinC can also act as transcriptional repressors in both yeast and human cells (Hengartner et al., 1998 ; Mo et al., 2004). It has been suggested that premature phosphorylation of the CTD by Cdk8/CyclinC might prevent the formation of the initiation complex (Hengartner et al., 1998). In human cells, Cdk8/CyclinC can phosphorylate CyclinH resulting in inactivation of the TFIIH kinase activity towards the CTD (Akoulichev et al., 2000). Moreover, Cdk8/CyclinC can also phosphorylate other gene-specific transcription factors decreasing their stability and preventing subsequent transcription activation (Chi et al., 2001 ; Nelson et al., 2003). Recent genome-wide analysis of Cdk8 and core Mediator binding in yeast showed that the kinase is present at nearly all Mediator binding sites independently of the promoter activity (Zhu et al., 2006 ; Andrau et al., 2006). The role of Cdk8/CyclinC at active genes remains elusive. It is possible that core Mediators are recruited to promoter to activate gene expression, whereas the kinase module is required to dampen or shut down transcription of the same gene (Zhu et al., 2006). Alternatively, the kinase activity of Cdk8/CyclinC might be involved in the formation of the Mediator scaffold complex at the promoter, and possibly in transcription initiation (Liu et al., 2004).

Subsequently to promoter clearance and Ser-5 phosphorylation by TFIIH, the CTD of RNAPII becomes phosphorylated at Ser-2. The Cdk9 kinase, associated with either T-type cyclins (CyclinT1, T2a, T2b) or CyclinK, is the major enzyme responsible for this modification during transcription and form the core of the positive transcription elongation factor b (P-TEFb) (Peng et al., 1998a,b ; reviewed by Marshall and Grana, 2006) (Fig. 2B). P- TEFb was originally identified based on its ability to overcome transcriptional pausing during the early elongation phase in vitro (Marshall and Price, 1992 ; Marshall and Price, 1995 ; Marshall et al., 1996). The mechanism by which P-TEFb stimulates transcription elongation is dependent on its kinase activity towards the CTD and the negative elongation factors NELF and DSIF (DRB-sensitivity-inducing factor) that promote RNAPII pausing (Marshall et al.,

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1996 ; Wada et al., 1998 ; Yamaguchi et al., 1998). This mechanism will be described in further details in another section of the introduction. The specificity of P-TEFb kinase towards Ser-2 in vivo was first demonstrated by RNA interference experiments in C. elegans (Shim et al., 2002). More recently, the used of the specific P-TEFb inhibitor flavopiridol has also demonstrated that P-TEFb primarily phosphorylates Ser-2 at most actively transcribed genes in Drosophila (Lam et al., 2001 ; Ni et al., 2004). The kinase activity of P-TEFb is regulated by both post-translational modifications of Cdk9 and interaction with various transcriptional activators or repressors (reviewed by Garriga and Grana, 2004 ; Zhou and Yik, 2006). Cdk9 autophosphorylation at several serine and threonine residues is required for both its nuclear localization (Herrmann and Mancini, 2001 ; Napolitano et al., 2003) and its interaction with transcriptional regulators such as the Tat transactivator of the HIV-1 virus (Garber et al., 2000 ; Fong and Zhou, 2000). Interestingly, Cdk9 autophosphorylation can be inhibited by TFIIH at the HIV-1 promoter, whereas the release of TFIIH during elongation coincides with Cdk9 autophosphorylation (Zhou et al., 2001). Similarly to Cdk7, Cdk9 phosphorylation at the activating residue Thr-186 is required for P-TEFb kinase activity, but is also involved in its assembly within the 7SK snRNP inactivating complex (Chen et al., 2004 ; Li et al., 2005). A recent study has now established that Thr-186 is an autophosphorylation site in vitro (Baumli et al., 2008). In human cells, the 7SK snRNP complex plays an important role in the regulation of P-TEFb activity, and is thought to be the major factor involved in P-TEFb inhibition (Zhou and Yik, 2006). Conversely, free active P- TEFb molecules are mainly associated with the bromodomain protein Brd4, which directs P- TEFb recruitment to active promoters (Yang et al., 2005 ; Jang et al., 2005). The pools of transcriptionally active and inactive P-TEFb are maintained in a dynamic equilibrium in human cells, and can be converted into each other in response to stress signals (Yang et al., 2005). An overview of this mechanism of P-TEFb regulation is provided in the second part of the introduction. Other P-TEFb repressors have been identified in selective tissues. In C.

