Role of co-activators and promoter architecture in transcription of yeast protein-coding genes

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Thesis

Reference

Role of co-activators and promoter architecture in transcription of yeast protein-coding genes

BRUZZONE, Maria

Abstract

Transcription initiation is a complex, energy consuming process that requires the massive and coordinated activity of many proteins often organized in big multi-subunit complexes. The General Transcription Factors (GTFs) and the TATA-binding protein (TBP) orchestrate the formation of the so-called pre-initiation complex (PIC), a key step of transcription initiation required for RNA Polymerase II (RNAPII) association to promoters. Although the PIC is made by the GTFs, many other complexes generally referred to as transcriptional co-activators participate in the formation of the PIC. Transcriptional co-activator complexes include chromatin remodelers, Histone AcetylTransferases (HATs) and the Mediator complex. In yeast, it is has been reported that two main HAT complexes, NuA4 and SAGA, and the Mediator complex have a global role in transcription. However, though, it is well-known that histone acetylation is associated to high transcription and that Mediator plays a central function in PIC assembly, the deployment of NuA4, SAGA and Mediator at yeast genes is still elusive. Here, we investigate the contribution of NuA4, SAGA [...]

BRUZZONE, Maria. Role of co-activators and promoter architecture in transcription of yeast protein-coding genes. Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5230

DOI : 10.13097/archive-ouverte/unige:107041 URN : urn:nbn:ch:unige-1070414

Available at:

http://archive-ouverte.unige.ch/unige:107041

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

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UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de biologie moléculaire Professeur David Shore

Role of co-activators and promoter architecture in transcription of yeast protein-coding genes

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Maria Jessica Bruzzone

de

Albissola Marina (Italié)

Thèse n° 5230 Atelier ReproMail

2018

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Acknowledgements

This thesis is the fruit of a long PhD work that I did in the David Shore laboratory. In these 6 years, I had the opportunity to learn much more of what I could ever imagine and for this I am extremely grateful to all the people who have contributed to my development, as a researcher and as a person.

I am truly grateful to my supervisor David Shore who has allowed me to work independently on different challenging, exciting projects. Thanks to David for his wise advices and for made me feel trusted every day.

I am grateful to Profs. Françoise Stutz and Thomas Schalch for agreeing to be part of my thesis advisory committee. A special thanks to Thomas for his support in many occasions during my PhD.

Thanks to Profs. Françoise Stutz and Laszlo Tora for accepting to be part of my thesis committee and for reading this thesis. Thanks to the IG3 foundation for founding part of my PhD.

Thanks to our collaborators Sebastian Grunberg and Gabriel Zentner for sharing with us their ideas and results. Thanks to Steve Hahn for his advice. A super thanks to Joachim Grisenbeck for his great support and his contagious enthusiasm.

I am extremely grateful to all the past and present members of the Shore lab for their advices, criticisms and for sharing many unforgettable moments. My special thanks is for the people who shared with me a long journey in the Shore lab. Thanks to Britta and Slawek who have introduced me in the lab. A special thanks to Slawek, for his constant help and support, for his many advices, for the funny and serious chats and for listening every morning the detailed stories of my night dreams.

Thanks to Maksym for his kindness and for sharing with me his knowledge in many different fields.

Thanks to Ben, for his honesty, his contagious craziness, for the many scientific discussions and for sharing with me the “never say no” disease. It is wonderful and inspiring to work with such extraordinary people and for this I am extremely grateful.

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Thanks to Christiane for her friendship and for teaching me everything I know about protein purification and crystallization. Thanks to Rocio for sharing with me a lot of fun and the best Tequila in the world. Horizontal thanks to all the members of the “Texeira lab and friends” group and to the fantastic “GBC”, especially to Lukas, Lyudmill and Bernard. Thanks to Tata Giselle, she knows why.

Thanks to Diego for his friendship and sweet heart and for sharing with me the wonderful “pastiere”

prepared by his mum. Thanks to Stefano, for being a terrible colleague but, unexpectedly, a good friend and for all the discussions on Carver and De Andrè that gave sense to many meaningless days.

Thanks to Chiara to be always so close despite the kilometers that divide us. Thanks to Manuele to be a special friend every day. Thanks to my sister and my parents for their love and support. Thanks to Margot for everything. Thanks to Tiziana for the love and the happiness she brings in my life every day.

A special thanks to Paul, my wonderful cat.

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Summary

Transcription initiation is a complex, energy consuming process that requires the massive and coordinated activity of many proteins often organized in big multi-subunit complexes. The General Transcription Factors (GTFs) and the TATA-binding protein (TBP) orchestrate the formation of the so- called pre-initiation complex (PIC), a key step of transcription initiation required for RNA Polymerase II (RNAPII) association to promoters. Although the PIC is made by the GTFs, many other complexes generally referred to as transcriptional co-activators participate in the formation of the PIC.

Transcriptional co-activator complexes include chromatin remodelers, Histone AcetylTransferases (HATs) and the Mediator complex. In yeast, it is has been reported that two main HAT complexes, NuA4 and SAGA, and the Mediator complex have a global role in transcription. However, though, it is well-known that histone acetylation is associated to high transcription and that Mediator plays a central function in PIC assembly, the deployment of NuA4, SAGA and Mediator at yeast genes is still elusive.

Here, we investigate the contribution of NuA4, SAGA and Mediator in RNAPII association at all yeast genes through rapid nuclear depletion of key complex subunits. We reveal that Gcn5, the HAT of the SAGA complex, is modestly but equally involved in transcription of all yeast genes, while Esa1, the HAT of the NuA4 complex, is most strongly required for transcription of certain groups of genes.

Curiously, we show that nuclear depletion of Med17, an essential subunit of the Mediator complex, strongly affects transcription of a small subset of genes characterized by a well-conserved TATA box, highlighting a previously underappreciated connection between Mediator complex and TBP. Our analysis reveals that three combinations of co-activator deployment are used to generate high transcription levels and that transcription of two groups of so called “house-keeping” or “growth- promoting genes” is particularly dependent on Tra1, a shared component of NuA4 and SAGA. Our work on Ribosomal Protein (RP) gene and Ribosome Biogenesis (RiBi) gene transcription discloses the importance of the transcription factors Ifh1 and Sfp1 in recruiting Tra1 at these promoters.

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Furthermore, a deep investigation of transcription factors binding at RP gene promoters highlights the existence of a hierarchy, with Rap1 being required for the association of the other TFs. Our study also reveals that RP gene promoters, as well as the promoters of many other highly transcribed genes, are characterized by the presence of one or more unusually unstable MNase-sensitive nucleosomes referred to as fragile nucleosomes. Furthermore, our work discovers the main role of Rap1 and of other pioneer transcription factors in establishing nucleosome fragility and promoter architecture. Finally, we investigate the biophysical properties of fragile nucleosomes and provide evidence of their nucleosomal nature.

Altogether the work presented in this thesis elucidates important features of yeast promoters and provides new insights into the mechanisms that drive eukaryotic transcription.

