Thesis
Reference
Role of the nuclear pore in transcription regulation of inducible genes in Saccharomyces cerevisiae
TEXARI, Lorane
Abstract
This study provides evidence for a new role for the NPC and the NPC-associated SUMO-protease Ulp1 in transcription regulation of two inducible genes, GAL1 and HXK1.
Interestingly, thanks to the complexity of GAL1 activation, we could show that Ulp1 is involved in the initial derepression step of GAL1 and not in its transcription activation, suggesting an early role of Ulp1 upon GAL1 induction. Furthermore, we showed that Ssn6 is a target of Ulp1. Ssn6 is sumoylated and acts as a repressor of GAL1. Our current model is that Ssn6 bound to the GAL1 promoter is relocalized to the NPC at an early stage of galactose induction. This allows the desumoylation of Ssn6, and probably other gene bound factors, by Ulp1 leading to the full derepression and activation of GAL1. Interestingly, we also found that Ulp1 is required for GAL1 localization at the NPC.
TEXARI, Lorane. Role of the nuclear pore in transcription regulation of inducible genes in Saccharomyces cerevisiae. Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4622
URN : urn:nbn:ch:unige-332452
DOI : 10.13097/archive-ouverte/unige:33245
Available at:
http://archive-ouverte.unige.ch/unige:33245
Disclaimer: layout of this document may differ from the published version.
Professeur Françoise Stutz
Role of the nuclear pore in transcription regulation of inducible genes in Saccharomyces cerevisiae
THÈSE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Lorane TEXARI de
Gonesse, France
Thèse n°4622
Genève
Atelier de reproduction de l’Université de Genève 2013
J’aimerais remercier tout d’abord Karsten Weis et David Shore d’avoir accepté d’être les membres de mon jury.
J’aimerais également remercier les membre de mon TAC meeting, David Shore et Robbie Loewith pour m’avoir suivi et conseillé durant ma thèse.
J’aimerais remercier Françoise Stutz de m’avoir acceptée dans son laboratoire, il y a tout juste quatre ans. Ces années passées au sein de son laboratoire ont été très enrichissantes. J’ai rencontré et travaillé avec un grand nombre de personnes et je voudrais remercier toutes ces personnes pour leur gentillesse, leur aide et leur bonne humeur. J’aimerais remercier en particulier les garçons du laboratoire, Andrea, Manuele et Noël qui ont amené un peu de testostérone et surtout leur joie de vivre et leur bonne humeur. Ce fut un plaisir de partager toutes ces années avec eux.
J’aimerais également remercier Evelina et Elisa G pour nos grandes discussions scientifiques. Je remercierai également une nouvelle venue, Valentina pour les bons moments passés ensemble à notre dernière EMBO conférence. Le meilleur pour la fin… J’aimerais remercier du fond du cœur, deux collègues mais surtout amies Mariana et Géraldine. Géraldine, ces trois ans et demi passés à tes côtés ont été un véritable bonheur tant d’un point de vue professionnel que personnel. Tu es une technicienne extraordinaire et tu as été un véritable soutien et pilier pour moi durant ma thèse. Mariana, tu es la meilleure « Master student » que j’ai eu (même si je n’en ai eu qu’une…). Tu es arrivée durant la dernière année de ma thèse et tu m’as sauvée… Tu as appris très vite les ficèles du métier (les inductions galactose et les centaines d’extraction d’ARN et RT-qPCR qui suivent) et tu as été d’une aide précieuse. En effet, ta dextérité et ta rapidité m’ont énormément aidé dans le sprint final pour le papier, mais je me rappellerai surtout de ta joie de vivre, de ton enthousiasme et de ton soutien dans les moments difficiles.
J’aimerais également remercier mes collaborateurs, Marian Groot Koerkamp et Franck Holstege avec qui nous avons fait les Microarrays. Benoit Palancade pour les nombreuses discussions scientifiques autour de notre protéine favorite, Ulp1.
J’aimerais remercier Pablo Echeverria pour son aide dans l’utilisation de cytoscape.
Thomas, Sébastien G, Chris et Ratheesh qui ont fait partie de ma vie professionnelle et personnelle durant ces quatre ans. J’aimerais remercier également mes amis biologistes : Cyril M, Kim, Chrystelle, Elodie P, Guillaume, Adrien et tous les autres.
J’aimerais également remercier tous mes amis non biologistes qui m’ont soutenu et amusé durant toutes ces années ou plus… Merci à mon amie de toujours Mélissa, à Romain, Marie, Lucie, Robin, Anne-Sophie, Elodie M et tous les autres…
J’aimerais remercier ma belle famille de m’avoir accepté dans leur famille et pour leur soutien.
La dernière partie de mes remerciements est dédiée à ma famille. J’aimerais leur dire à quel point leur soutien et leur amour a été nécessaire pour moi et pour survivre à ces quatre années de dur labeur. Daphné, je te remercie d’avoir toujours été présente dans les moments difficiles mais également dans les moments les plus importants de ma vie. Je n’aurai pas pu rêver d’une meilleure sœur que toi. Nous avons réussi à garder notre amour mais aussi nos disputes de petites filles et j’espère que nous nous aimerons et nous soutiendrons toujours comme nous l’avons toujours fait. Papa, maman, je ne sais pas par où commencer. Je vais donc commencer par le début… Depuis toujours vous avez tout fait pour moi, j’ai sélectionner un petit échantillon… 1) l’enfance : passer des heures en voiture pour m’amener au cours de piano, solfège, gymnastique, tennis, etc… 2) l’adolescence : passer des heures sous la pluie (quand ce n’était pas de la grêle) en attendant la fin des régates ; m’amener en plein milieu de l’Espagne lors de la pire des canicules recensée durant ces vingt dernières années (sans climatisation dans le voiture) … 3) Ma vie loin de vous : me loger sur Bordeaux pour m’éviter les trajets quotidiens ; me loger à Paris car j’avais décidé de partir encore plus loin, et enfin traverser la France pour m’amener moi et mes dix tonnes d’habits et objets personnels en Suisse. Je sais que vous avez fait de nombreuses concessions pour que Daphné et moi soyons les plus heureuses possibles.
Je vous remercie du fond du cœur et je peux vous dire que vous avez réussi !! Sans vous, je ne serai pas là… Oui, je sais, vous êtes mes parents donc forcément sans vous je ne serai pas là… mais je ne serai jamais arrivé là où je suis aujourd’hui (presque Docteure) sans vous !!
amour, d’être tout simplement toi… Sans ton soutien et ton amour, je ne serai certainement pas là où j’en suis aujourd’hui.