elegans, the PIE-1 protein (pharynx and intestine in excess protein 1) globally represses RNAPII transcription in early embryonic germ cell precursors through interaction with CyclinT1 (Zhang et al., 2003). This repressor contains a single CTD-like heptapeptide repeat in which the serines at positions 2 and 5 are substituted with alanines, a feature essential for PIE-1-mediating transcription silencing (Zhang et al., 2003). P-TEFb inhibition in the primordial germ cells of Drosophila has also been demonstrated, and involves the interaction of Cdk9 with the Pgc (polar granule component) repressor (Hanyu-Nakamura et al., 2008).

The complexity of P-TEFb regulation in different cell-types reflects its potential requirement

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at multiple stages of the transcription cycle and its role in the timely regulation of gene expression. A schematic summary of the kinase activities presented above is shown in figure 3A.

1.3.2.2. Regulation of CTD phosphatases.

During the transcription cycle, a change of the phosphorylation pattern of the CTD is necessary for accurate mRNA synthesis and recycling of the RNAPII (reviewed by Meinhart et al., 2005). Dephosphorylation of Ser-2 and Ser-5 is accomplished by specific phosphatases, which modify sequentially the phosphorylation status of the CTD within the elongation complex. Fcp1 (TFIIF-associating CTD phosphatase 1) is a evolutionarily conserved protein that is globally required for gene expression (Kobor et al., 1999 ; Lin et al., 2002b). Fcp1 is a component of the PIC and interacts with both general transcription factors such as TFIIF and TFIIB, and the phosphorylated RNAPII CTD (Chambers and Dahmus, 1994 ; Chambers et al., 1995 ; Chambers and Kane, 1996 ; Archambault et al., 1997, 1998 ; Kobor et al., 1999 ; Kamada et al., 2003 ; Nguyen et al., 2003). During transcription in yeast, Fcp1 localized at the promoter regions as well as within the coding sequences (Cho et al., 2001), and can stimulate transcription elongation independently of its CTD phosphatase activity (Cho et al., 1999 ; Mandal et al., 2002). Fcp1 was shown to preferentially dephosphorylates Ser-2 in vivo, and its mutation results in increased level of Ser-2 phosphorylation at active genes (Cho et al., 2001 ; Hausmann and Shuman, 2002). However, in vitro studies gave conflicting results as Fcp1 was shown to dephosphorylate both Ser-2 and Ser-5 with equal efficiency (Cho et al., 1999 ; Lin et al., 2002a) or specifically Ser-5 (Kong et al., 2005). To date, Fcp1 is the only identified phosphatase that directs Ser-2 dephosphorylation, but its substrate specificity can potentially switch towards Ser-5 when disassembled from the TEC. As mention earlier, Fcp1 binding to the CTD can be inhibited by the peptidyl-prolyl isomerase Pin1 resulting in transcriptional arrest (Xu et al., 2003). On the other hand, Fcp1 phosphorylation by CK2 was shown to enhance its interaction with TFIIF and to stimulate its CTD-phosphatase activity in vitro (Palancade et al., 2002 ; Friedl et al., 2003 ; Abbott et al., 2005).

The Ssu72 phosphatase is a highly conserved and essential factor involved in mRNA transcription and 3' end processing. Ssu72 (suppressor of sua7-1 clone 2) was initially identified in a yeast genetic screen as enhancer of TFIIB mutation (Sun and Hampsey, 1996).