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Résumé

L'initiation de la transcription est un processus complexe et énergivore qui nécessite l'activité massive et coordonnée de nombreuses proteines, souvent organisées en larges complexes. Les Facteurs Généraux de Transcription (FGT) et la “TATA binding protein” (TBP) orchestrent la formation du complexe de Pré-Initiation (PIC), étape clé de l'initiation de la transcription nécessaire à l'association de l’ARN Polymerase II (RNAPII) aux promoteurs. Bien que le PIC soit constitué par les FGT, de nombreux autres complexes, généralement appelés co-activateurs transcriptionnels, participent à la formation du PIC. Les complexes de co-activateurs transcriptionnels comprennent les remodeleurs de la chromatine, les Histone AcetylTransférases (HATs) et le complexe Mediator. Chez la levure, il a été rapporté que les deux principaux complexes HATs, NuA4 et SAGA, et le complexe Mediator ont un rôle global dans la transcription. Il est bien connu que l'acétylation des histones est associée à une transcription élevée et que Mediator joue un rôle central dans l'assemblage PIC, mais l’implication de NuA4, SAGA et Mediator dans la transcription des gènes de levure reste encore évasive.

Ici, nous étudions la contribution de NuA4, SAGA et Mediator dans l'association RNAPII à tous les gènes de levure après la déplétion nucléaire rapide des sous-unités clés de complexes. Nous révélons que Gcn5, le HAT du complexe SAGA, est impliqué dans la transcription de tous les gènes de levure, tandis que Esa1, le HAT du complexe NuA4, est le plus fortement requis pour la transcription de certains groupes de gènes. Curieusement, nous montrions que la déplétion nucléaire de Med17, sous-unité essentielle du complexe Mediator, affecte fortement la transcription d'un petit sous- ensemble de gènes caractérisés par une « TATA box» bien conservée, mettant en évidence une connexion précédemment sous-estimée entre le complexe Mediator et TBP. Notre analyse révèle que trois combinaisons de déploiement de co-activateurs sont utilisées pour générer des niveaux de transcription élevés et que la transcription de deux groupes de gènes dits " house-keeping " ou "de

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croissance" est particulièrement dépendante de Tra1, une composante commune de NuA4 et de SAGA. Nos travaux sur le gène de la protéine ribosomique (RP) et la transcription du gène de la biogénèse du ribosome (RiBi) révèle l'importance des facteurs de transcription Ifh1 et Sfp1 dans le recrutement de Tra1 chez ces promoteurs. En outre, une étude approfondie des facteurs de transcription se liant aux promoteurs de gènes de la RP mis en évidence l'existence d'une hiérarchie d’assemblage, Rap1 étant nécessaire pour l'association des autres TF. Notre étude révèle également que les promoteurs de gènes RP, ainsi que les promoteurs de nombreux autres gènes hautement transcrits sont caractérisés par la présence d'un ou plusieurs nucléosomes MNase-sensibles anormalement instables, appelés nucléosomes fragiles. De plus, notre travail a permis de découvrir le rôle principal de Rap1 et d'autres facteurs de transcription pionniers dans l'établissement de la fragilité des nucléosomes et de l'architecture des promoteurs. Enfin, nous étudions les propriétés biophysiques des nucléosomes fragiles et fournissons des preuves de leur nature nucléosomique.

Dans l'ensemble, le travail présenté dans cette thèse élucide les caractéristiques importantes des promoteurs de levure et fournit de nouvelles connaissances sur les mécanismes qui permettant la transcription eucaryote.

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Introduction

Transcription, the key of the central dogma of biology

The central dogma of biology states that the genetic information encoded in a piece of DNA defined as a gene is read in a molecule of RNA (transcription) and then translated into a functional protein (translation) (Crick, 1958) (Figure 1). Though nowadays we know that not all the RNA molecules are designated to be translated into proteins and that RNA molecules can be retro-transcribed into DNA, the key point of the dogma is still transcription. Transcription is the process by which a gene is copied into a molecule of RNA by enzymes called RNA polymerases that use the DNA as template to assemble a new molecule of RNA. In the seventies, early chromatographic studies led to the discovery of three eukaryotic RNA polymerases dedicated to the transcription of different classes of RNAs (Roeder and Rutter, 1969). RNA Polymerase I (RNAPI) transcribes the 25S ribosomal RNA (rRNA), RNA Polymerase II (RNAPII) transcribes all messenger RNAs (mRNAs) and some small non- coding RNAs (ncRNAs) and RNA Polymerase III (RNAPIII) transcribes transfer RNAs (tRNAs), the 5S rRNA and other small ncRNAs. Though they transcribe different classes of genes, the mechanisms by which the three polymerases work have strong similarities. First, all the three polymerases require the work of additional proteins to initiate transcription. Some of these proteins, generally called General Transcription Factors (GTFs), are used by all the three polymerases (for example the TATA Binding Protein, TBP see below) while some others are polymerase-specific. Second, though the three polymerases are composed by a different number of subunits (14 for RNAPI, 12 for RNAPII and 17 for RNAPIII), some of these are co-shared between two or all three polymerases (reviewed in (Thomas and Chiang, 2006)). Importantly, the shared subunits (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) reside all in the polymerase core of the three enzymes, suggesting similar mechanisms of transcription. Furthermore, recent structural studies (reviewed in (Vannini and Cramer, 2012)) revealed that the polymerase core of the three enzymes is structurally and functionally conserved.

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Indeed, the three RNAPs are characterized by a ten-subunit catalytic core and by a polymerase stalk.

In the course of this thesis, we will focus on RNAPII transcription and thus we will now describe RNAPII structure.

Figure 1. The central dogma of biology.

RNAPII transcription

RNAPII composition and structure

Similarly to the other RNAPs, RNAPII catalytic core is composed of 10 subunits, the RNAPII-specific subunits Rpb1, Rpb2, Rpb3, Rpb9, Rpb11 and the common subunits Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12. All the subunits are essential for cell viability excluding Rpb9 that together with the C-ribbon of the GTF TFIIS is involved in RNA cleavage. The active catalytic center of the enzyme resides in the two subunits Rpb1 and Rpb2. Importantly, the C-terminal domain (CTD) of Rpb1, though does not have any catalytic activity, is crucial in different phases of the transcription process (see below). Two RNAPII-specific subunits, the non-essential protein Rpb4 and Rpb7, constitute the dissociable stalk of the polymerase and, though not required for transcription in vitro, are needed for in vivo transcription. Crystal structure of both the whole RNAPII (Figure 2) and of the RNAPII core revealed that RNAPII is organized in four structural modules, the core, the clamp, the shelf and jaw lobe (Armache et al., 2003; Bernecky et al., 2016; Bushnell and Kornberg, 2003; Cramer et al., 2001).

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Importantly, these four modules are extremely mobile and conformation of the RNAPII changes during the different phases of the transcription cycle.

Figure 2. Mammalian RNAPII structure as reported in (Bernecky et al., 2016).