1 Summary and Résumé en français ... 1
1.1 Résumé en français ... 3
1.2 Summary ... 7
2 Introduction ... 9
2.1 Organization of the nucleus ... 11
2.1.1 General introduction on the nucleus and its organization ... 11
2.1.2 The Nuclear Pore Complex (NPC) ... 14
2.1.2.1 Structure of the NPC ... 14
2.1.2.2 NPC assembly into the nuclear envelope ... 16
2.1.2.3 The role of the NPC in import/export of cargo proteins ... 18
2.1.2.1 Role of the NPC in mRNA export ... 19
2.1.2.2 Role of the NPC in quality control and genome stability ... 22
2.2 Transcription ... 23
2.2.1 General introduction on gene expression ... 23
2.2.2 Transcription of RNA Pol II-‐dependent genes ... 24
2.2.2.1 Co-‐transcriptional mRNP formation via CTD phosphorylation ... 25
2.2.2.1.1 Transcription and mRNP formation ... 25
2.2.2.1.2 Recruitment of Export Factors during transcription ... 27
2.2.3 Role of Chromatin during Transcription ... 28
2.2.4 GAL1 gene transcription regulation ... 29
2.3 Role of nuclear organization in transcription regulation ... 34
2.3.1 Role of the nuclear envelope in transcription regulation ... 34
2.3.2 Role of the nuclear pore complex in transcription regulation ... 36
2.3.2.1 Gene gating in yeast ... 36
2.3.2.2 Role of nucleoporins in transcription regulation in higher eukaryotes ... 38
2.3.2.3 Gene gating can induce DNA replication stress ... 40
2.4 Sumoylation ... 41
2.4.1.1 General introduction on sumoylation ... 41
2.4.1.2 SUMO pathway at the NPC ... 43
2.4.1.3 Known roles of Ulp1 ... 44
2.5 Aim of my PhD thesis work ... 47
3 Results ... 49
3.1 The nuclear pore regulates GAL1 gene transcription by controlling the localization of the SUMO protease Ulp1 ... 51
3.2.2 Microarray analyses ... 86
3.2.2.1 Other genes are regulated at the NPC by Ulp1 ... 86
3.2.2.2 The majority of the 43 genes are inducible ... 87
3.2.2.3 Genes found in this study are enriched in Tup1 and SAGA dependent genes and TATA box-‐containing genes ... 89
3.2.2.4 Relationship between Transcription Factors and Ulp1 ... 91
3.2.2.4.1 Transcription factors associated with the 43 genes of interest are enriched for sumoylation ... 91
3.2.2.4.2 The top 20 transcription factors could be targets of Ulp1 ... 93
3.2.2.4.3 Bridge proteins could also be targets of Ulp1 ... 96
3.2.2.4.4 A desumoylated Gcn5 mutant shows increased GAL1 and HXK1 mRNA levels ... 98
4 General Discussion ... 101
4.1 Role of the NPC in transcription regulation in S. cerevisiae ... 103
4.1.1 The dual role of the pore in transcription regulation of GAL1 ... 103
4.1.2 Ulp1, a new player in the derepression of GAL1 and other inducible genes ... 103
4.1.3 Ulp1 could reconcile two different views on the role of the NPC in transcription regulation ... 105
4.2 Conserved role of nucleoporins in transcription regulation in higher eukaryotes ... 106
4.3 Role of the sumoylation state of proteins involved in transcription regulation ... 108
4.3.1 Role of sumoylation of Ssn6-‐Tup1 complex in transcriptional regulation 108 4.3.2 Other sumoylated proteins could be implicated in transcription regulation at the NPC ... 109
4.3.2.1 The SAGA component Gcn5: a putative target of Ulp1 ... 110
4.3.2.2 Sumoylation and chromatin: What are the links? ... 110
4.3.2.3 Kinases: putative targets of Ulp1 ... 112
4.4 Could Ulp1 be involved in mRNA export? ... 113
5 References ... 115
6 Annexes ... 139
1. Summary and Résumé en français
1.1 Résumé en français
Le pore nucléaire est un élément essentiel pour la cellule permettant la communication entre le contenu du noyau et le reste de la cellule, le cytoplasme, grâce à son rôle dans le trafic de protéines et d’ARN. Lors de leur activation transcriptionnelle, certains gènes de levure ont été trouvés associés aux pores nucléaires. Le rôle de cette nouvelle localisation à l’intérieur même du noyau est mal comprise et est même sujet à controverse dans le domaine. Le phénomène de relocalisation des gènes semble être spécifique aux gènes inductibles. La relocalisation des gènes requiert plusieurs composants du pore nucléaire, des facteurs de transcription mais également des protéines impliquées dans l’export des ARNs messagers. Deux protéines, Mlp1 et Mlp2, constituant la partie nucléaire du pore, ont également été impliquées dans la relocalisation des gènes.
Mon travail de thèse a été de comprendre le rôle de l’architecture nucléaire dans l’expression des gènes chez la levure, et plus particulièrement, le rôle des composants du pore tels que Mlp1 et Mlp2 dans la transcription de deux gènes inductibles, GAL1 et HXK1.
Dans un premier temps, nous avons montré que l’ancrage artificiel du gène GAL1 aux pores nucléaires provoquait une augmentation du taux de transcrits GAL1 en condition de répression. Ce résultat suggère que le pore nucléaire est un compartiment favorisant l’activation des gènes.
Nous avons décidé d’étudier le rôle des protéines Mlp1 et Mlp2 dans la régulation transcriptionnelle des gènes GAL1 et HXK1, car une étude précédente avait démontré que ces dernières interagissaient avec le promoteur du gène GAL1. Afin d’étudier le rôle de ces deux protéines dans la transcription, nous avons délété les gènes MLP1 et MLP2 et observé que la cinétique de dérépression de GAL1 était plus rapide et que le gène HXK1 était également moins réprimé.
Un des nombreux rôles des protéines Mlp1 et Mlp2 est d’ancrer Ulp1 aux pores. Ulp1 est une SUMO-protéase qui permet d’enlever la modification post- traductionnelle SUMO, un court peptide attaché à certaines protéines. Sachant que la délétion des gènes MLP1 et MLP2 affecte la localisation d’Ulp1 aux pores nucléaires,
nous avons décidé d’étudier le rôle de la délocalisation d’Ulp1 dans la régulation transcriptionnelle des gènes GAL1 et HXK1. Pour ce faire, nous avons construit un mutant Ulp1 dans lequel le domaine responsable de la localisation aux pores est délété (ΔNulp1-GFP). Tout comme un mutant Δmlp1/2, le niveau d’ARN de GAL1 et HXK1 est augmenté dans ΔNulp1-GFP. De plus, l’ancrage artificiel d’Ulp1 aux pores nucléaires dans une souche ayant perdu les protéines Mlp1 et Mlp2 restore une cinétique de dérépression du gène GAL1. Ce résultat confirme que la localisation d’Ulp1 aux pores est nécessaire pour obtenir une expression sauvage du gène GAL1.
A l’inverse, le gène GAL1 est surexprimé lorsque Ulp1 est ancré artificiellement au locus GAL1 après quatre heures d’induction en galactose.
Tous ces résultats suggèrent que la proximité entre Ulp1 et le gène GAL1 est nécessaire pour favoriser la dérépression du gène GAL1. Notre hypothèse est que les protéines Mlp1 et Mlp2 participent à la régulation transcriptionnelle qui a lieu au niveau des pores en favorisant la désumoylation de protéines associées au gène via Ulp1.
Pour tester cette hypothèse, nous avons recherché une cible potentielle d’Ulp1 qui serait impliquée dans la régulation transcriptionnelle des gènes GAL1 et HXK1.
Nous avions deux protéines candidates : Ssn6 et Tup1. Ces deux protéines sont sumoylées et impliquées dans la répression des gènes GAL1 et HXK1. De plus, une ancienne étudiante du laboratoire, Patricia Vinciguerra, avait trouvé ces deux protéines dans un crible double hybride cherchent des interactions avec le domaine C- terminal de Mlp2, suggérant une interaction entre Ssn6/Tup1 et la protéine Mlp2.