Ssu72 interacts with RNAPII and TFIIB at the promoter in both yeast and human cells (Wu et al., 1999 ; Pappas and Hampsey, 2000 ; Dichtl et al., 2002 ; St-Pierre et al., 2005). Ssu72

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possesses intrinsic phosphatase activity in vitro, and its mutation in yeast increases RNAPII pausing (Dichtl et al., 2002 ; Ganem et al., 2003 ; Meinhart et al., 2003). Subsequently, Ssu72 was shown to dephosphorylate the CTD at Ser-5 in vitro, and depletion of Ssu72 in vivo results in accumulation of Ser-5 phosphorylated RNAPII (Krishnamurthy et al., 2004). The yeast Ssu72 also has a phosphatase-independent function during transcription. It is a component of the cleavage and polyadenylation factor (CPF) complex and is involved in transcription termination (Dichtl et al., 2002 ; He et al., 2003 ; Nedea et al., 2003 ; Steinmetz and Brow, 2003).

In higher eukaryotes, a family of small CTD phosphatases (SCPs) was also found to mediate the selective dephosphorylation of Ser-5 on the CTD (Yeo et al., 2003). Scp1, one of the SCP family members, can affect the rate of transcription activation from a number of promoters and, like Fcp1, its phosphatase activity is enhanced by TFIIF (Yeo et al., 2003).

Inactivation of Scp1 stimulates transcription in vitro and induces derepression of neuronal genes in the human neuronal stem cells (Yeo et al., 2003 ; Yeo et al., 2005). As observed for Fcp1, Scp1 activity can be inhibited in vitro by Pcif1 (PDX-1 C terminus-interacting factor 1), a protein that directly binds to the phosphorylated CTD in a manner similar to that of Pin1 (Hirose et al., 2008).

During transcription elongation, Ser-5 is actively dephosphorylated by Ssu72 and/or Scp1 (Fig. 3A), which coincides with the release of the 5’ end processing factors, whereas Ser-2 phosphorylation leads to the recruitment of the 3’ mRNA processing factors (Komarnitsky et al., 2000 ; Cho et al., 2001). Following transcription termination, dephosphorylation of Ser-2 by Fcp1 is finally required for the regeneration of the hypophosphorylated CTD allowing RNAPII to initiate the next round of transcription (Cho et al., 1999).

1.3.3. Transcription elongation and pre-mRNA processing.

Transcriptional pausing in early elongation is viewed as a checkpoint allowing the timely addition of the 5’ pre-mRNA cap prior to the exit of RNAPII into productive elongation (reviewed by Orphanides and Reinberg, 2002 ; Pei and shuman 2002). RNAPII pausing is promoted by the assembly of negative elongation factors to the TEC and can stimulate capping activity along with Ser-5 phosphorylation of the CTD. The kinase activity of P-TEFb is next required to alleviate RNAPII pausing and to regulate downstream events of pre-mRNA processing through phosphorylation of the CTD at Ser-2. In addition to the

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Figure 3A. Regulation of RNAPII phosphorylation by CTD kinases and phosphatases.

During transcription initiation, the CTD is primarily phosphorylated at Ser-5 by TFIIH. The kinase activity of TFIIH is regulated by phosphorylation of both Cdk7 and CyclinH. CyclinH phosphorylation by CK2 has positive effect on TFIIH while its phosphorylation by Mediator inhibits the kinase activity of TFIIH. CTD phosphorylation at Ser-2 by P-TEFb stimulates transcription elongation. The kinase activity of P-TEFb is also regulated through phosphorylation of the Cdk9 subunit. During elongation, Ser-5 is first dephosphorylated by Scp1 and/or Ssu72, while Ser-2 dephosphorylation by Fcp1 is required for RNAPII recycling.