The three phases of RNAPII transcription

Transcription is a complicated and energy-consuming process that can be divided in three main phases, initiation, elongation and termination. During initiation RNAPII together with the GTFs binds the promoter of a gene, recognizes the Transcription Start Site (TSS) and starts to transcribe few nucleotides. If RNAPII transcribes at least nine nucleotides, it moves downstream from TSS with a mechanism named “promoter escape” or “promoter clearance” and elongation takes place. On the other hand, stop of RNAPII before having transcribed at least nine nucleotides leads to RNAPII slippage, backtracking, or arrest and finally to abortive transcription. Once RNAPII escapes from the promoter, it moves along the gene body together with many elongation factors transcribing the whole gene (elongation) (Cheung and Cramer, 2012). However, important studies showed that in metazoans RNAPII tends to pause between 20 and 100 nucleotides downstream of TSS (Core and Lis, 2008; Core et al., 2008; Lis, 1998). Though the functional importance of RNAPII promoter proximal

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pausing is still matter of investigation, it is now well demonstrated the existence of promoter- proximal pausing factors (such as NELF and DSIF) as well as factors involved in RNAPII release (such as PAF1) (Adelman and Lis, 2012). In yeast, RNAPII promoter proximal pausing has never been observed, however, some works reported the presence of poised RNAPII on promoters in stationary phase cells (Radonjic et al., 2005). When RNAPII reaches the Transcription Termination Site (TTS), elongation factors are released from RNAPII, termination factors are recruited and termination takes place. Importantly, transition from a phase to the next one is strictly regulated and the CTD of Rpb1 plays a crucial role in this transition (Figure 3).

Figure 3. First steps of the transcription cycle (Koch et al., 2008).

The Rpb1 C-terminal domain

The big essential RNAPII central subunit Rpb1 is characterized by a long C-terminal Domain (CTD).

The CTD is made of tandem low complexity heptarepeats of the sequence YSPTSPS. The number of repeats varies between species and increases from lower to higher eukaryotes (from 5 repeats in Plasmodium yoelii to 52 in vertebrates). The increase in the number of repeats is followed by an in increase in the number of repeats that do not match the perfect consensus. In the budding yeast S.cerevisiae the CTD has 26 repeats, 19 of which with a perfect consensus (reviewed in (Harlen and Churchman, 2017; Zaborowska et al., 2016)). Though eight repeats are sufficient for viability of yeast

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cells growing in rich medium, the minimal number of repeats required for viability increases under stress conditions (West and Corden, 1995). The CTD does not have any catalytic activity but it has crucial regulatory functions and the last 10 residues at the very C-terminus of it are required for stabilization of the whole RNAPII (Chapman et al., 2004). Structural and biochemical studies revealed that the CTD is largely unstructured and highly modified, mostly phosphorylated. Indeed, the serines in position 2, 5 and 7 can be phosphorylated as well as the tyrosin in position 1 and the threonine in position 4, while the prolines (position 3 and 6) can be isomerized. Furthermore, glycosylation, ubiquitinylation, and methylation of multiple residues have also been reported (Kelly et al., 1993; Li et al., 2007; Sims et al., 2011). Mass spectrometry analysis showed that not all the repeats of one CTD are modified at the same time and that it is rare to observe two modifications on one repeat.

Phosphorylation is the most well-studied modification of the CTD and its importance is underscored by the fact that mutations of tyrosine 1, serine 5 or serine 2 are lethal in budding yeast (West and Corden, 1995) and mutations of Tyr1 and Ser2 lead to cold-sensitive phenotype in S.pombe (Schwer and Shuman, 2011). Phosphorylation of the serines is highly dynamic during the transcription cycle and it is crucial for transition from one phase to the next one. The dynamic behavior of the CTD is revealed also by the structure of CTD that when unphosphorylated forms a compact spiral, while when phosphorylated forms a more extended tail (Meinhart and Cramer, 2004; Meinhart et al., 2005).

RNAPII is recruited unmodified on promoters and is then modified. The unmodified form of RNAPII has high affinity for the transcription co-activator Mediator (discussed further in this Introduction).

Phosphorylation of Ser5 reduces the affinity of RNAPII for Mediator and favors RNAPII promoter escape (Jeronimo and Robert, 2014; Wong et al., 2014). The importance of this phenomenon is underscored by the observation that mutation of Ser5 is lethal in yeast as well as in metazoans.

Phosphorylation of Ser5 is important also for mRNA capping (Schwer and Shuman, 2011) and splicesome recruitment (Harlen et al., 2016). Furthermore, phosphorylated Ser5 interacts with the NuRD (Set1/COMPASS) complex and it is also involved in transcription termination (Vasiljeva et al.,

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2008). While phosphorylation of Ser5 is maximal around the TSS, phosphorylation of Ser2 is mostly in the gene body and phosphorylation of Ser7 is both in the gene body and at 3’ of the gene. Both phosphorylated Ser2 and Ser7 interact with elongation and termination factors. Importantly, the pattern of CTD phosphorylation is well conserved between yeast and metazoans with the exception of Tyr1 phosphorylation that in yeast is maximal at the Poly-Adenilation Site (PAS) while in metazoans is mostly around the TSS (Harlen and Churchman, 2017; Zaborowska et al., 2016) (Figure 4). The kinases and the phosphates that place and remove the phosphorylation mark on the different serines are known. Kin28 (CDK7) phosphorylates Ser5 and Ser7, while Bur1 and Ctk1 (CDK9) phosphorylate Ser2 (reviewed in (Egloff et al., 2012)). Curiously, a subunit of the Mediator complex, CDK8, phosphorylated the CTD in vitro, but whether this happens also in vivo is still controversial (reviewed in (Galbraith et al., 2010)). Considering the phosphates, Ssu72 dephosphorylates all the serines and specific phosphates exist for Ser5 (Rtr1) and Ser2 (Fcp1) (Egloff et al., 2012).

Figure 4. Chromatin Immunoprecipitation (ChIP) profiles of phosphorylated residues of Rpb1 CTD along the gene body in humans and in budding yeast. PAS: Poly-Adenylation Site (Harlen and

Churchman, 2017)

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Transcription initiation

Transcription initiation is the most regulated phase of the transcription cycle and the rate-limiting step of the whole transcription process. It is a highly energy-consuming process that requires the efforts of many different proteins and enzymatic activities often organized in big multisubunit complexes named General Transcription Factors (GTFs). The first step of transcription initiation is the formation of the so called Pre-initiation Complex (PIC) followed by separation of the DNA strands (a process named “DNA melting”), formation of the transcription bubble, positioning of the active center of RNAPII near the ssDNA emerging strand and RNA synthesis ((Plaschka et al., 2016a) and reviewed in (Cheung and Cramer, 2012)). The molecular mechanisms and the structural details of the whole initiation process have been investigated for decades and are now well known. Here, we are going to describe first the GTFs composing the PIC and then how PIC assembly takes place.

General transcription factors are required for transcription initiation

The GTFs are multisubunit complexes that participate in PIC formation and are required for recruitment and activity of RNAPII (Orphanides et al., 1996; Roeder, 1996). Here we briefly introduce them in the order in which they are recruited on promoters.

TBP, the key of PIC assembly

The TATA Binding Protein (TBP, also named Spt15 in yeast) is a small essential protein that represents the center of the whole PIC. It is considered integral part of TFIID as well as of the co-activator complex SAGA (see below). TBP has a saddle-like structure and binds the DNA minor groove at an AT- rich sequence named TATA box having the consensus TATAWAWR (Basehoar et al., 2004; Hahn et al., 1989a, b). The TATA box is located around 30 nucleotides upstream of the TSS in human, while in yeast the distance between the TATA box and the TSS is variable but generally bigger than in human.

