Reading chromatin

Abstract

Chromatin-modifying complexes typically contain signature domains that either have catalytic activity or recognize and bind to specific histone modifications such as acetylation, methylation and phosphorylation. Despite tremendous progress in this area, much remains to be learned about the mechanistic functions of less well-characterized signature domains. One such module is the evolutionarily conserved YEATS domain, found in a variety of chromatin-modifying and transcription complexes from yeast to human. Three yeast proteins contain a YEATS domain, including Yaf9, a subunit of both the histone variant H2A.Z deposition complex SWR 1-C and the histone acetyltransferase complex NuA4. The three-dimensional structure of the YEATS domain from Yaf9 was solved recently, revealing the existence of three distinct structural regions. One region is characterized by a shallow groove that might constitute a potential acetyl-lysine binding pocket, raising questions about possible protein interaction partners of the Yaf9 YEATS domain.

Introduction

In eukaryotic cells, DNA is packaged together with histone proteins into repeating units of nucleosomes, which constitute the basic building block of chromatin. Chromatin is the principal substrate for many cellular processes including transcription, DNA replication, recombination and repair as the constituent enzymes and associated factors need to access the DNA template in the context of chromatin.⁴² Histone variants, posttranslational histone modifications and ATP-dependent chromatin remodeling events all contribute to creating distinct structural and functional chromatin “neighborhoods.” The dynamic addition and removal of posttranslational modifications to histones is one important mechanism by which accessibility to chromatin and ultimately DNA is regulated.⁴³ Chemical groups attached to histones include acetylation, methylation, phosphorylation, ubiquitylation, sumoylation and others.¹ Rather than occurring and functioning independently, histone modifications often act in a spatially and temporarily coordinated manner and intersect closely with multisubunit complexes that provide “writer,” “eraser” and “reader” functions.^2,3 The writer and eraser functions reside in subunits containing catalytic activity that add or remove the mark on histones, while the reader function frequently requires signature domains that specifically recognize and bind to these marks.³ Chromatin-regulating complexes can act in step-wise and/or combinatorial fashion and engage in extensive crosstalk in order to regulate chromatin structure and chromatin accessibility for transcription factors and associated proteins.^3,4 Improper target recognition by chromatin readers can have severe consequences, potentially leading to human disease, making these proteins attractive targets for therapeutic intervention.⁵ The better characterized families of reader pockets include bromodomains, chromodomains, PhD fingers and PWWP domains, to name a few.⁶ Although these domains clearly provide important chromatin-binding functions through their interaction with histone marks, several intriguing protein domains present in chromatin-modifying complexes have been less explored and largely lack structural and functional information. One of these lesser-known protein domains is the YEATS domain, named after the first proteins recognized to contain this module (Yaf9, ENL, AF9, Taf14, Sas5).⁷ The primary amino acid sequence of the YEATS domain is evolutionarily conserved from yeast to human and is found in proteins belonging to a variety of chromatin-modifying complexes and transcription factors. In Saccharomyces cerevisiae, three proteins (Yaf9, Taf14 and Sas5) contain a YEATS domain, whereas the most prominent YEATS-proteins in humans, GAS41, ENL and AF9, are all linked to human cancers.⁷ Here we discuss the recently solved structure of the Yaf9 YEATS domain and its potential functions in light of its similarity to histone chaperone Asf1 and its ability to bind histones.

Yaf9 in Transcription and DNA Repair

Yaf9 is a subunit of both the ATP-dependent chromatin remodeling complex SWR1-C, which deposits histone variant H2A.Z into chromatin and the essential histone acetyltransferase complex NuA4, which acetylates H2A.Z among other substrates.^8–13 H2A.Z has roles in a variety of processes, including transcriptional regulation and DNA damage response.^14,15 Yaf9 is required for deposition of H2A.Z in vitro as well as in vivo,^16,17 suggesting it facilitates the exchange of H2A with H2A.Z in some way. Moreover, the shared module of subunits between NuA4 and SWR1-C, including Yaf9, might participate in recruiting these two complexes to chromatin by binding nucleosomes.¹⁸ The current model of extensive crosstalk between NuA4 and SWR1-C converging on H2A.Z suggests that in an initial step, acetylation of histone H4 by NuA4 is required for deposition of H2A.Z by SWR1-C into chromatin and subsequently, NuA4 acetylates H2A.Z itself.^9,10,19,20 Through its ability to recognize and bind acetylated H4, the bromo-domain protein Bdf1 has an important role in the recruitment of SWR1-C.^20–23 However, other domains in SWR1-C subunits likely also partake in this process, with Yaf9 being an attractive candidate due to its presence in the shared module and its ability to bind histones.^16,18 Specifically, it might be the YEATS domain of Yaf9 that mediates this process, as it is required for H2A.Z deposition at specific promoters.¹⁶

In addition to functioning in transcription, both NuA4 and SWR1-C have important roles during the DNA damage response.⁴⁴ The central signaling pathway activated in response to DNA damage caused by double strand breaks involves the phosphorylation of H2A.X at C-terminal serine residues.²⁴ Arp4, which, analagous to Yaf9, is present in the shared subunit module between NuA4 and SWR1-C, physically interacts with phosphorylated H2A.X, thereby recruiting NuA4 to acetylate H4 at double strand breaks.^25,26 Subsequently, the SWR1-C and INO80 complexes are recruited to displace phosphorylated H2A.X.^27,28 Therefore it is tempting to speculate that Yaf9, as a member of the shared module, may also participate in the recruitment of SWR1-C to DNA damage sites. Alternatively, Yaf9 might facilitate the spatial and temporal coordination of NuA4 and SWR1-C activities at the break site.

