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.
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Indeed, steady-state mRNA analysis revealed minimal changes in Ubp8-deleted cells in both yeast and human (Bonnet et al., 2014; Lenstra et al., 2011).
Furthermore, the DUB subunit Sus1 is also part of the TREX complex and is located in the inner face of the Nuclear Pore Complex (NPC) involving thus the DUB module and SAGA also in mRNA export (Garcia-Oliver et al., 2012; Rodriguez-Navarro et al., 2004).
Finally, it is important to mention that the SLIK/SALSA complex is involved in stress response and specifically in the retrograde response pathway that signals mitochondrial dysfunction to the nucleus. Indeed, it has been shown that SLIK/SALSA induces expression of the genes that allow the usage of acetate as carbon source upon mitochondrial stress (Jiang et al., 2016; Pray-Grant et al., 2002).
In the next paragraph, we will describe the role of the HAT module in transcription regulation.
The view on Gcn5 role in transcription has been recently challenged
The role of Gcn5 in transcription has been defined more than ten years ago by the pioneer work of Pugh’s lab (Huisinga and Pugh, 2004). Huisinga and Pugh measured steady-state mRNA levels in delta-gcn5 yeast cells showing that just 10% of the yeast genes were strongly affected in absence of Gcn5. The result of this analysis led them to conclude that Gcn5 is important for transcription of a limited number of genes that they defined as “SAGA-dominated genes”. These genes are primarily stress-induced and contain a consensus TATA box sequence in their promoters (see also below in this Introduction) (Bhaumik, 2011; Huisinga and Pugh, 2004). Importantly, the first attempts to measure Gcn5 binding in the genome of different organisms revealed few binding sites consistently with the reduced number of affected genes in gcn5-delta yeast cells (Bian et al., 2011; Krebs et al., 2011;
Venters et al., 2011; Vermeulen et al., 2007; Weake et al., 2011).
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Recently, this restricted view of Gcn5 function has been challenged using more direct measures of transcription. Specifically, two works from Tora’s lab showed reduced level of transcription of almost all yeast genes in delta-gcn5 cells implicating a role of Gcn5 and SAGA in the activation of nearly all yeast genes (Baptista et al., 2017; Bonnet et al., 2014). These findings were also supported by the observation that Gcn5, similarly to NuA4, binds to UASs of most yeast genes (Baptista et al., 2017;
Bonnet et al., 2014; Kuang et al., 2014; Ohtsuki et al., 2010). As for Esa1, it has been reported Gcn5 binding to coding sequences where it promotes nucleosome eviction and interacts with phosphorylated RNAPII (Govind et al., 2007; Qiu et al., 2016).
Finally, Gcn5 regulates transcription also in an indirect way. Indeed, it acetylates many non-histone proteins (Downey et al., 2015) and among these some important regulators of transcription such as Snf2 the catalytic subunit of the remodeling complex Swi/Snf (Kim et al., 2010). Notably, it has been shown that acetylation of Snf2 by Gcn5 regulates its binding to nucleosomes during stress response (Dutta et al., 2014).
Recruitment of SAGA complex to the genome
An important point that still needs to be fully addressed regarding Gcn5 is how Gcn5 is recruited to promoters. The global role of Gcn5 in yeast transcription clashes with the reduced number of Gcn5 molecules in the cell. A possible explanation for this apparent inconsistency comes from a recent report that investigated the mobility of Gcn5 in the nucleus and revealed that the interaction of Gcn5 with chromatin is transient and very dynamic (Vosnakis et al., 2017). However, the mechanisms that drive Gcn5 association to promoters are still elusive. Many subunits of the SAGA complex, including Gcn5, contain protein domains that bind to modified nucleosomes. For examples, Gcn5 and Spt7 have both bromodomains, Sgf29 has a Tudor domain, Ada2 has both a SANT and a SWIRM domain and Spt8 has a WD40 (reviewed in (Lee and Workman, 2007)). Furthermore, as for Esa1, Gcn5
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binding to the UAS in the context of SAGA is promoted by the recruitment module of the complex represented by the protein Tra1 that interacts with specific TFs. Importantly, Gcn5 is also part of the ADA complex that lacks any recruitment module (Grant et al., 1997). However, how and in which conditions the ADA complex can be recruited to the UASs still needs to be fully investigated.