Nous avons ensuite montré que la délocalisation d’Ulp1 affecte le niveau de sumoylation de Ssn6 et Tup1 et que des mutants de Ssn6, dans lesquels la sumoylation est altérée, montrent une augmentation de l’expression des gènes GAL1 et HXK1. La restauration de la sumoylation de Ssn6 corrèle avec une restauration du niveau de transcrits de GAL1 et HXK1, ce qui suggère un rôle de la sumoylation de Ssn6 dans la régulation transriptionnelle de GAL1.
Ce travail a permis de mettre en évidence le rôle du pore nucléaire dans la dérépression des gènes GAL1 et HXK1 grâce à la désumoylation de Ssn6 via Ulp1, lors de la relocalisation des gènes aux pores. Ce travail a récemment était publié dans le journal Molecular Cell.
La deuxième partie de mon travail de thèse a été de trouver d’autres gènes régulés par Ulp1. Pour cela, nous avons examiné l’expression des gènes de façon globale dans les souches Δmlp1/2, ΔNulp1-GFP et type sauvage par Microarrays en collaboration avec le laboratoire de Frank Holstege. Nous sommes actuellement en train d’analyser les résultats et de valider certains candidats, mais il semblerait que Ulp1 régule préférentiellement des gènes inductibles. Ces résultats consolident notre modèle selon lequel les gènes inductibles relocalisent aux pores nucléaires afin d’être déréprimés par un mécanisme impliquant la désumoylation de protéines clés par Ulp1.
1.2 Summary
The Nuclear Pore Complex (NPC) is essential for the communication between the nucleoplasm and the cytoplasm because of its role in protein and RNA trafficking.
Intriguingly, transcription activation of some yeast genes correlates with their repositioning to the Nuclear Pore Complex. The role for this mechanism is still poorly understood and controversial. NPC gene association mainly concerns highly transcribed and inducible genes and depends on NPC constituents, transcription factors and components of the mRNA processing and export machineries. The NPC nuclear basket Mlp1/2 proteins have also been involved in this process.
My PhD work consists in understanding the role of nuclear architecture in gene expression and more specifically the contribution of the nuclear basket proteins such as Mlp1 and Mlp2 in transcription regulation of GAL1 and HXK1, two glucose repressed and inducible genes.
First, we showed that artificial tethering of the GAL1 gene to the NPC increases GAL1 mRNA levels in repressive condition, suggesting that the NPC environment promotes gene expression. Because Mlp1/2 proteins have been shown to interact with the GAL1 promoter, we decided to study the role of these two proteins in GAL1 and HXK1 transcription regulation. We observed that in a strain lacking both MLP1 and MLP2, the kinetics of GAL1 gene derepression were enhanced and the HXK1 gene was also derepressed.
Mlp1/2 proteins are known to restrict the key SUMO-protease, Ulp1 at the NPC. We therefore decided to study whether delocalization of Ulp1 from the NPC could be involved in the effect of MLP1/2 deletion on the expression of GAL1 and HXK1. We designed a Ulp1 mutant in which the NPC targeting domain is deleted (ΔNulp1-GFP) and found that this mutant enhanced GAL1 derepression kinetics and increases HXK1 mRNA levels, similarly to what we observed in a Δmlp1/2 mutant.
Furthermore, artificial re-anchoring of Ulp1 at the NPC in ∆mlp1/2 strain restored normal GAL1 derepression kinetics, confirming a role for the NPC localization of Ulp1 in normal GAL1 derepression. Conversely, artificial tethering of Ulp1 to the GAL1 locus increased GAL1 mRNA levels after 4 hours of galactose induction.
Taken together, these results suggest that the proximity between the GAL1 gene and Ulp1 enhances GAL1 derepression kinetics. We hypothesized that Mlp
proteins, through the action of the SUMO-protease Ulp1, could participate in transcription regulation at the NPC by desumoylating factors implicated in repression/activation.
To test this hypothesis, we searched for potential Ulp1 targets involved in GAL1 and HXK1 transcription regulation. Ssn6 and Tup1 were two candidates as they have been shown to be sumoylated and implicated in the repression of both GAL1 and HXK1. Moreover, a former student in the laboratory, Patricia Vinciguerra, found these two proteins in a two-hybrid screen with the C-terminal domain of Mlp2 as bait, suggesting a direct or indirect physical interaction between Ssn6/Tup1 and Mlp2.
We then showed that delocalization of Ulp1 from the NPC affected the sumoylation levels of Ssn6. Moreover, Ssn6 mutants with decreased sumoylation showed an increase in GAL1 and HXK1 mRNA levels. The rescue of Ssn6 sumolyation was accompanied by restoration of wild-type GAL1 and HXK1 mRNA levels. Thus, this work highlights the role of NPC-bound Ulp1 in GAL1 and HXK1 derepression through its ability to modulate the level of Ssn6 sumoylation when genes relocate to the nuclear periphery. This work was recently published in Molecular Cell.
The second part of my work aimed at finding additional genes regulated by Ulp1. For this purpose, in collaboration with the laboratory of Frank Holstege, we performed microarray analyses of transcripts from Δmlp1/2, ΔNulp1-GFP and WT strains. We are currently analyzing the data and trying to validate some of the candidates, but it seems that Ulp1 may regulate preferentially inducible genes. This result would reinforce our model in which Ulp1 tethered at NPCs would participate in the derepression of inducible genes when they relocate to NPCs.
2. Introduction
2.1 Organization of the nucleus
2.1.1 General introduction on the nucleus and its organization
The nucleus was the first organelle to be discovered in eukaryotic cells and described in 1802 by Franz Bauer. It stores most of the deoxyribonucleic acid (DNA) of the cells and is delimited by the nuclear envelope (NE) formed by two membrane layers called inner (INM) and outer nuclear membranes (ONM). More than 60 years ago, electron microscopy studies revealed that these two membranes were continuous with the endoplasmic reticulum (ER) membrane (Watson, 1955) and (Figure 1).
However, although lipids freely diffuse between the NE and the ER, proteins found in the INM and ONM are different from those found in the ER membrane, as reviewed in (Hetzer, 2010; Hetzer et al., 2005).
The nucleolus, which is not delimited by a membrane, is a sub-compartment of the nucleus (Figure 1). Its major functions are transcription of ribosomal ribonucleic acids (rRNA) and ribosome biogenesis.
Figure 1: Structural organization of the yeast nucleus.
The spindle pole body is found at one side of the nucleus and the nucleolus on the opposite side. Centromeres (attached to the spindle pole body) and telomeres are located at the nuclear periphery. The nuclear envelope is composed of a two-layer membrane, which is continuous with the endoplasmic reticulum membrane. The nuclear pore complexes (NPCs) are inserted within the nuclear envelope (NE) and mediate nucleo-cytoplasmic exchanges.