Both Scp1 and Fcp1 phosphatase activity can be prevented by association of Pcif1 and Pin1 to the CTD respectively.

Figure 3B. Regulation of RNAPII promoter-proximal pausing and capping processes.

RNAPII pausing is promoted by assembly of DSIF and NELF to the elongation complex. CTD phosphorylation at Ser-5 allows recruitment of the RT/GT enzymes at the 5’ end of the nascent transcript and stimulates their capping activities. The MT enzyme is next recruited to the CTD along with P-TEFb, and CTD phosphorylation at Ser-2 by P-TEFb might stimulate the capping activity of the MT enzyme. P-TEFb also phosphorylates the Spt5 subunit of DSIF and NELF-E resulting in dissociation of the NELF complex and the release of RNAPII into productive elongation.

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phosphorylation status of RNAPII, specific regions of the CTD were shown to stimulate distinct processes, so that the N-terminus heptapeptide repeats are mainly involved in the addition of the 5’ cap, while the C-terminal repeats (heptapeptides 27 to 52 in human) regulate pre-mRNA splicing and 3’ end processes (Fong and Bentley, 2001 ; Fong et al., 2003). The structure of the CTD thus plays a central role in regulating mRNA processing, which occurs most efficiently co-transcriptionally, and acts as a loading platform for many processing factors (reviewed by Hirose and Manley, 2000 ; Shatkin and Manley, 2000 ; Proudfoot et al., 2002).

1.3.3.1. Transcription factors involved in RNAPII promoter-proximal pausing.

The current model of RNAPII pausing at the promoter-proximal region implies the interplay of three protein complexes, NELF (negative elongation factor), DSIF (DRB sensitivity inducing factor) and P-TEFb (Cheng and Price, 2007) (Fig. 3B). NELF and DSIF were originally identified on their ability to promote RNAPII pausing and function together to inhibit transcription elongation in vitro (Wada et al., 1998 ; Yamaguchi et al., 1999 ; Renner et al., 2001). DSIF is a heterodimer composed of Spt4 and Spt5, which directly interact with RNAPII and a variety of factors involved in transcription and chromatin remodeling including TFIIF, TFIIS, Spt6, FACT, Chd1 and the PAF complex (reviewed by Sims et al., 2004). By contrast, NELF does not bind RNAPII by itself but requires the preassembly of DSIF to the TEC (Yamaguchi et al., 2002). Human NELF consists of four subunits NELF-A, B, C or D and E, where NELF-C and D arise from the same mRNA transcript through alternate translation (Yamaguchi et al., 2001 ; Narita et al., 2003). The ability of NELF to inhibit elongation notably involves the interaction of NELF-E with the nascent transcript after synthesis of a 20-30 nucleotides pre-mRNA (Yamaguchi et al., 2002 ; Rao et al., 2008).

In vivo studies in Drosophila have shown that DSIF and NELF colocalize at the promoter region of pausing RNAPII, while only Spt5 is associated with transcriptionally active genes (Kaplan et al., 2000 ; Andrulis et al., 2000 ; Wu et al., 2003b). In addition, depletion of NELF reduced the level of paused polymerases in both Drosophila and human cells (Wu et al., 2003b ; Wu et al., 2005 ; Aida et al., 2006 ; Muse et al., 2007). Depletion of DSIF has more variable effects on gene expression and reflects its dual role during transcription elongation (Hartzog et al., 1998 ; Wu et al., 2003b). Notably, DSIF was shown to antagonize the negative effect of the ISWI (imitation switch) chromatin remodeling factor in yeast and to prevent premature termination during elongation (Bourgeois et al., 2002 ;

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Rondon et al., 2003 ; Morillon et al., 2003). Recent genome-wide analysis of NELF binding in Drosophila reveals that this factor is associated with paused RNAPII at thousands gene promoters and can be considered as a general transcription factor in higher eukaryotes (Muse et al., 2007 ; Gilmour, 2009).

