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).