In eukaryotes, DNA has to be compacted in order to fit inside the nucleus. For this purpose, the double stranded DNA is subjected to different levels of compaction (Hubner et al., 2013). The first condensation step is the wrapping of 147 base pairs (bp) of DNA around the nucleosome to form a 10nm diameter fiber. Nucleosomes are formed by histone octamers, composed of two H3-H4 dimers that are flanked on each side by one H2A-H2B dimer (Kurat et al., 2013). Variants exist for some of these histones (see below). Histone proteins can be grouped in two classes according to their expression during the cell cycle. The first class constitutes histones that are replication-dependent (RD), i.e. predominantly expressed during replication (S- phase). Up-regulation of histone expression during late G1 and S phase is required at each doubling to package the newly synthetized DNA. Interestingly, depletion of histone H4 during S phase leads to lethality (Kim et al., 1988). The RD class includes histones that form the nucleosome core composed of H2A, H2B, H3 and H4 as well as the histone linker Hho1p (homologous to H1 in mammals). The second class contains replication-independent (RI) histones, i.e. whose expression is low and constant throughout the cell cycle. Histones that constitute this class are histone variants of H2A and H3 called H2A.Z, H2A.X and H3.3.
The core histones can be modified post-translationally resulting in open or closed chromatin conformations, depending on the modification and the histone residue modified. Chromatin structure has been involved in many DNA-linked processes such as transcription, DNA recombination, DNA repair, replication, kinetochore and centromere formation, as reviewed in (Li et al., 2007). In this Introduction, I will focus on post-translational modifications that occur during transcription (see Section 2.2.3 “Role of Chromatin during transcription”).
In the classical textbook view, the 10nm diameter nucleosomal fiber forms superhelical turns generating the 30nm fiber, which is further compacted through formation of loops and hyperhelical turns of looped DNA to give rise to the condensed chromosome. However, physics combined with biological data challenged this view (Fudenberg and Mirny, 2012). Even if the model of how chromosomes are condensed is still unclear, it is well established that during interphase, mammalian chromosomes occupy distinct nuclear regions called chromosome territories, which have been extensively studied and reviewed (Hubner et al., 2013). In yeast, the concept of chromosome territories has also been proposed based on the observation
that intra-chromosomal interactions occur more frequently than inter-chromosomal interactions (Duan et al., 2010; Rodley et al., 2009). However, in contrast to mammals, the sub-nuclear localization of yeast chromosomes appears driven mainly by the localization of centromeres and telomeres at the nuclear periphery (Figure 1).
Indeed in yeast, centromeres are attached to the microtubule-organizing center, called the spindle pole body (SPB, persisting during interphase in yeast but not in mammalian cells), which localizes opposite to the nucleolus, and telomeres are tethered at the nuclear periphery and clustered in 4 to 6 distinct foci. This nuclear organization was demonstrated using fluorescence in situ hybridization (FISH) and immunofluorescence for telomeric and centromeric binding proteins (Gotta et al., 1996; Jin et al., 1998). Moreover, it has been shown in vivo that the relative positions of subtelomeric regions inside the nucleus are determined by chromosomal arm length and nuclear volume (Therizols et al., 2010), illustrated in (Figure 1). The consequence of this organization is that two telomeres that are at the same distance from their corresponding centromere have more chances to interact together than telomeres present on two chromosomes with different arm length. This result supports a previous study from the laboratory of Susan Gasser (Schober et al., 2008), where the authors developed a chromosome swap technique in which endogenous chromosome arms are swapped, inducing changes in chromosome arm length. They demonstrated that conversion of unequal arm length to equal arm length increased inter- chromosomal telomere-telomere interactions.
Taken together, all these results suggest that chromosome positioning inside the nucleus is not random and that inter-chromosomal interactions are governed by physical constraints such as chromosome length, centromere attachment to the SPB and nuclear crowding.
The communication between the nucleus and the cytoplasm requires nuclear pore complexes (NPCs), large aqueous channels inserted within the nuclear envelope (NE) (Figure 1). NPCs act as a “customs house” controlling import and export in and out of the nucleus. In the following part, I will focus on the structure of NPCs, their assembly into the NE and how they regulate import and export of different classes of molecules.
2.1.2 The Nuclear Pore Complex (NPC) 2.1.2.1 Structure of the NPC
The NPC is a large complex, of 60 to 125 MDa in mammals and 40 to 60 MDa in yeast. It contains around 30 different proteins called nucleoporins.
Nucleoporins are present in a multiple of eight copies in a single pore, as reviewed in (D'Angelo and Hetzer, 2008). There are about 200 NPCs per nucleus in yeast and as much as 2000 in mammalian cells. Even if the size and the number of NPCs differ between species, the overall architecture and function of the NPC are conserved from yeast to mammals. The NPC can be subdivided into several parts (Figure 2), each containing a specific class of nucleoporins.
The first class is composed of pore membrane proteins called Poms, which form the membrane ring of the NPC (Figure 2). Four Pom proteins have been described: Pom33, Pom34, Pom152 and Ndc1 and have been hypothesized to participate in NPC assembly and insertion into the nuclear envelope thanks to their trans-membrane alpha helices and to their interaction with core components (Aitchison and Rout, 2012; Chadrin et al., 2010; Flemming et al., 2009; Madrid et al., 2006; Makio et al., 2009; Onischenko et al., 2009).
The proteins of the second class are part of the NPC core scaffold formed by two inner and two outer rings (Figure 2). The inner rings are composed of the four nucleoporins (Nups) Nup188, Nup192 and the homologous proteins Nup170 and Nup157. The outer rings are composed of seven nucleoporins identified by the Hurt laboratory that are part of the Nup84 complex: Nup133, Nup120, Nup145C, Nup85, Nup84, Seh1 and Sec13 (Flemming et al., 2009; Lutzmann et al., 2002; Siniossoglou et al., 2000; Siniossoglou et al., 1996). The Hurt laboratory showed by electron microscopy that the Nup84 complex forms a Y-shaped structure. Interestingly, mutations in Nup84 complex components affect different nuclear processes such as mRNA and pre-ribosomal RNA export and also lead to NPC clustering in discrete foci in the nuclear envelope (Doye et al., 1994; Goldstein et al., 1996; Li et al., 1995).
Another class of nucleoporins representing more than a third of all Nups is the well-studied FG-Nups (Figure 2). Some of these FG-Nups will be used in this study:
Nsp1, Nup42, Nup49 and Nup60. As suggested by their name, they contain a repetitive motif that consists of multiple repeats of phenylalanine-glycine (FG) pairs,
spaced by around 20 polar amino acids. Interestingly, despite the fact that the FG repeats are conserved, the spacer sequences evolved more rapidly than other nucleoporins (Denning and Rexach, 2007). Moreover, the FG repeat-containing regions of FG-Nups have been proposed to form long and flexible unstructured filaments (Denning et al., 2003). Lack of structural constraint could explain the high evolution of this region. It has been proposed that FG-Nups also have small structured domains such as coiled-coil, α-helices, β-sandwich and even RNA recognition motifs (RRM) (Alber et al., 2007; Devos et al., 2006). The FG-repeats of a fraction of FG- nucleoporins are proposed to fill the central channel acting as a selective barrier controlling nucleo-cytoplasmic trafficking (Aitchison and Rout, 2012).
Additional nucleoporins constitute the cytoplasmic filaments extending from the NPC into the cytoplasm. These comprise Nup82 and the FG-Nups Nup159 and Nup42 (used in this study) proposed to function in the late stages of export by contributing to the release of cargos from the NPC as reviewed in (Aitchison and Rout, 2012).
On the other side of the NPC, several nucleoporins form the nuclear basket protruding into the nucleoplasm and consisting of the FG-Nups Nup1 and Nup60.
Nup60 participates in the association of the myosin-like proteins Mlp1 and Mlp2 with the NPC (Feuerbach et al., 2002). During my PhD work, I have aimed at understanding the role of these nuclear basket components in transcription regulation.