Structure and Function of the Yaf9 YEATS Domain

In light of the diverse roles of Yaf9, the recently determined three-dimensional structure of the Yaf9 YEATS domain¹⁶ provides an important step forward in understanding the function of Yaf9 and specifically its YEATS domain in these processes. The YEATS domain consists of a beta-sandwich characteristic of the Immunoglobulin (Ig) fold family with 8 antiparallel β-strands capped on one end by 2 short α-helices, and contains three distinct structural features.¹⁶ First, a highly conserved cleft is located on the end of the Ig fold opposite the two capping helices. Second, a relatively shallow groove near the N- and C-terminal termini of the YEATS domain is formed in part by the capping helices. Third, a patch rich in conserved charged residues lies between the cleft and the putative peptide-binding groove. Structure-function analysis of the most evolutionarily conserved amino acid residues located in these distinct regions revealed that the YEATS domain of Yaf9 is required for efficient deposition of the histone variant H2A.Z at specific promoters, global H2A.Z acetylation and resistance to genotoxic stressors.¹⁶

Interestingly, the overall structure of the YEATS domain of Yaf9 is similar to that of the histone chaperone Asf1, and this structural similarity is congruent with Yaf9's ability to bind histones H3 and H4 in vitro,¹⁶ a function that has been well established for Asf1.^29–31 Yaf9's connection to Asf1 in yeast extends even further as simultaneous deletion of the two genes encoding them causes dramatic growth defects when compared to the two single deletions, suggesting that these two proteins are involved in a similar process. Yeast strains carrying an unacetylable form of H3K56ac, the histone modification linked to Asf1, have a similar synthetic growth defect in the absence of YAF9,¹⁶ indicating that the genetic interaction between YAF9 and ASF1 involves acetylation on H3K56. Taken together, Yaf9's structural and genetic connection to Asf1 and its ability to bind histones H3 and H4 lend support to the hypothesis that the YEATS domain is a chromatin-binding module (i.e., reader) perhaps through interacting with nucleosomes. This might facilitate SWR1-C dependent deposition of H2A.Z during transcription and/or the DNA damage response, in both cases potentially involving coordination with NuA4.

Potential Binding Targets of the Yaf9 YEATS Domain

If the Yaf9 YEATS domain were to function as a reader in the context of recruiting its resident complexes to chromatin, one might speculate that it should have a typical “reader pocket” present in signature domains such as bromodomains or chromodomains.³ In contrast to these typical domains, there currently is no evidence for Yaf9 or other YEATS-domain proteins having the ability to bind to any modified histones. However, based on a more in-depth analysis of the Yaf9 YEATS domain structure, we suggest that Yaf9 has the capability to bind acetylated histones, consistent with a bonafide reader function. One of the distinguishing structural features in the YEATS domain is a shallow groove with a deep hydrophobic pocket that might function as a peptide-binding region.¹⁶ Comparative structure analysis of Yaf9 and Asf1 provides an intriguing hint for the function of this pocket and its potential role in YEATS-domain target recognition. Superposition of the two folds, Yaf9 YEATS domain and Asf1, correctly co-aligned the topology of the β-strands based on their switched-Ig fold configuration (Fig. 1A). Moreover, Asf1 engages the C-terminal tail of histone H4 in the same orientation and position as the hydrophobic groove of Yaf9 bound to the N-terminal segment of one of its three partner protomers (Fig. 1B).^29–31 In the crystal, the deep pocket within the groove bound a peptide such that an isoleucine was placed directly over the entrance into the pocket (Fig. 1C). Modeling showed that the pocket was deep enough and was of sufficient dimension to accommodate an acetyl-lysine (Fig. 1D).

Figure 1.