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The TBP DNA binding domain (TAND1) has nanomolar affinity for the TATA box though few base- specific TBP-DNA contacts were observed. Binding of TBP to the DNA is facilitated by TFIIA and TFIIB and leads to a 90° bending of the DNA required for assembly of the PIC (Chasman et al., 1993; Kim et al., 1993a; Kim et al., 1993b; Nikolov et al., 1995). As previously said, TBP binding to the DNA is modulated by TFIID and also by two others negative regulators, Mot1 and NC2, that bind to TBP preventing its interaction with DNA (Zentner and Henikoff, 2013).

TFIIB, a bridge between RNAPII and TBP

In yeast, TFIIB is not a multisubunit complex but it is represented by the protein Sua7. Sua7 plays an essential role in promoter recognition and it helps in arranging the RNAPII in the proper configuration to start transcription. Indeed, TFIIB binds to the region of DNA flanking the TATA box (the DNA motif bound by TBP, see below) and it interacts directly with TBP through its C-terminal domain (Imbalzano et al., 1994). On the other hand, Sua7 N-terminal domain interacts with RNAPII lobe creating thus a bridge between TBP and RNAPII. The importance of TFIIB in promoter recognition is highlighted by the fact that metazoan promoters are often characterized by the presence of the TFIIB specific Recognition Elements (BRE) that is directly bound by TFIIB. TFIIB is also required for transcription starting; once the RNA molecule is longer than 12-13 nucleotides, TFIIB is released (Cheung and Cramer, 2012).

TFIIA, a RNAPII-specific TFIIB partner

Similarly to TFIIB, TFIIA binds to TATA box flanking regions and is involved in promoter recognition and stabilization of the DNA-TBP complex (Imbalzano et al., 1994). TFIIA is a heterodimer composed in budding yeast by the proteins Toa1 and Toa2 and specifically involved in RNAPII-dependent

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transcription. Though it is not required for basal transcription, it is important for transcription of some classes of genes (Ozer et al., 1998a).

TFIID, promoter recognition and TBP activity

TFIID is a big (around 1.2 MDa) complex found often associated to TBP. Indeed, it is composed of 14 subunits named TBP Associated Factors (TAFs). Some of these Tafs are also part of another multisubunit complex involved in transcription initiation, the SAGA complex (discussed later in this Introduction). In addition to these 13 Tafs, metazoans present also some cell-type and developmental-stage specific Tafs.

Tafs are characterized by several histone fold domains (Gangloff et al., 2000) and early studies proposed a histone core-like structure for TFIID (Hoffmann et al., 1996; Selleck et al., 2001). The recent cryo-EM structure of TFIID revealed that the complex is characterized by a high flexibility (Louder et al., 2016; Nogales et al., 2017; Nogales et al., 2016) and encounters several conformational changes (Cianfrocco et al., 2013). Importantly, the new structural studies have not supported the proposal of a histone core-like structure and showed instead that TFIID has a three- lobe horseshoe-like structure (Bieniossek et al., 2013; Brand et al., 1999; Cianfrocco et al., 2013;

Leurent et al., 2002; Louder et al., 2016).

TFIID plays a major role in promoter recognition and in modulating TBP activity. Taf1 and Taf2 recognize and bind to TSS flanking regions in yeast and to TFIID-specific elements in metazoans, like the Motif Ten Elements (MTEs) and the Downstream Promoter Elements (DPEs) in fly and human and the human-specific Downstream Core Element (DCE) (Lee et al., 2005). Furthermore, the yeast TFIID- associated protein Bdf1 and Bdf2 (and the Taf1 subunit in human (Jacobson et al., 2000)) contain two bromodomains, known to bind specific acetylated lysines on histone H4 (Matangkasombut et al., 2000). Taf1 has multiple interactions with TBP. The N-terminus of Taf1 interacts with the DNA

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binding domain of TBP (TBP TAND1 domain) inhibiting TBP binding to TATA box. Furthermore, Taf1 competes with TFIIA for binding to the TAND2 domain of TBP. When transcription takes place TFIIA displaces TFIID stabilizing DNA-TBP interaction (Anandapadamanaban et al., 2013; Kokubo et al., 1993; Liu et al., 1998; Ozer et al., 1998b). Furthermore, it has been shown that several Tafs interact with specific TFs both in yeast (for example Taf4 and Taf5 have been shown to interact with Rap1) and in human where they facilitate enhancer-promoter interaction (Layer and Weil, 2013; Papai et al., 2010).

TFIIF, PIC stabilization and RNAPII recruitment

TFIIF is involved in the early steps of PIC assembly. It directly interacts with RNAPII core favoring its recruitment on the DNA. Indeed, 50% of RNAPII in the cell is associated with TFIIF (Rani et al., 2004).

It is a heterodimer of the two subunits TFIIF-alpha and TFIIF-beta (Tfg1 and Tfg2 in yeast corresponding to the human RAP74 and RAP30). In yeast a third TFIIF subunit, Tfg3, exists, though it is not essential for transcription and it is also part of other multisubunit complexes (Henry et al., 1994). TFIIF is important for RNAPII recruitment PIC stabilization and RNAPII promoter escape (Cheung and Cramer, 2012).

TFIIE, a bridge between RNAPII and TFIIH

TFIIE is a heterodimer of the proteins TFIIE-alpha and TFIIE-beta, in yeast Tfa1 and Tfa2. TFIIE has a very flexible structure characterized by an anchoring domain that binds to RNAPII clamp domain and makes a bridge between RNAPII and TFIIH. Its primary function is indeed favoring the interaction between TFIIH and RNAPII (Okuda et al., 2000).

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TFIIH is a big multisubunit complex organized in three structural modules with three distinct enzymatic activities. A first module is represented by the helicase Ssl2 (mammalian XPB). A second module contains a second helicase, Rad3 (mammalian XPD) and the proteins Tfb1 (p62), Tfb2 (p52), Tfb4 (p34), Tfb5 (p8) and Ssl1 (p44). A third module is composed of the kinase Kin28 and the proteins Ccl1 and Tfb3 (CDK7, cyclin H and MAT1 in human). TFIIH is not only involved in transcription initiation but also in DNA repair (reviewed in (Compe and Egly, 2012)). However, while a complete TFIIH complex is required for transcription, a TFIIH complex lacking the kinase module is still functional in nucleotide excision DNA repair. The two helicases of the complex differ for the directionality of their activity, with Ssl2 working 3’-5’ and Rad3 working in 5’-3’, and for their involvement, in transcription for Ssl2 and in DNA repair for Rad3. Ssl2 opens 11 nucleotides of DNA and favors formation of the transcription bubble (Compe and Egly, 2016). Furthermore, Kin28 phosphorylates Ser5 on the Rpb1 CTD and this allows RNAPII promoter escape and transcription elongation. A fundamental role in this process is played by the co-activator Mediator that, as observed in recent structural studies, is in close proximity to TFIIH (Plaschka et al., 2016b) (see below).