Intriguingly, experiments in higher eukaryotes have revealed that certain nucleoporins have a short residence time at the NPC and rapidly exchange between the nucleoplasm and the nuclear periphery (Griffis et al., 2002; Rabut et al., 2004;
Tran and Wente, 2006). Some nucleoporins, such as the yeast Nup2 were described to be preferentially soluble proteins (Aitchison and Rout, 2012; Dilworth et al., 2001).
Moreover, experiments performed in Drosophila and mammalian cells indicate that some nucleoporins may be involved in transcription regulation within the nucleoplasm (see Section “2.3.2.2 Role of nucleoporins in transcription regulation in higher eukaryotes”). These results complicate the understanding of the importance of the NPC in different processes since some nucleoporins may have additional roles away from the NPC.
Figure 2: Composition and structure of the nuclear pore complex (NPC).
Distribution of nucleoporins within yeast and vertebrate NPCs. The NPC is subdivided into several compartments: the transmembrane ring, composed of Pom proteins; the core scaffold formed by two inner rings sandwiched by two outer rings.
FG-Nups are localized in the central channel of the NPC and seem to be linked to the core scaffold through the linker Nups: Nup82 and Nic96. Additional FG-Nups are part of the cytoplasmic filaments and nuclear basket emanating from the pore.
Illustration taken from (Strambio-De-Castillia et al., 2010).
2.1.2.2 NPC assembly into the nuclear envelope
Metazoans and S. cerevisiae present different types of mitosis. While higher eukaryotes are characterized by an open mitosis that involves nuclear envelope breakdown, S. cerevisiae undergoes a closed mitosis, during which the nuclear envelope remains intact (Arnone et al., 2013). Consequently, the process of NPC assembly and insertion into the NE varies between these organisms. In metazoans two different mechanisms of NPC assembly into the NE occur depending on the cell-cycle stage (Doucet et al., 2010) and reviewed in (Doucet and Hetzer, 2010). NPC assembly at the end of mitosis has been extensively studied (Fernandez-Martinez and Rout, 2009) and reported to be different from the de novo assembly of NPCs during interphase, for which much less is known. At the end of open mitosis, the nuclear
envelope is reformed around segregated chromosomes. This mechanism is mediated by Nup107/160, which is recruited by ELYS/Mel28 that binds chromosomes (Fernandez-Martinez and Rout, 2009; Hetzer and Wente, 2009; Kutay and Hetzer, 2008; Onischenko and Weis, 2011). Interestingly, orthologs of ELYS/Mel28 have not been found in yeast, suggesting that this complex is only required for NPC reassembly following open mitosis (Liu et al., 2009). In S. cerevisiae, only the de novo NPC assembly into the NE has been reported since the NE remains intact during mitosis. One could speculate that insertion of NPCs into the NE in S. cerevisiae is comparable to the NPC assembly during interphase in metazoans and could be an ancient mechanism kept during evolution. Xenopus egg extracts have been instrumental in the study of NPC assembly into the NE. Based on electron microscopy observations, Goldberg and colleagues have shown that spacing between INM and ONM decreases at new putative de novo NPCs, suggesting that interaction of INM and ONM proteins could trigger formation of new NPCs (Goldberg et al., 1997). To identify proteins involved in the de novo NPC assembly, a genetic screen was performed in S. cerevisiae (Ryan et al., 2003). In this study, the authors found that the small GTPase Ran was involved in this mechanism. A few years later, the same laboratory reported that the karyopherin Kap95, which is responsible for the nuclear import of NLS-containing proteins (see below), was involved in NPC assembly (Ryan et al., 2007). Furthermore, the pore membrane proteins Pom33, Pom34, Pom152 and Ndc1 have been shown to be critical for NPC assembly (Chadrin et al., 2010; Flemming et al., 2009; Madrid et al., 2006; Makio et al., 2009;
Onischenko et al., 2009). Several membrane proteins involved in lipid homeostasis such as Brr6 and Apq12 have also been reported to be linked to NPC biogenesis (Hodge et al., 2010; Scarcelli et al., 2007). Moreover, mutations or depletion of specific ER proteins resulted in NPC assembly defects, suggesting a role for these proteins in the de novo NPC assembly (Dawson et al., 2009). In particular, Rtn1, a member of the reticulon family, and Yop1 involved in NPC biogenesis, have been reported to interact genetically with the Poms, several nucleoporins and the Nup84 complex (Dawson et al., 2009). Finally, several NPC subunits such as Nup133, Nup120, Nup85, Nup170 and Nup188 contain a membrane curvature-sensing motif (ALPS) that could be required to stabilize the highly curved pore membranes, as reviewed in (Doucet and Hetzer, 2010; Jaspersen and Ghosh, 2012).
Although several proteins have been implicated in this process, the exact and sequential events of de novo NPC assembly into the NE are still poorly understood and puzzling, and many questions remain unanswered.
2.1.2.3 The role of the NPC in import/export of cargo proteins
Many cellular components, including messenger RNAs (mRNAs) and pre- ribosomes, have to be exported from the nucleus into the cytoplasm. At the same time, many proteins have to be imported inside the nucleus. Imported proteins are involved in nuclear processes such as transcription, DNA replication, DNA repair and DNA packaging. Since nuclear pores contain a large aqueous channel, ions and small molecules can diffuse, while larger proteins or complexes need to be actively imported or exported through the NPCs. This active transport requires energy and an appropriate signal carried by the transported cargo protein: a nuclear localization sequence (NLS) for import and a nuclear export sequence (NES) for export. These nuclear transport signals are recognized by transport receptors called karyopherins (Figure 3). In yeast, there are 14 known karyopherins subdivided into importins and exportins (Aitchison and Rout, 2012). Karyopherins direct their cargoes through the NPC by sequential interaction with nucleoporin FG-repeat domains within the central channel of the NPC. An essential regulator of the interaction between karyopherins and their cargoes is the small GTPase Ran, which binds karyopherins in its GTP- bound form (Figure 3). While Ran-GTP binding to importins promotes the dissociation of cargoes, its binding to exportins has the opposite effect and stimulates their interaction with specific substrates. Importantly, the Ran GEF (Guanine nucleotide Exchange Factor) exchanging GDP for GTP on Ran is a strictly nuclear enzyme, while the Ran GAP (GTPase Activating Protein) is cytoplasmic (Figure 3).
The asymmetric distribution of these two essential Ran regulators establishes a steep RanGTP gradient between the nucleus and the cytoplasm. The high levels of RanGTP in the nucleus favour the dissociation of import complexes and stimulate the formation of export complexes in this compartment, while the presence of the RanGAP stimulates GTP hydrolysis by Ran on the cytoplasmic side ensuring release of export substrates at this location and recycling of Ran-GDP into the nucleus through interaction with Ntf2 (Figure 3). A number of experiments indicate that the
Ran-regulated terminal step of import and export provides the energy and is a major determinant of transport directionality while the sequential interaction of karyopherins with FG-repeat domains contributes to the facilitated diffusion of transport complexes within the central channel of the NPC (Aitchison and Rout, 2012). Interestingly, interaction between cargo-receptor complexes and FG-Nups seems to be a general mechanism for the transport of cellular components. Indeed, the mRNA export receptor Mex67 (see below) and the protein Ntf2 responsible for the import of Ran- GDP also interact with FG-Nups (Weis, 2003).