1.3.3.2. Recruitment of capping enzymes at paused RNAPII.

Eukaryotic mRNAs are modified at their 5’ end by the addition of a m7GpppN cap catalyzed by the sequential action of three enzymatic activities, RNA 5’-triphosphatase (RT), guanylyl-transferase (GT), and (guanine-N7) methyltransferase (MT)(reviewed by Hirose and Ohkuma, 2007). In yeast, these proteins are encoded by three different genes, whereas in metazoans the RT and GT activities are mediated by a single protein. As mention above, capping occurs co-transcriptionally and requires both binding of DSIF/NELF to the polymerase and phosphorylation of the CTD at Ser-5 (Fig. 3B). The GT domain of the capping enzyme interacts with and is activated by the Ser-5-phosphorylated CTD (McCracken et al., 1997a ; Ho and Shuman, 1999 ; Pei et al., 2001 ; Moteki and Price, 2002 ; Fabrega et al., 2003). In addition, the Spt5 subunit of DSIF directly interacts with the RT and GT proteins and stimulates RNA guanylylation in both yeast and mammals (Wen and Shatkin, 1999 ; Pei and Shuman, 2002). The recruitment of these capping enzymes was also shown to disable the repressive effect of NELF (Mandal et al., 2004).

The relief of NELF-mediated transcription inhibition coincides with the recruitment of P-TEFb (Yamaguchi et al., 1999). Beside its CTD kinase activity, P-TEFb was shown to phosphorylate NELF-E resulting in dissociation of the NELF complex from the TEC (Fujinaga et al., 2004). In addition, phosphorylation of Spt5 by P-TEFb is also required to convert its activity to positive transcriptional regulator (Kim and Sharp, 2001 ; Yamada et al., 2006). Following P-TEFb action, the TEC enters into productive elongation phase and the removal of Ser-5 phosphorylation by specific phosphatases leads to the dissociation of the RT/GT enzymes from the CTD (Schroeder et al, 2000). In this aspect, the MT enzyme remains bound to the TEC throughout the transcription unit, suggesting that Ser-2 phosphorylation might also contribute to the recruitment of capping enzymes (Komarnitsky et al, 2000 ; Schroeder et al, 2000). Studies in fission yeast indicate that bindingof RT (Pct1) and GT (Pce1) to the CTD is optimal when both Ser-2 and Ser-5 are phosphorylated (Pei et al., 2001), and that P-TEFb directly interacts with RT and MT (Pcm1) in vivo (Pei and Shuman, 2002 ; Pei et al, 2003 ; Pei et al., 2006). A recent report has now established that P-

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TEFb is recruited to the CTD through its interaction with the MT enzyme, and that MT depletion in yeast compromises the Cdk9 kinase activity towards the CTD resulting in promoter-proximal stalling (Guiguen et al., 2007). The stepwise recruitment of the capping enzymes along with P-TEFb ensures that the kinase is not recruited to chromatin before capping has occurred, thus making sure that uncapped transcripts are not elongated.

1.3.3.3. Pre-mRNA splicing and transcription elongation.

In higher eukaryotes, the excision of intronic sequences by the splicing machinery is also known to occur co-transcriptionally in vivo (reviewed by Jurica and Moore, 2003 ; de Almeida and Carmo-Fonseca, 2008). Pre-mRNA splicing takes place within a large spliceosome complex composed of small nuclear ribonucleoprotein particles (snRNPs) and a variety of non-snRNP auxiliary proteins including the serine/arginine-rich (SR) protein family (Zhou et al., 2002 ; Chen et al., 2007). As mention earlier, the CTD plays an integral role in the splicing process and CTD truncation inhibits both capping and splicing activities during transcription in vivo (McCracken et al., 1997b ; Fong et al., 2003 ; Rosonina and Blencowe, 2004). Conversely, the phosphorylated CTD can activate pre-mRNA splicing by itself even in absence of transcription in vitro (Hirose et al., 1999 ; Zeng and Berget, 2000). In vivo study using Xenopus oocytes demonstrated that CTD phosphorylation by Cdk7 and Cdk9 is actually required for co-transcriptional splicing, and points out to the role of Ser-2 and Ser-5 phosphorylation in this process (Bird et al., 2004).