Interaction of the Yaf9 YEATS domain hydrophobic groove with its potential target. (A) Structural comparison of the Yaf9 YEATS domain and Asf1 core. Stereo superposition of the Yaf9 YEATS domain on Asf1. The ribbon diagram is colored gray for Yaf9 and green for Asf1. (B) Docking of the Asf1/histone H3–H4 complex onto Yaf9. Note that the N-terminal tail/hydrophobic groove interaction formed between symmetry-related Yaf9 protomers (cyan sticks) spatially and directionally overlaps with the interaction seen between the C-terminal tail of histone H4 (magenta ribbon) and Asf1. In both instances, the peptide from the binding partner docks into an inter-sheet groove and pairs by the last (“h”) strand of the Ig fold. (C) Close up of the Yaf9 YEATS domain hydrophobic groove (surface) and its interaction with the N-terminal segment of adjoining protomer (cyan sticks). An isoleucine in the peptide sits over a deep hole in the floor of the groove. (D) The hole in the hydrophobic groove (surface) of the Yaf9 YEATS domain is sufficiently wide and deep to accommodate a modeled acetyl-lysine residue (white sticks). The modified lysine was modeled using the preferred rotamer library available in PYMOL.⁴¹

Therefore, Yaf9's putative peptide-binding pocket could function as a reader module that targets specific acetyl-lysines on histones to trigger subsequent chromatin changes. While this is an attractive model, the ability of the Yaf9 YEATS domain to bind acetyl-lysine is clearly speculative and requires detailed analyses with direct peptide-binding assays. However, based on its biological functions and genetic interactions, it is tempting to consider which acetyl-lysine Yaf9 may bind to. One hint comes from work describing a requirement of Asf1/H3K56ac for chromatin assembly and checkpoint recovery after DNA repair, leading to inactivation of the DNA damage checkpoint and cell survival.³² Given the involvement of NuA4 and SWR1-C in the cellular response to DNA double strand breaks, Yaf9, through its YEATS domain, may recognize and bind to H3K56ac during the DNA repair process.

Another possibility is that Yaf9, analogous to Bdf1, binds to acetylated H4 to recruit SWR1-C and NuA4 during transcription to specific promoters to deposit and acetylate H2A.Z, respectively. Compromising Yaf9 YEATS domain function results in loss of H2A.Z at specific promoters,¹⁶ consistent with the YEATS domain reading certain histone marks in these regions and thereby helping to recruit the SWR1-C to these regions.

Functional Conservation of the YEATS Domain

It is reasonable to extrapolate our hypothesis of the Yaf9 YEATS domain being chromatin readers in yeast to human YEATS domain-containing proteins as this domain is not only structurally but also functionally conserved through evolution. The closest relative of Yaf9 is the human protein GAS41, which is amplified in glioblastomas and astrocytomas.^7,33,34 GAS41 is a subunit of the human TIP60 and SRCAP complexes, which are the equivalents of NuA4 and SWR1-C in yeast.^35–38 Interestingly, the GAS41 YEATS domain can substitute for the Yaf9 YEATS domain in yeast,¹⁶ providing direct evidence for functional conservation. This makes it likely that the GAS41 YEATS domain also binds histones, although this has not yet been tested to our knowledge. It will be interesting to reconcile this model with GAS41 serving as a co-activator for mammalian transcription factors AP-2 and p53,^38,39 specifically as it relates to YEATS-mediated chromatin recognition proposed here. Regardless, histone binding might be a broader and general function of YEATS domains. The human YEATS protein ENL, which is one of the most common fusion partners of the mixed lineage leukemia (MLL) protein in human leukemias, binds to histones H3 and H1 in vitro through its YEATS domain.⁴⁰

Conclusion

It is well established that chromatin-modifying complexes have writer, eraser and reader functions that reside in multiple signature domains. We hypothesize that YEATS domains, found in a variety of chromatin-modifying and transcription-related complexes, generally may act as a reader of histone marks to recruit its resident complexes to specific chromatin neighborhoods during cellular processes that the particular YEATS protein is involved in. Specifically, for Yaf9's YEATS domain in yeast, we speculate that it might bind acetylated-lysine residues on histones, thereby facilitating the recruitment of NuA4 and SWR1 complexes to specific chromatin neighborhoods during DNA repair or transcription (Fig. 2). Future work will determine the relevant biochemical target of the Yaf9 YEATS domain beyond our speculation, and clarify its role in the recruitment of ATP-dependent chromatin remodeling and histone acetyltransferase activities at specific promoters during transcription or at DNA repair sites. Although the YEATS domain may have one specific target, the combination of all reader, writer and eraser domains in a complex, along with other regulators such as transcription factors, probably is responsible for targeting it to specific chromatin neighborhoods. There is still much to learn about the YEATS domain and its function, but the first structure of it in yeast is a good step forward that may encourage future studies of YEATS domains in other proteins and organisms.

Figure 2.

Model highlighting the SWR1-C and NuA4 complexes with their respective “reader” and “writer” subunits containing signature domains that bind to acetyl- or methyl-groups on histones. The SWR1-C subunit Bdf1 has bromodomains that bind to acetylated histones and NuA4 subunits Yng2 and Esa1 have a PhD finger and chromodomain, respectively, that bind to methylated histones.³⁷ Note that the YEATS domain of Yaf9 binding to acetyl groups on histones is speculative in nature and requires further experiments to provide support. Furthermore, it is possible that the subunits bind to the same nucleosome rather than multiple nucleosomes as shown.