Figure 3: The small GTPase Ran regulates the interaction of karyopherins with Import/Export cargoes and confers transport directionality through the NPC.
For more details, see the main text. Illustration taken from (Aitchison and Rout, 2012).
2.1.2.1 Role of the NPC in mRNA export
Another role for the NPC is to export mRNA from the nucleus to the cytoplasm. This process is mediated through interactions between nucleoporins and export factors or adaptors required for mRNA export. In contrast to protein export, mRNA export through the NPC channel is Karyopherin- and Ran-independent but
requires the evolutionarily conserved and essential export receptor Mex67 (Segref et al., 1997). Mex67 temperature sensitive mutants exhibit rapid nuclear accumulation of polyA mRNA when shifted to 37°C indicating an essential role in mRNA export (Segref et al., 1997; Zenklusen et al., 2001). Interestingly, Mex67 shuttles between the nucleus and the cytoplasm and directly interacts with FG-nucleoporins, suggesting a direct role in mediating export of mRNA through the NPC channel (Segref et al., 1997; Strasser et al., 2000; Strawn et al., 2001).
Mex67 interacts poorly with RNA and several adaptor proteins, including Yra1, Nab2, Sub2 and Npl3, have been described to tether Mex67 to mRNAs. Yra1 (yeast RNA annealing proteins 1) interacts with mRNA and Mex67 in vivo and yra1 mutants accumulate poly(A) RNA in the nucleus (Strasser and Hurt, 2000; Zenklusen et al., 2001). Yra1 is a non-shuttling nuclear protein implying that Yra1 has to dissociate from the mRNA before export through the NPC. Our laboratory proposed that ubiquitination of Yra1 promotes its dissociation from the mRNA (Iglesias et al., 2010). The same study indicated that Yra1 facilitates the interaction of Mex67 with the adaptor RNA binding protein Nab2. Interestingly, Nab2 was also described to associate with the nuclear basket through interaction with Mlp1 (Green et al., 2003;
Vinciguerra et al., 2005). The adaptor protein Npl3 also functions in mRNA export through direct interaction with Mex67 helping its loading on mRNA (Gilbert and Guthrie, 2004). As for Yra1, mutations in Npl3 lead to nuclear retention of poly(A) RNA (Lee et al., 1996). In addition, Npl3 plays a role independent of Mex67 in the export of the large 25S ribosomal subunit by interacting with the NPC component Nup60 (Hackmann et al., 2011). To date, Npl3 is the most extensively studied shuttling mRNA export factor/adaptor (Tutucci and Stutz, 2011).
The TREX2 complex, composed of Sac3, Thp1, Sus1 and Cdc31 is part of the nuclear pore-associated mRNA export machinery (Garcia-Oliver et al., 2012; Kohler and Hurt, 2007; Rodriguez-Navarro and Hurt, 2011). Deletion of Nup1 leads to the delocalization of Sac3, suggesting a role for this nucleoporin in tethering TREX2 at the NPC. In addition, Sac3 physically interacts with Mex67 and loss of Sac3 results in an mRNA export defect (Fischer et al., 2002). Together these observations suggest a role for TREX2 in linking mRNA biogenesis and export through nuclear pores (for more details see below, Section “2.2.2.1.2 Recruitment of Export Factors during transcription”).
Another NPC-associated factor required for mRNA export is the shuttling DEAD box RNA helicase Dbp5 (also called Rat8), which preferentially associates with the cytoplasmic face of the NPC through interaction with Nup159 (Folkmann et al., 2011). Dbp5 is required for a late step of mRNA export by promoting the dissociation of Mex67 and Nab2 from mRNAs, allowing the release of transcripts for translation and the recycling of export factors back into the nucleus (Lund and Guthrie, 2005; Montpetit et al., 2011; Tran et al., 2007; Weirich et al., 2006)
Altogether these studies show that the NPC is not only a hole in the nuclear membrane, but that nucleoporins play key roles in mRNA export, both during docking of mRNP complexes to the nuclear face of the NPC as well as in regulating the interaction of mRNA export factors/adaptors with mRNAs (Figure 4).
Figure 4: Factors involved in mRNA export through the NPC.
Illustration taken from (Strambio-De-Castillia et al., 2010). See main text for more information.
2.1.2.2 Role of the NPC in quality control and genome stability
More than a channel, the NPC appears as a sub-compartment that may concentrate factors involved in multiple functions serving as a platform for processes as diverse as transcription, mRNA surveillance, DNA repair of double strand breaks (DSB) and maintenance of telomere length, as discussed in (Aitchison and Rout, 2012) and (Figure 5).
Some NPC proteins have been implicated in mRNA surveillance and quality control. It has been shown that loss of Mlp1, Mlp2 or the Mlp-bound protein Pml39 leads to the leakage of intron-containing transcripts into the cytoplasm (Galy et al., 2004; Palancade et al., 2005; Vinciguerra et al., 2005). Interestingly, deletion of MLP1 and MLP2 has also been shown to alleviate the mRNA biogenesis and export defect of a yra1 mutant strain (yra1-8) (Vinciguerra et al., 2005), suggesting that Mlp proteins contribute to the retention of malformed mRNPs.
NPC components have also been involved in telomere tethering at the nuclear envelope. Indeed, loss of Nup84 subunits reduces the association of telomeres with the nuclear periphery (Therizols et al., 2006). Mlp1 and Mlp2 were also suggested to participate in telomere anchoring (Galy et al., 2000), however a subsequent study did not confirm this observation and indicated that loss of Mlp1 and Mlp2 primarily results in increased telomere length (Hediger et al., 2002). However, the exact molecular basis of this phenotype is still unknown.
Loss of Nup84 complex components also decreases the efficiency of DSB repair at subtelomeric regions indicating a role for the NPC in genome stability (Therizols et al., 2006). Moreover, DSB DNA repair is impaired in Δmlp2 (Galy et al., 2000) and DNA damage is increased in strains lacking pore components such as Nup60, Mlp1/2 or the Nup84 complex (Palancade et al., 2007). Interestingly, a recent study showed that damaged DNA is recruited to NPCs in a Nup84-dependent manner further suggesting a role for this compartment in DNA repair (Nagai et al., 2008).
Finally, Δmlp1/2 and Δnup60 strains show irregularly shaped colonies due to clonal lethality. This phenotype is linked to an increase in the number of 2µ circles.
These DNA circles are naturally present in budding yeast at 50-100 copies per cell, but affect viability when present in higher amounts (Zhao et al., 2004a). Thus, another
function of the yeast NPC may be to protect cells from lethality by regulating the number of 2µ circles.
An additional role of the NPC in transcription regulation will be addressed later in this Introduction (Section 2.3).
Figure 5: The NPC as a coordination platform for various nuclear processes.
Numerous factors and complexes involved in mRNA biogenesis and export, mRNA surveillance and remodeling, as well as DNA repair and sumoylation are associated with nuclear pore complexes. From (Aitchison and Rout, 2012).
2.2 Transcription
2.2.1 General introduction on gene expression
Eukaryotic gene expression is a multistep process, which involves transcription of the DNA template into mRNA molecules, export of these mRNAs into the cytoplasm and translation into proteins. In eukaryotes, transcription is carried
out by 3 different RNA polymerases: RNA Pol I, Pol II, and Pol III that produce different classes of transcripts (Vannini and Cramer, 2012).