An overview of PIC assembly

When it became clear that transcription initiation required the involvement of many different protein complexes, many efforts were put in order to reveal how these complexes interact among each other and with RNAPII. Two hypothetical models of PIC assembly were proposed in the years, the holoenzyme model and the stepwise model. The holoenzyme model, supported by the relative easiness of co-purify RNAPII together with the GTFs, proposed that RNAPII and the GTFs interact together in a “DNA-free environment” and the whole so pre-assembled PIC is then loaded onto the

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DNA (Keaveney and Struhl, 1998; Koleske and Young, 1995; Thompson and Young, 1995). Conversely, the stepwise model proposed that assembly of the PIC takes place on gene promoters in several sequential steps (Buratowski et al., 1989). Biochemical and, more recently, structural studies from different research groups show that the stepwise model is actually the correct one (Hahn and Young, 2011). In the 90’s the first crystal structures of single subunits of the GTFs were available and in a decade were followed by the high resolution structures of the whole complexes. Great works in this field were performed by the Kornberg’s and Cramer’s labs that in the last eight years successfully obtained the structure of the minimal PIC (He et al., 2013; Plaschka et al., 2015) and defined important structural and mechanistic details of the stepwise assembly of the PIC. The main steps of PIC assembly are shown in Figure 5 and briefly described here.

PIC assembly starts with (1) interaction between the RNAPII core and TFIIF. The non-core RNAPII subunits Rpb4 and Rpb7 stabilize this interaction. (2) The TFIIF-RNAPII complex binds to a complex made by TBP bound to the TATA box, TFIIB and TFIIA. This minimal assembly of PIC is named core initiation complex. (3) TFIIE and TFIIH bind to the core initiation complex making the initially Transcribing Complex (iTC). TFIIH promotes DNA melting and formation of the transcription bubble around 20 nucleotides downstream of the TATA box. (4) RNAPII starts to synthetize RNA. (5) When the nascent RNA is long eight nucleotides, phosphorylation of Ser5 on RNAPII CTD favors RNAPII promoter escape and recruitment of the elongation factors Spt4 and Spt5. Elongation takes place (Sainsbury et al., 2015).

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Figure 5. Stepwise PIC assembly (Sainsbury et al., 2015).

Though the cited structural studies have given us a complete and detailed picture on PIC assembly, some important open questions remain (Figure 6). First, some mechanistic details on DNA opening and on nucleotide addition cycle still need to be clarified. Second, the role and the position of TFIID in PIC assembly have still not be investigated. Third, all the in vitro PIC reconstitution studies used as DNA template a canonical promoter sequence containing a well-defined TATA box though 80% of

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yeast promoters do not have a TATA box (see later in this Introduction). Thus, how PIC is assembled on promoters lacking a TATA box still needs to be explored. Furthermore, it is important to remind that these biochemical and structural studies describe the assembly of a minimal form of PIC that is competent for transcription in vitro but not necessarily in vivo. Indeed, in vivo assembly of the PIC is complicated by the presence of many obstacles that are absent in vitro, first of all, nucleosomes.

Figure 6. PIC structure as reported in (Plaschka et al., 2015).

Yeast promoter architecture is shaped by pioneer transcription factors and chromatin remodelers

In yeast the promoter is defined as the region of 400 or 500 bp upstream of the TSS. Many authors divide the yeast promoter in two parts, the core promoter, the region 200 bp upstream of the TSS, comprising the TATA box and where the PIC assembles, and the Upstream Activator Sequence (UAS) where the TFs and other complexes involved in transcription initiation bind. More importantly than the division in core promoter and UAS, promoter architecture in yeast is defined by the chromatin landscape and specifically by the position of the nucleosomes on the promoter. Indeed, position of

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the so called +1 nucleosome defines the correct position of the TSS (Kubik et al. manuscript under revision). Furthermore, while in yeast +1 nucleosome overlaps with position of the TSS, in human it is further downstream (Kaplan et al., 2009; Yuan et al., 2005). Upstream of +1 nucleosome, where the core promoter is and where PIC assembly takes place, there is a region of DNA defined as Nucleosome Depleted Region or Nucleosome Free Region (NDR or NFR) (Lieleg et al., 2015). The view of this region as completely nucleosome-depleted has been recently challenged by our and other works (Chereji et al., 2016; Ishii et al., 2015; Knight et al., 2014; Moyle-Heyrman et al., 2013; Voong et al., 2016; Xi et al., 2011). Indeed several lines of evidence suggest the existence of a nucleosome- like particle referred to as Fragile Nucleosome (FN) in this region. Whether these particles are really nucleosomes or not and the biological significance of their presence on many promoters will be discussed further in the Results and in the Discussion sections of this thesis.

Nucleosomes are generally considered as impediments for DNA-related processes such as transcription as they wrap DNA around the histone core and make it less accessible to proteins and enzymes. Thus, in vivo, transcription requires the activity of many different proteins and protein complexes that deal with nucleosomes, such as Pioneer transcription Factors and chromatin remodelers. The Pioneer Transcription Factors (PTFs) or General Regulatory Factors (GRFs) are TFs that have a role in positioning nucleosomes, such as the human Oct4, Klf4, Sox2 (Soufi et al., 2015) and the yeast Rap1, Reb1, Abf1 and Tbf1 (Ganapathi et al., 2011; Morse, 2007) (Figure 7). Chromatin remodelers are proteins, often organized in big multi-subunits complexes, which remodel, position, move, evict or modify nucleosomes. Though different families of chromatin remodelers exist and are conserved between yeast and human, in yeast only RSC is essential for cell viability (Chaban et al., 2008).

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Figure 7. (A) Canonical yeast promoter architecture as revealed by MNase-Seq. (B) PTFs and RSC

generate a Fragile Nucleosome (FN) in the NFR of highly transcribed yeast genes. Modified from (Kubik et al., 2017a).

Transcription co-activators

Aside of chromatin remodelers, several other protein complexes are required for transcription in vivo. These complexes are generally referred to as transcription co-activators. Among the transcription co-activators there are the Mediator complex and the complexes with an enzymatic activity that introduce post-translation modifications of the histone tails. Indeed, one mechanism used by cells to counteract the repressive property of nucleosomes is represented by post- translational modifications of histone tails, such as histone acetylation (Kouzarides, 2007). Histone tails are highly modified and they can be acylated, acetylated, methylated, phosphorylated and ubiqutinated. Importantly, these modifications are reversible and are placed and removed by specific enzymes. The most studied histone modification in transcription is histone acetylation (Kouzarides 2007). In this thesis we will focus on two kinds of transcription co-activators, Histone Acetyltransferases (HATs) and the Mediator complex.

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Histone acetylation and its importance in transcription

Histone acetylation occurs at lysine residues on the amino-terminal tails of the histones (Allfrey and Mirsky, 1964; Phillips, 1963) and it takes place through the antagonistic action of two classes of enzymes, Histone Acetyl Transferases (HATs) and Histone De-Acetylases (HDACs).

