The structure of the NuA4 HAT complex comprises two large globular domains joined by multiple connections (). One of these domains is mostly formed by Tra1, the largest NuA4 subunit (433 kDa MW, accounting for ~43% of the mass and ~48% of the volume of the 1 MDa complex) (). Presumably, most of the remaining subunits, including the Piccolo catalytic subcomplex, are localized in the second NuA4 domain. This is confirmed by the results from antibody and gold-cluster labeling of the Epl1 Piccolo subunit (), and it is further supported by a diminished size and large-scale changes in the structure of the non-Tra1 domain in images of the ΔPiccolo NuA4 mutant, which comprises all NuA4 subunits except those found in the Piccolo subcomplex. Incubation of NuA4 with NCPs results in NCP density adjacent to the non-Tra1 NuA4 domain, again indicating that Piccolo is located in that portion of NuA4 (). The structure of the non-Tra1 NuA4 domain indicates that Piccolo and two other NuA4 subunit complexes identified by biochemical analyses, Eaf5–Eaf7–Eaf3 and Eaf2–Yaf9–Act1–Arp4 (), are in close physical proximity10
. It has been reported that deletion of the Eaf1 scaffold protein10
results in a partial NuA4 complex that lacks only Tra1 and Eaf115
, suggesting that Eaf1 must form most the interface between the two globular NuA4 domains. Despite biochemical evidence that Eaf1 provides most of the contacts that keep together the Piccolo, Eaf3–Eaf5–Eaf7, and Eaf2–Yaf9–Act1–Arp4 subcomplexes10,14
, it seems plausible from their relatively compact arrangement in the NuA4 structure that additional contacts among these three subcomplexes could account for their observed association in cell extracts in the absence of Eaf115
, which is not detected in affinity-purified complexes10,14
Our results also provide structural insight into the interaction of a HAT complex with the NCP. The absence of any large cavities in the NuA4 structure and the direct observation of NCP binding to NuA4 () indicate that interaction of NuA4 with the nucleosome occurs at the periphery of NuA4. This is in contrast with what we observed for the RSC chromatin-remodeling complex, which binds the nucleosome in an internal cavity where extensive interactions take place42
. NuA4 binds the nucleosome in a non-enveloping fashion through limited, localized contacts. The nimble interaction between NuA4 and the NCP might explain how NuA4 could operate in a condensed chromatin environment (e.g.
the 30 nm fiber). Relaxation of heterochromatin packing following histone tail acetylation by NuA4 may alleviate steric inhibition and provide a platform for bromodomain interaction, allowing complexes like RSC to bind the nucleosome and carry out physical remodeling of chromatin. Future biochemical studies of RSC activity on condensed and relaxed chromatin templates, and in the absence and presence of histone acetylation, could provide evidence to test this hypothesis.
We used a number of binding assays to further investigate the interaction between NuA4 (or Piccolo) and the NCP. Mobility shift assays showed that, on its own, Esa1 made no or only negligible stable contacts with the NCP, whereas Yng2 and Epl1 made observable binding contacts (). The interaction between Epl1 and the NCP was the strongest, and deletion analysis showed that the conserved N-terminus of Epl1 was mainly responsible for interaction with the NCP. Further deletion analysis showed that Epl1 residues 51–71 are required for the binding interaction between Piccolo and the NCP (). We also obtained evidence that NuA4 association with chromatin in vivo is primarily dependent on the presence of the Piccolo subcomplex (), further supporting the role of Epl1 as the primary chromatin-binding subunit of NuA4. In our model, the chromatin-specific role of Yng2 in stimulating acetylation would be explained by correctly orienting the binding of Epl1–Esa1 to the nucleosome, leading to productive co-localization of the Esa1 catalytic site and H4 or H2A histone tails.
These binding assay results are consistent with our structural observations. EM analysis of a recombinant Piccolo–NCP complex allowed us to determine its 3D structure, which we partially interpreted by docking of an atomic resolution model of the NCP. Piccolo has been reported to bind the H4HFD through the Esa1 HAT subunit39
. However, in order to account for the increased catalytic efficiency of Piccolo over Esa1 alone (an increase of ~2–3 orders of magnitude) the authors of that study propose that the Piccolo subunits Epl1 and Yng2 might also contact the nucleosome. Our EM structure of the Piccolo–NCP complex shows a large Piccolo–NCP interface comprising multiple contacts, primarily on one face of the histone octamer, that includes the H4HFD but also extends to possibly include other histones and DNA. This observation is consistent with the hypothesis that Yng2 and/or Epl1 make additional contacts with the nucleosome. Additionally, we note that the Piccolo–NCP interaction we report occurs in the absence of the Yng2 PHD domain, providing structural evidence supporting the hypothesis that the binding interaction between Piccolo and the NCP does not require this domain.
Further insight into Piccolo subunit organization derived from EM analysis of a number of Piccolo variants is also consistent with the description of Piccolo–NCP contacts we propose. Differences between the structures in projection of different Piccolo deletion variants (Supplementary Figs. 5 through 7
) suggest that at least some portions of Esa1 are localized in the central region of the Piccolo structure, distal to the area of NCP interaction. On the other hand, portions of Epl1 (and perhaps also the Yng2 PHD domain) are in close proximity to the nucleosome-binding region of Piccolo. These structural observations are based on comparison of 2D structures calculated from images of particles preserved in stain (a low resolution technique), and comparison between the projection structures of different Piccolo variants highlights regions where density disappears upon subunit deletion, providing only indirect evidence for localization of specific subunits. Nonetheless, taken together with our other structural and biochemical observations, these results are consistent with a model for Piccolo–NCP interaction where Epl1 plays a primary role in binding the NCP, Yng2 stabilizes this interaction and positions the NCP for optimal catalysis, and Esa1 plays a lesser (though catalytically relevant) role in binding and is primarily involved in catalysis.
Finally, one of the most intriguing observations from this study is an uncanny resemblance between the structures of NuA4 and SAGA that extends well beyond what could be explained by the presence of the common Tra1 subunit. The SAGA subunits (Taf5, Taf6, Taf9, Taf10, and Taf12) that presumably comprise part of the SAGA domain that corresponds structurally to the non-Tra1 portion of NuA433
share no sequence homology with NuA4 subunits. A search for subunit interaction partners shared by NuA4 and SAGA components failed to identify any. Still, NuA4 and SAGA acetylate nucleosomes and often act in concert in the same environment inside the nucleus19
. Furthermore, it has been recently shown that NuA4 and SAGA are both recruited to the phosphorylated C-terminal domain of elongating RNA polymerase II15
. Structural similarities between the two complexes that extend beyond Tra1 may represent convergent evolution of structure highlighting common aspects of two different solutions to a shared set of general molecular functions. Alternatively, it is possible that NuA4 and SAGA may, at some point during their shared roles in DNA transactions, bind to a common interface (perhaps a particular chromatin architecture or another large protein complex) that dictates their structural similarity.