RNA Pol I transcribes most of the ribosomal RNAs, which represent the major part of total RNA (over 50% of cellular transcripts). Ribosomal RNAs are transcribed in the nucleolus.
RNA Pol III transcribes the 5S rRNA, transfer RNA (tRNA) and other small RNAs. RNA Pol III recognizes two promoter elements located within the genes.
Generally, Pol III transcribed genes are described as “Housekeeping genes” since their transcription is required throughout the cell cycle and in most environmental conditions.
RNA Pol II transcribes all messenger RNAs i.e protein-coding genes, as well as a number of non-coding snRNAs and snoRNAs. The aim of my PhD was to understand the role of the NPC in the transcription regulation of inducible RNA Pol II-dependent genes in yeast, introduced in the next section.
2.2.2 Transcription of RNA Pol II-dependent genes
The promoters of RNA Pol II-dependent genes contain essentially a combination of three DNA sequence elements in yeast: the upstream activating sequence (UAS), the TATA box and the Initiation Element, as reviewed in (Struhl, 1989).
The UAS is a short, 10 to 30 bp sequence located 100 to 1500 bp upstream of the transcription start site (TSS). This sequence usually gives the specificity of a given promoter or set of promoters for the same activator. This is the case for the GAL promoters that share a similar UAS bound by the activator Gal4. The UAS is the major regulatory element for genes that are induced under specific conditions such as our gene of interest GAL1 (see below). Another element in the promoter of some genes is the TATA box. TATA boxes are localized 40 to 120 bp upstream of the TSS and bound by the TATA Binding Protein (TBP), which is important for the positioning of RNA Pol II at the TSS. Only 10% of yeast genes contain a TATA box and mostly correspond to highly regulated SAGA dependent genes. The 90% TATA- less genes mainly consist of constitutively expressed genes. While SAGA promotes
TBP binding at TATA box genes, TFIID is required for TBP binding to TATA-less genes (Huisinga and Pugh, 2004; Venters and Pugh, 2009).
RNA Pol II is composed of 12 subunits. RNA synthesis is catalyzed by the largest subunit Rpb1 and requires also several co-factors, including TFIIA, TFIIB, TFIID, TFIIE, TFIIH and TFIIF. All these co-factors, RNA Pol II and the Mediator form the pre-initiation complex (PIC) (Liu et al., 2013).
2.2.2.1 Co-transcriptional mRNP formation via CTD phosphorylation 2.2.2.1.1 Transcription and mRNP formation
During transcription, precursor mRNAs (pre-mRNAs) undergo several sequential maturation steps, such as 5’ capping, splicing (in some cases) and 3’ end formation, consisting of cleavage and polyadenylation. This co-transcriptional maturation involves the loading of proteins onto nascent transcripts leading to the production of processed and export competent messenger ribonucleoprotein complexes (mRNPs). Importantly, co-transcriptional recruitment of processing and export factors is regulated by dynamic phosphorylation of the C-terminal domain (CTD) of the largest RNA Pol II subunit Rbp1 along the transcription cycle (Buratowski, 2009; Perales and Bentley, 2009).
The CTD is formed of repeats of the heptamer sequence Tyr1-Ser2-Pro3- Thr4-Ser5-Pro6-Ser7. The three major sites of phosphorylation on the heptad repeats are Serine 2, Serine 5 and Serine 7. In S. cerevisiae, genome-wide chromatin immunoprecipitation (ChIP) analyses of the different phosphorylated forms of CTD revealed that Ser5P and Ser7P are enriched at the 5’ end of genes and mostly within 150 nucleotides downstream of the TSS, whereas Ser2P is found at the 3’ end of genes with a peak at 600-1000 nucleotides downstream of the TSS (Mayer et al., 2010). These modifications regulate the ability of the CTD to interact with proteins involved in the initiation of transcription, RNA processing and chromatin modifications (Buratowski, 2009).
During the early events of transcription, the pre-initiation complex (PIC) is assembled and the Mediator binds the non-phosphorylated CTD at the promoter. This association stimulates the CTD kinase activity of TFIIH and CTD is phosphorylated
at Serine 5. Ser5P disrupts the binding between CTD and Mediator and thus releases RNA Pol II (Sogaard and Svejstrup, 2007). Interestingly, the enzyme required to cap the mRNA interacts directly with the Ser5 phosphorylated CTD of RNA Pol II and allows co-transcriptional capping of mRNA (Fabrega et al., 2003). Capping of pre- mRNA consists in the attachment of a 7-methylguanosine (m7G) to the 5’ end of pre- mRNA. For this purpose, three enzymes are recruited through Ser5P CTD. These three enzymes are the triphosphatase Cet1, the guanylyltransferase Ceg1 and the 7- methyltransferase Abd1 (Bentley, 2005). The (m7G) cap is then rapidly bound by the cap-binding complex composed of Cbp20 and Cbp80, which is replaced by the translation initiation factor eIF4E once the mRNP reaches the cytoplasm (Tutucci and Stutz, 2011).
During elongation, a transition from Ser5 phosphorylation to Serine2 phosphorylation of RNA Pol II CTD occurs. However, the mechanism responsible for this transition is still not well defined. As reviewed by Buratowski (Buratowski, 2009), the phosphatase Rtr1 (and perhaps also Rtr2) could decrease Ser5P at an early stage of elongation, while another phosphatase, Ssu72, may remove the remaining Ser5 phosphorylation during transcription termination. In contrast, phosphorylation of Ser2 is well characterized and involves, in budding yeast, the two kinases Bur1 and Ctk1. Interestingly, Bur1 has been reported to interact directly with Ser5P (Qiu et al., 2009), suggesting a recruitment of this kinase on CTD through Ser5P and highlighting the sequential phosphorylation events that occur during elongation.
Termination of transcription of most coding genes is coupled to mRNA 3’ end cleavage and polyadenylation (Kuehner et al., 2011). Interestingly, several proteins required for these two linked maturation steps have been shown to interact with Ser2 phosphorylated CTD. For instance Pcf11, described to play a role in RNA cleavage and polyadenylation, has been shown to preferentially interact with Ser2P (Meinhart and Cramer, 2004). Following cleavage of the nascent mRNA at the poly(A) site, the 3’ fragment is degraded by the 5’ to 3’ exonuclease Rat1, resulting in transcription termination (Brannan et al., 2012; Luo and Bentley, 2004; Mischo and Proudfoot, 2013). Rat1 is recruited to RNA Pol II through Rtt103, which is known to bind the Ser2P of the CTD (Kim et al., 2004). These examples illustrate that maturation of nascent transcripts occurs co-transcriptionally and involves the sequential recruitment of factors in a process regulated by RNA Pol II CTD phosphorylation.