It has been recognized for a long time that histone acetylation influences several DNA-related processes including DNA replication, repair and transcription. Specifically, it is well-known that acetylation of histone tails exerts a role on transcription and that a positive correlation exists between transcription of a gene and histone acetylation at its promoter (Allfrey et al., 1964; Dion et al., 2005). In higher eukaryotic cells, conditions that lead to massive perturbations of gene expression, like developmental cues and cancer, are accompanied by modifications in the histone acetylation pattern on enhancers and promoters (Gong et al., 2016; Kinnaird et al., 2016; Podobinska et al., 2017). In yeast, variation of growing conditions and environmental stress induce changes in gene expression that go together with alterations in the histone acetylation state of gene promoters (Kuang et al., 2014; Weiner et al., 2015). It has been proposed that the histone acetylation levels are a sensor of the metabolic state of the cell. Indeed, metabolism changes the cellular concentration of acetyl-coenzyme A (Acetil-CoA) the donator of acetyl groups for HATs. Furthermore, in the cell NAD+

levels increase when cellular nutrient levels are low and this activates some HDACs (like the sirtuins) that deacetylate various proteins including histones (Yu and Auwerx, 2009).

Histone acetylation has been proposed to promote transcription through two different non-exclusive mechanisms. (1) Acetylated proteins, including histones, are recognized and bound by specific protein domains called bromodomains (Dhalluin et al., 1999). Proteins containing bromodomains are the readers of the acetylation mark. So far, in yeast 8 proteins containing bromodomains have been identified (Gcn5, Snf2, Spt7, Sth1, Rsc1, Rsc2, Bdf1, Bdf2). Significantly, all these proteins are nuclear proteins having a positive role in transcription regulation (Josling et al., 2012). Recently, another

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protein domain, the YEATS (Yaf9, ENL, AF9, Taf14 and Sas5) domain, has been identified as an acetyl reader specific for acetylated Lys9 of histone H3, expanding so the putative number of histone acetylation readers (Li et al., 2014). (2) Acetylation of lysines on histone tails neutralize the positive charge of histones decreasing their affinity for DNA and disfavoring so chromatin organization in secondary and tertiary structures and making the DNA more accessible to the binding of TFs, members of the PIC or other transcriptional co-activators that do not contain bromodomains (Garcia- Ramirez et al., 1995; Wang and Hayes, 2008) (Figure 8). However, the importance of histone acetylation for transcription activation and PIC assembly is still very controversial and among the limited number of proteins containing bromodomains identified so far in yeast only one, Sth1, is essential (Bdf1 and Bdf2 deletions are synthetically lethal). Furthermore, evidence of the in vivo role of histone acetylation in chromatin de-compaction are still missing.

Figure 8. Histone acetylation and transcription (Verdin and Ott, 2015).

HATs, the writers

HATs are the enzymes that acetylate the histone tails. They belong to the big class of Lysine Acetyl Transferases (KATs) and indeed many of them acetylate also non-histone proteins, among these many TFs and other nuclear proteins, but also some cytoplasmic or mitochondrial proteins. HATs are structurally characterized by a central catalytic core, where acetyl-CoA finds place, flanked by N-

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terminal and C–terminal regions important for histone or substrate binding and specificity. Indeed, HATs have a strong specificity for specific lysine residues on histone tails. HATs are classified in three main families (GNAT, MYST and orphan) depending on the structure of the catalytic core and the catalytic mechanism of action (Yuan and Marmorstein, 2013).

GNATs (Gcn5-related N-acetyltransferases) are a family of HATs that includes Gcn5, PCAF (p300/CBP- associated factor), Elp3, Hat1, Hpa2 and Nut1. The catalytic core of GNATs is characterized by a short (160 residues) highly conserved motif, named motif A, important for Acetyl-CoA binding. Many GNATs have also a C-terminal bromodomain. The catalytic mechanism of acetylation involves the formation of a ternary complex between the HAT, the histone and a molecule of Acetyl-CoA. A conserved glutamic acid residue in the motif A (E173 for Gcn5) is crucial for the reaction (Marmorstein and Trievel, 2009).

The MYST family of HATs comprises the human MOZ (Sas3 in yeast), Ybf2, Sas2, Tip60/Esa1 and the human MORF and HBO1. They are characterized by the presence of cysteine-rich zinc finger domains important for HAT activity and N-terminal chromodomains. The catalytic core is longer than the GNAT one (around 250 residues). Furthermore, some also have the motif A typical of GNATs. They acetylate histones through a “ping-pong” mechanism (see below) where a cysteine residue of the HAT is acetylated first and then it transfers the acetyl group to a lysine residue on the histone tail (Marmorstein and Trievel, 2009).

All the HATs that do not belong to the GNAT and the MYST families made up the family of orphan HATs such as p300/CBP and Rtt109. They are characterized by structural heterogeneity and by the presence of a big HAT catalytic domain (up to 500 residues for the human p300/CBP). The heterogeneity of structural domains in the catalytic core reflects also different catalytic mechanisms.

The most studied one is the “hit and run” mechanism of p300/CBP (Liu et al., 2008).

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HDAC are the enzymes that remove the acetyl group from histone tails. Similarly to HATs, also HDACs are able to de-acetylate also non-histone proteins. Contrary to HATs, HDACs are characterized by a lower level of substrate specificity and some of them are redundant among each other. They are classified in three main groups. Group I (Rpd3/ HDAC1, Hos1, Hos2) and Group II (Hda1, Hos3) are all Zinc-dependent, while group III are NAD+ dependent. Sirtuins, among the most studied HDAC in both yeast and human, belong to group III. Sir2, Hst1 and Hst2 de-acetylate both histones and non-histone proteins, while Hst3 and Hst4 de-acetylate specifically H3K56 (de Ruijter et al., 2003).

Esa1 and NuA4

NuA4 composition and structure

Esa1 (Tip60/KAT5 in human) is the only HAT essential for cell viability in yeast. Esa1 is a member of the MYST family of HATs (Kouzarides, 2007) and it is mainly responsible of acetylation of histones H4 and H2A (Boudreault et al., 2003; Xu et al., 2016). It is part of the 13-subunit complex NuA4 (Allard et al., 1999). Esa1, Yng2, Eaf6 and Epl1 constitute the catalytic core of the NuA4 complex also named Piccolo-NuA4. Piccolo-NuA4 exists in the nucleus as free complex or in association with the other subunits of NuA4. Among these, Eaf1 (Esa1-associated factor 1) represents the scaffold protein required for assembly of the four functional modules of the complex, Piccolo, Tra1, Eaf3/5/7 (also named TINTIN) and the Arp4/Act1/Swc4/Yaf9 module (Boudreault et al., 2003). Furthermore, while Eaf1 is the only subunit exclusively found in NuA4, all the other subunits are associated with different complexes involved in transcription regulation. For example, the essential protein Tra1 is found in both the NuA4 and the SAGA complexes, Act1 and Arp4 are also associated with the chromatin

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remodeler INO80 and Eaf2 (Swc4), Yaf9, Arp4 and Act1 are components also of the SWR1 remodeler (Figure 9).

Figure 9. NuA4 complex. The red asterisk labels the subunits essential for cell viability. Modified from (Chittuluru et al., 2011).