Several studies have previously reported a direct connection of the phosphorylated CTD with the splicing apparatus (Chabot et al., 1995 ; Vincent et al., 1996 ; Mortillaro et al., 1996 ; Kim et al., 1997). Particularly, SR and SR-like proteins such as SCAFs (SR-like CTD associated factors) directly interact with the phosphorylated CTD and are good candidates for linking transcription and splicing (Kornblihtt et al., 2004). SR proteins play a key role in spliceosome assembly in both yeast and human cells, and might be pre-loaded onto the CTD upon transcription (Shen and Green, 2004, 2006). Interestingly, P-TEFb was also found to interact with some splicing factors during transcription of the HIV-1 virus, suggesting that the kinase may participate in the coupling of transcriptional elongation with pre-mRNA splicing.

These factors include Tat-SF1, a homologue of the yeast splicing factors CUS2 (Yan et al., 1998 ; Fong and Zhou, 2000), and SKIP (splicing-associated c-Ski-interacting protein) which is an essential component of activated spliceosomes (Bres et al., 2005). Recent in vitro analysis in yeast have also demonstrated that formation of the spliceosomal complex occurs

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co-transcriptionally and requires the pre-assembly of the capping components (Gornemann et al., 2005 ; Lacadie and Rosbash, 2005). The early assembly of the splicing machinery during transcription is thought to protect the newly synthesized pre-mRNA from nuclease degradation and/or splicing inhibitors, and to enhance splicing efficiency (Das et al., 2006 ; Hicks et al., 2006). Finally, the kinetics of transcription elongation was shown to influence the selection of the splice sites (de la Mata et al., 2003 ; Kornblihtt et al., 2004). Notably, the Brahma subunit of human SWI/SNF chromatin remodeling complex can regulate alternative splicing by promoting RNAPII pausing near the alternative splice sites (Batsche et al., 2006).

1.3.3.4. Transcription termination and 3’end pre-mRNA processing.

Most eukaryotic mRNA possess a poly(A) tail at their 3’ extremity, which is formed by endonucleolytic cleavage of the transcript followed by addition of a poly(A) tail (reviewed by Danckwardt et al., 2008 ; Mandel et al., 2008). These reactions are catalyzed by multiprotein complexes which bind specific elements at the 3’ end of the pre-mRNA. The polyadenylation signal, located 10-30 nucleotides upstream of the cleavage site, is bound by the cleavage/polyadenylation specific factor (CPSF), while the downstream sequence element (DSE) is recognized by the cleavage-stimulating factor (Cstf) (Fig. 4). Other RNA elements upstream of the cleavage site have been identified to anchor the 3’ end-processing machinery or to recruit other components such as the cleavage factor CFIm. After assembly of the multimeric complexes at their elements and endonucleolytic cleavage by CPSF, a nuclear poly(A) polymerase (PAP) synthesizes the 3’ end poly(A) tail.

Similarly to capping and splicing, transcription termination and 3’ end processing is regulated by multiple interactions with the elongating polymerase, and CTD truncation results in 3’ end processing defects (McCracken et al., 1997b ; Fong et al., 2003 ; Rosonina and Blencowe, 2004). Several components of both CPSF and Cstf complexes directly interact with the phosphorylated CTD (Barilla et al., 2001 ; Licatalosi et al., 2002 ; Proudfoot et al., 2002 ; Rosonina et al., 2006). Recently, it was demonstrated that Ser-2 phosphorylation is critically required for proper 3’ processing of the transcripts. The loss of Ser-2 phosphorylation caused by Cdk9 (Ctk1) mutation in yeast (Ahn et al., 2004) or following flavopiridol inhibition of P-TEFb in Drosophila (Ni et al., 2004) leads to inefficient mRNA processing. Intriguingly, the authors found that inactivation of the Cdk9 kinase activity had little effects on transcription elongation, while the recruitment of 3’ processing factors was impaired resulting in rapid degradation of the transcripts. Thus it was proposed that Ser-2