2.2.2.1.2 Recruitment of Export Factors during transcription
Many export factors are recruited during transcription. During elongation, RNA Pol II interacts with the THO complex composed of Hpr1, Mft1, Thp2 and Tho2. Deletion of any of those components has been shown to affect transcription elongation and mRNA export (Gomez-Gonzalez et al., 2011; Jimeno and Aguilera, 2010; Luna et al., 2012). Moreover, the THO component Hpr1 has been implicated in the recruitment of the export adaptors Yra1 and Sub2 (Zenklusen et al., 2002) as well as of the export receptor Mex67 through a direct RNA-independent interaction (Dieppois et al., 2006). Association of the THO complex to the export adaptors Yra1 and Sub2 forms the TREX complex, proposed to couple transcription and mRNA export, as reviewed in (Rodriguez-Navarro and Hurt, 2011; Tutucci and Stutz, 2011) (Figure 6). Yra1 is also recruited to transcribing genes through the RNA Pol II associated 3’ processing factor Pcf11 (Johnson et al., 2009). The export adaptor Npl3 similarly associates with transcribing RNA Pol II, promoting transcription elongation, splicing and preventing termination (Bucheli and Buratowski, 2005; Dermody et al., 2008).
Figure 6: Assembly of export competent mRNPs is tightly coupled to transcription.
The mRNA export machinery (THO/TREX) is recruited during early and late events of transcription via the RNA Pol II CTD. Illustration taken from (Tutucci and Stutz, 2011).
2.2.3 Role of Chromatin during Transcription
Chromatin structure and organization are highly dynamic and tightly regulated through a variety of mechanisms including histone modifications, histone variant incorporation, chromatin remodeling and histone eviction, as reviewed in (Li et al., 2007). Here, I will focus on histone modifications.
Histones can be post-translationally modified by methylation on their arginine residues and by phosphorylation on their serines and threonines. Moreover, the lysines can be subjected to numerous modifications such as the well-studied methylation and acetylation but also ubiquitination and sumoylation. These modifications are tightly linked to transcription regulation (Figure 7). Acetylation of H3 and H4 as well as methylation of H3K4 are associated with active transcription and thus linked to euchromatin, whereas methylated H3K9 and H3K27 are found in repressed genes and condensed chromatin (heterochromatin) (Figure 7). Interestingly, some modifications are enriched at particular positions on the gene. For example, H3K4 methylation seems to follow a defined pattern throughout the open reading frame (Figure 7), with H3K4 trimethlyation being enriched at the 5’ of the gene while H3K4 monomethylation is more prominent at the 3’ end. Moreover, sumoylation of H2A, H2B and H4 have been reported to be involved directly in repression of transcription (Nathan et al., 2006) (Figure 7). Indeed, the authors observed a very strong correlation between loss of H2B sumoylation (due to multiple KA mutations) and derepression of inducible genes such as GAL1.
All these data, suggest that histone modifications can be transcription dependent and conversely can help or affect transcription.
Figure 7: Correlation between transcription and distribution of histone modifications.
The distribution of histone modifications over transcription units was determined by genome-wide analyses (curves) or at specific genes (squares). Histone marks are correlated with transcription activation/repression on the right. The data are based on yeast genes except for H3K9me and H3K27me (which are not present in yeast).
Illustration taken from (Li et al., 2007).
2.2.4 GAL1 gene transcription regulation
Our model gene for this study is the GAL1 gene. This gene encodes the galactokinase protein required for the first step of galactose catabolism. GAL1 is part of the GAL gene family required for growth with galactose as carbon source. This family encodes proteins divided into two groups: those involved in the uptake and catabolism of galactose (Gal1, Gal10, Gal7 and Gal2) and whose expression is tightly controlled by the carbon source, and those involved in the transcriptional regulation of the first group (Gal80, Gal3 and Gal4). GAL1 gene transcription is induced by galactose but the molecular events leading to GAL gene transcription differ depending on the growth conditions before induction. When cells are cultured in the presence of glucose, the GAL1 gene is fully repressed, while it is in a pre-induced state when cells are grown in raffinose. Thus, switching cells from raffinose to galactose results in
rapid gene activation. In contrast, when shifting cells from glucose to galactose, a first step of derepression is necessary before activation. As discussed previously, UAS elements are important regulators of gene expression and are also found at the GAL1 promoter. The Gal4 activator is known to bind to the GAL UAS elements as a dimer (Johnston, 1987; Lohr et al., 1995). The GAL UAS sequence is composed of two CGG repeats spaced by 11 nucleotides (Liang et al., 1996).
In the repressed state (i.e. in medium containing glucose), Gal4 is bound to GALUAS but its activity is repressed through interaction with the inhibitory protein Gal80 reviewed recently in (Weake and Workman, 2010). It has been shown in vitro that binding of Gal80 to Gal4 impairs its interaction with TBP (Wu et al., 1996) and the SAGA co-activator complex (Carrozza et al., 2002) (Figure 8). When GAL1 is activated, i.e. in the presence of galactose, the cytoplasmic protein Gal3 relieves Gal80 inhibition on Gal4. However the exact mechanism by which Gal3 interacts and acts on Gal80 is not yet known (Traven et al., 2006). Gal4 activates transcription by interacting with several co-activators. Many studies have shown that Gal4 interacts with; 1) components of the PIC such as TBP and TFIIB (Melcher and Johnston, 1995;
Wu et al., 1996), 2) components of the SAGA complex such as Tra1 and Spt20, an essential component for the integrity of SAGA (Bhaumik and Green, 2001; Bhaumik et al., 2004; Larschan and Winston, 2001), 3) the nucleosome remodeling complex SWI/SNF (Yudkovsky et al., 1999) and 4) Mediator components such as Gal11 and Srb4 (Jeong et al., 2001; Koh et al., 1998) (Figure 8). Moreover, ChIP analyses proposed that SAGA is the first complex that binds to the GAL gene promoters but its role in the recruitment of other co-activators such as the Mediator is still a matter of debate and discussed in (Traven et al., 2006). However, it has been shown that recruitment of TBP to Gal4-dependent genes requires some components of the SAGA and Mediator complexes (Bhaumik and Green, 2001, 2002; Larschan and Winston, 2001, 2005), suggesting that TBP binding is SAGA- and Mediator-dependent.
Furthermore, ChIP analyses suggest that the Mediator and Pol II but not the SAGA complex are required to recruit efficiently SWI/SNF to the GAL1 gene promoter (Lemieux and Gaudreau, 2004). However, this analysis was performed in cells induced to express GAL1 by switching cultures from raffinose to galactose. We could speculate that SAGA, and particularly the histone acetyl transferase Gcn5 subunit, is required for the recruitment of the SWI/SNF complex only when chromatin is
condensed, i.e. when starting from the repressed state. Several studies support this model. Indeed, the SWI/SNF complex and Gcn5 are essential for GAL1 expression in Δhtz1 (coding for the histone variant H2A.Z), in which chromatin is less permissive to transcription (Santisteban et al., 2000). Furthermore, SWI/SNF and Gcn5 are required to fully induce GAL1 during mitosis when the chromatin is condensed (Krebs et al., 2000). Finally, the induction of another inducible gene, PHO8, is based on sequential events implicating first Gcn5 and then SWI/SNF (Gregory et al., 1999). Taken together, all these studies suggest that the SAGA complex may recruit SWI/SNF only when GAL1 is activated from a fully repressive state.
Figure 8: Model of GAL1 gene transcription regulation by the Gal4-Gal80 pathway.
Schematic representation of proteins previously shown to interact with Gal4. Protein complexes are labeled in white and complex components or proteins are labeled in black.
Gal4-Gal80 are not the only regulators of GAL genes. It has been shown that the general co-repressors Ssn6 (also called Cyc8) and Tup1 are also important players in the regulation of GAL gene expression. The Ssn6-Tup1 co-repressor, which is