First insights into the architectural organization of the NuA4 complex came from the first cryo-EM structure of the whole complex (Chittuluru et al., 2011). This study revealed the presence of two big domains, one entirely occupied by the gigantic Tra1, joined by thin connections. A more recent study described the cryo-EM structure of Piccolo-NuA4 and of a sub-complex of NuA4 containing Tra1, Eaf1, Eaf5, Act1 and Arp4 (Wang et al., 2018). In agreement with early biochemical studies (reviewed in (Doyon et al., 2004)), Wang et al. showed that Eaf1 is characterized by strong structural plasticity and interacts with subunits of the four sub-modules of NuA4 confirming its central role in maintenance of the assembly and the architecture of NuA4. Indeed, Eaf1 interacts with Eaf5 through its N-terminal domain and with Actin, Arp4 and Epl1 through its HAS domain. Furthermore, the structure revealed also multiple interactions between Eaf1 SANT domain and Tra1 C-terminus (Figure 10). Importantly, though this recent study elucidated the molecular interactions between the different sub-modules of NuA4 and expanded our view on Piccolo and NuA4 assembly, many questions regarding the function of the different sub-modules still need to be addressed.

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Figure 10. NuA4 complex scheme and structure as reported in (Wang et al., 2018).

Esa1 acetylates histones H2A and H4 through a double recognition mechanism

Despite the crystal structure of Esa1 was solved more than fifteen years ago (Yan et al., 2000; Yan et al., 2002), only a recent structural study described in detail the molecular mechanism by which Esa1 acetylates preferentially histones H2A and H4 (Xu et al., 2016). Esa1 structure revealed the presence of two domains, the TUDOR domain responsible of the interaction with the nucleosome and the HAT domain where the catalytic pocket is located. The catalytic pocket is made of the Histone Binding Loop (HBL) and the Catalytic Loop (CataL) containing the crucial catalytic residue Glu338 (Berndsen et al., 2007). The observation that the Piccolo-NuA4 acetylates histones also in absence of the other modules of NuA4 (Boudreault et al., 2003) revealed that NuA4 interaction with the nucleosome takes place in the periphery of the complex where Piccolo resides. In 2016 Xu et al. solved the crystal structure of a minimal form of the Piccolo containing Esa1 HAT domain, Epl1 EpcA domain, the ING domain of Yng2 and full length Eaf6, showing that Epl1 is the central organizer of Piccolo architecture interacting both with the Esa1 catalytic pocket (with its C-terminus) and with the nucleosome (with its N-terminus). Furthermore, in the same study the cryo-EM structure of this minimal form of Piccolo associated with a nucleosome core particle (NCP) was also reported. The structure revealed a modest interaction between NuA4 and the histones (contrary to what happens for Gcn5, see below)

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in agreement with the relatively low binding affinity of Esa1 for the histones (Berndsen et al., 2007).

Importantly, this study showed that Esa1 acetylates specifically histones H2A and H4 thanks to a double recognition mechanism. The first mechanism of recognition is based on sequence preference.

Indeed, Esa1 catalytic pocket preferentially binds to the GK/AK motif that is characterized by the presence of a small residue (glycine or alanine) at position -1. However, the GK/AK motif is common and frequent in the tails of all histones and thus it cannot explain the specificity for H2A and H4 that it is instead driven by a space recognition mechanism. Histone H4 tail as well as histone H2A tail are indeed projected on the dish face of the nucleosome towards Esa1 while the N-terminal tails of the other histones are unreachable by Esa1. Notably, the preference of Esa1 for certain lysine residues of H4 and H2A reflects their distance to Esa1 (with H4K5, K8 and K16 being the closest ones to Esa1).

Furthermore, the study showed that Epl1 plays the crucial role of orienting the catalytic site of Esa1 towards H4 and H2A underscoring thus its essentiality for cell viability.

Esa1 role in transcription and its binding to the genome

Though the importance of Esa1 is underscored by the fact the Esa1/Tip60 is essential for viability in both yeast and higher eukaryotes, its precise role in transcription regulation is still elusive.

Importantly, the lethal phenotype of Esa1-null yeast cells enforced a large use of different Esa1- temperature sensitive mutants. Early studies used these mutants to measure steady-state mRNA levels at non-permissive temperature and hinted to a specific role of Esa1 in transcription of specific classes of genes such as the Ribosomal Protein (RP) genes (Reid et al., 2000b; Rohde and Cardenas, 2003). On the other hand, more recent genome-wide studies pointed to a more global role of Esa1 in transcription regulation (Durant and Pugh, 2007). The question is Esa1 important for transcription of all yeast genes or just for specific groups is linked to another elusive point, the binding of Esa1 in the genome. Esa1 Chromatin Immunoprecipitation (ChIP) followed by high-throughput genome-wide sequencing showed that Esa1 binds the UAS of actively transcribed genes (Kuang et al., 2014), where

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it acetylates promoter nucleosomes. However, how Esa1 is recruited to promoters is still matter of investigation. It is thought that the mechanism by which Esa1 is recruited at promoters depends on whether Esa1 is associated just with the Piccolo or with the whole NuA4 complex. The protein Tra1, that is part of NuA4 but not of Piccolo, interacts with specific TFs (Gal4, Gcn4, c-Myc in human, see below) and thus favors the recruitment of Esa1 on specific target genes. The Piccolo lacks Tra1 as well as all the other subunits of NuA4 with a putative recruitment function (Boudreault et al., 2003), and it thus thought to bind promoters in an untargeted way. Furthermore, it is important to highlight that some subunits of the Piccolo complex and the NuA4 complex contain domains that act as readers of specific histone modifications; among these the PHD domain of Yng2 (Loewith et al., 2000), the chromodomain of Eaf3 (Joshi and Struhl, 2005) and the YEATS domain of Yaf9 (Wang et al., 2009). However, the role of some of these domains in NuA4 recruitment is controversial (Steunou et al., 2016).

It is important to underline that Esa1 role in transcription might be also mediated by its ability to acetylate also nuclear non-histone proteins such as chromatin remodelers regulating their activity (Downey et al., 2015). Furthermore, some authors also reported Esa1 binding in the ORF of active genes suggesting a possible role of Esa1 in transcription elongation (Ginsburg et al., 2009) that still needs to be demonstrated.

Aside of its role in transcription, Esa1 plays also a role in DNA repair and DNA damage response (Bird et al., 2002) in both yeast and human. Notably, mutations of Esa1 HBL domain or at the interaction interface of Epl1 and Esa1 enhance cell sensitivity to genotoxic agents such as MMS (Xu et al., 2016).

The recruitment of NuA4 to DNA damage sites is mediated in part by the Arp4/Act1 module (Downs et al., 2004). Furthermore, TRRAP the human homologous of the yeast Tra1 is also involved in DNA repair (Murr et al., 2006; Robert et al., 2006).

Finally, it is worth to mention that Esa1 acetylates also a subset of non-histone cytosolic substrates in both yeast and human, among these the septin proteins (Mitchell et al., 2011) and some important

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regulators of cell metabolism involved in signaling pathways responsive to nutrient availability and stress such as Pck1. Importantly, it has also been shown that acetylation of Pck1 regulates its enzymatic activity, inferring a role of Esa1 in cell metabolism (Lin et al., 2009).