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phosphorylation is necessary for accurate transcription termination and 3’ end processing, but appeared dispensable for transcription elongation of certain genes such as the intron-less Drosophila heat shock genes (Ni et al., 2004).

Transcription termination, which results in the dissociation of the elongation complex and the release of the mRNA, is intimately coupled with the 3’ processing machinery and requires additional factors that bind RNAPII and its CTD such as TTF2 (transcription termination factor 2) and Pcf11 (pre-mRNA cleavage complex 2 protein Pcf11) (Jiang et al., 2004 ; Zhang and Gilmour, 2006 ; West and Proudfoot, 2008). However, in contrast to the 3’

processing factors, the recruitment of these proteins does not depend on the phosphorylation status of the CTD.

As discussed above, transcription elongation is an integrated process that is tightly coupled to pre-mRNA processing, and the phosphorylation cycle of the RNAPII CTD occurs to coordinate these events. Extensive crosstalk between the pre-mRNA processing activities is also required for efficient transcription (Fig. 4). Indeed, some proteins that bind to the cap were shown to facilitate the recruitment of the splicing machinery at the cap-proximal splice site (Lewis et al., 1996 ; Colot et al., 1996). Similarly, many studies have reported a physical interaction of the cleavage/polyadenylation complexes with splicing factors, which stimulates both terminal intron splicing and 3’ end processing (Vagner et al., 2000 ; Li et al., 2001 ; McCracken et al., 2002 ; Awasthi and Alwine, 2003 ; Millevoi et al., 2006 ; Kyburz et al., 2006 ; Danckwardt et al., 2007). The cooperation between the different co-transcriptional mechanisms ensures the efficient production of mature mRNA, and the packaging of the pre- mRNA within the processing complexes is thought to protect the transcript from nuclease degradation and to prevent interactions with the DNA template during transcription (Li and Manley, 2006 ; Hicks et al., 2006).

1.3.4. Transcription elongation and chromatin remodeling.

The packaging of the DNA template into chromatin represents an important block to transcription elongation. The basic repeating unit of chromatin, the nucleosome, contains two copies of the four core histones H2A, H2B, H3 and H4 wrapped around by a 147 base pairs DNA. During transcription, RNAPII associates with multiple factors that promote changes in the chromatin structure. These factors include ATP-dependent chromatin remodelers, histone- modifying enzymes and histone chaperones (reviewed by Kulaeva et al., 2007 ; Li et al.,

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Figure 4. Schematic drawing of the 3’ end processing machinery in mammals (Danckwardt et al., 2008). The cleavage/polyadenylation complexes CPSF and CstF are recruited to the elongation complex through interaction with the Ser-2-phosphorylated CTD, and binds distinct RNA elements. CPSF recognizes the polyadenylation signal (AAUAAA) while CstF associates with the downstream sequence element (DSE). CPSF 73 catalyzes the endonucleolytic cleavage of the transcript at the cleavage site (CA) allowing the subsequent addition of the poly(A) tail by the poly(A) polymerase (PAP). Multiple stimulatory interactions between the pre-mRNA processes (capping, splicing and cleavage/polyadenylation) occur to coordinate their activities during transcription, and are indicated by an arrow (+).

The transcription elongation factor P-TEFb in &#039;Drosophila melanogaster&#039; : characterization of a mutation of the Cdk9 kinase and of the different activities of two co-factors

Thesis

Reference