Gcn5 and SAGA

Gcn5 is part of the SAGA complex

Gcn5 (Ada4), the first HAT to be identified (Brownell et al., 1996), is a non-essential HAT that belongs to the SAGA complex (Grant et al., 1997). Gcn5 acetylates almost exclusively histone H3 (Suka et al., 2001) through extensive interactions with the sequence flanking the target lysine residues (Rojas et al., 1999; Trievel et al., 1999). Gcn5 has a quite well-defined decreasing specificity for the lysines 14, 9 and 23, 18, 27 and 36 of histone H3 (Feller et al., 2015; Kuo and Andrews, 2013). Yeast Gcn5 has two human counterparts, PCAF (KAT2B) and GCN5 (KAT2A), and is part of both the SAGA and ATAC (Ada Two A containing) metazoan complexes (Guelman et al., 2006; Nagy and Pankotai, 2010; Wang et al., 2008).

The SAGA complex is a 21-subunit complex, organized in five distinct structural and functional submodules, the HAT module, the Deubiqutination (DUB) module, the structural module, the TBP module and the protein Tra1. The HAT module comprises Gcn5 and the proteins Ada2, Ada3 and Sgf29. Furthermore, Gcn5, Ada2, Ada3 and Sgf29 assemble together with the proteins Ahc1 and Ahc2 to make up a second Gcn5-containing HAT complex named the ADA complex (Eberharter et al., 1999). A second enzymatic activity of the SAGA complex is represented by the deubiquitinase Ubp8 that together with Sgf73, Sgf11 and Sus1 constitute the DUB module. The TBP, the DUB and the HAT modules are structurally connected by the structural core of the SAGA complex that is constituted by Ada1, Spt20, Spt7 and by several TAFs (Taf5/6/9/10/12) shared with the TFIID complex. Spt7 and the

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TAFs of the structural module interact with TBP and the other proteins (Spt3 and Spt8) of the TBP module. Finally, Tra1 also associates to the SAGA complex (reviewed in (Helmlinger and Tora, 2017)) (Figure 11).

The ADA complex is not the only SAGA-like Gcn5-containing complex. Indeed, a second variant of the SAGA complex exists in both yeast and metazoans. This SAGA variant, named SLIK (SAGA-like complex) or SALSA, lacks the protein Spt8, has a C-terminal truncating version of Spt7 deficient of the Spt8 interacting domain and contains the protein Rtg2. The controlled cleavage of Spt7 takes place in the cytoplasm by the cytoplasmic protease Pep4. The metazoan SAGA is highly similar to the yeast SLIK/SALSA complex but it also contains additional subunits involved in RNA splicing (Pray-Grant et al., 2002; Wu and Winston, 2002).

Figure 11. SAGA and ADA complexes. The red asterisk labels the subunits essential for cell viability.

Modified from (Lee et al., 2011).

SAGA structure revealed a highly organized submodular organization

The first low-resolution structural studies already revealed the highly organized submodular organization of the SAGA complex that appeared as a five-modular complex with an elongated shape

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(Wu et al., 2004). More recent studies confirmed the elongated shape of the SAGA complex and revealed new details of the structural architecture of the complex (Han et al., 2014; Lee et al., 2011).

Specifically, Han et al., performing cross-link and Mass Spectrometry analyses on a large collection of truncated mutants, revealed that the whole SAGA complex is organized around a central TFIID-like structure. This structural core of SAGA is composed of two copies of the heterodimers Taf6-Taf9 and Ada1-Taf12, by two copies of Taf5 and by the protein Spt20 that has a main role in the assembly of the whole SAGA complex. Spt7 has multiple interactions with this central core. Furthermore, this structural core is the center of a complicated network of interactions that involves also the TBP module and Tra1. Tra1 occupies a peripheral position in SAGA and the interface between Tra1 and the rest of the SAGA complex is minimal. While Wu et al. located Tra1 at the opposite site of Spt20, the work by Han et al. showed that Tra1 is in close proximity with Spt20 and Taf12 (see later in this Introduction). The DUB and the HAT modules are close to each other and interact with the central core TFIID-like structure. Specifically, for the HAT module the interaction between Ada3 C-terminus and several TAFs and Spt7 has been shown to regulate Gcn5 activity (Han et al., 2014). Furthermore, Ada2 is important for integrity of HAT module but not for the whole complex (Lee et al., 2011).

The main characteristic of the SAGA complex is its high flexibility and the ability to adopt different conformations, as recently shown (Setiaputra et al., 2015). The more recent structure of the complex revealed that the SAGA structure has a croissant-shape with a globular head, a torso region and a long tail. The globular head is made up by the TBP module and by Tra1. The torso has two distinct regions the shoulder and the joint. While the shoulder does not have any interaction with the tail and is occupied by Spt8 (thus, it is absent in SLIK/SALSA), the joint is constituted by the structural core and by the DUB module and connects the tail region with the rest of the complex. The tail region is entirely occupied by the HAT module and it is the most mobile region of the complex. Indeed, the position of the tail varies and this defines three different conformations of the SAGA complex, the donut, the arched and the curved conformation (Setiaputra et al., 2015) (Figure 12). However, what determines the conformational changes of the SAGA complex still needs to be investigated.

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Figure 12. Schematic representation of the SAGA complex and its conformations. Modified from (Setiaputra et al., 2015).

Multiple activities of the SAGA complex

The SAGA complex and its close-related ADA and SLIK/SALSA complexes are involved in several different aspects of transcription regulation. As described, the SAGA complex contains two modules with two different enzymatic activities, the DUB and the HAT modules. The DUB module catalytic subunit Ubp8 is a deubiqutinase that removes ubiquitin on lysine 123 of histone H2B (Henry et al., 2003). Monoubiquitination of H2BK123 is posed by the protein Rad6/Bre1 and is required for the recruitment of the histone methylase Set1. Set1 di- and tri-methylates lysine 4 on histone H3 promoting active transcription, while deubiquitination of H2BK123 by Ubp8 favors transcription elongation (Wyce et al., 2007). This interplay of histone modifications is called “trans-histone regulatory pathway” (Briggs et al., 2002) and it involves also the 19S proteasome (Ezhkova and Tansey, 2004). The importance of the trans-histone modification pathway for transcription regulation is still matter of investigation and many authors pointed to a minor role of Ubp8 in this process.

Role of co-activators and promoter architecture in transcription of yeast protein-coding genes

Thesis

Reference

Role of co-activators and promoter architecture in transcription of yeast protein-coding genes

Role of co-activators and promoter architecture in transcription of yeast protein-coding genes

Maria Jessica Bruzzone

Table of contents

Acknowledgements

Summary

Résumé

Introduction

Transcription, the key of the central dogma of biology

RNAPII transcription

RNAPII composition and structure

The three phases of RNAPII transcription

The Rpb1 C-terminal domain

Transcription initiation

General transcription factors are required for transcription initiation

An overview of PIC assembly

Yeast promoter architecture is shaped by pioneer transcription factors and chromatin remodelers

Transcription co-activators

Histone acetylation and its importance in transcription

Esa1 and NuA4

Gcn5 and SAGA