Histone acetyltransferase (HAT) enzyme complexes are key regulators of transcriptional control in all eukaryotic cells and have been linked to a diverse range of biological processes (reviewed in reference 61
). HAT acetylation of specific lysines of N-terminal histone tails is believed to relieve DNA-protein interactions as well as to serve as a molecular beacon to attract additional chromatin remodeling or modifying proteins and/or transcription factors (reviewed in reference 34
). In addition, the cellular processes regulated by HATs may not be governed solely by histone acetylation but, alternatively, may be mediated through the acetylation of nonhistone targets (reviewed in reference 23
). To fully grasp the cellular implications of HATs will require both a comprehensive understanding of their diverse biological roles and a detailed understanding of the proteins within the complexes.
In Saccharomyces cerevisiae
, the only essential HAT is the NuA4 complex. The known acetylation targets of NuA4 in vivo are histone H4 (1
) and the histone H2A variant Htz1 (2
). Similar to other HATs, NuA4 has been implicated in numerous nuclear events, including regulation of gene expression (reviewed in reference 18
), DNA repair (reviewed in references 18
), cell cycle progression (12
), and chromosome stability (31
). Though the various cellular roles of NuA4 are likely mediated through H4 or Htz1 acetylation, the molecular mechanisms by which histone acetylation achieves diverse cellular functions is largely unknown. As the Tip60 complex, the human homolog of the yeast NuA4 complex, has numerous nonhistone targets (51
), it is possible that some of the cellular functions of the yeast complex may be fulfilled through the acetylation of nonhistone substrates. Further, it is unlikely that all of the NuA4-mediated cellular functions have yet been defined. Indeed, NuA4 homologs in both humans and Caenorhabditis elegans
have been implicated in a much broader range of cellular functions, including cytoplasmic roles in cell signaling (35
), suggesting that there may be roles for the yeast complex outside the nucleus.
NuA4 is composed of 13 subunits, including the essential acetyltransferase subunit Esa1 (13
, 58; reviewed in reference 18
). Of the other 12 NuA4 subunits, 5 are essential for cell viability (Act1, Arp4, Epl1, Swc4, and Tra1) and the remaining 7 are not (Eaf1, Eaf3, Eaf5, Eaf6, Eaf7, Yaf9, and Yng2) (reviewed in reference 18
). Despite being the catalytic subunit, Esa1 on its own can target only free histones and is incapable of acetylating nucleosomes or chromatin (1
). As with other HAT enzyme complexes, the ability of Esa1 to target nucleosomes is dependent on complex formation. Esa1 has been isolated in two distinct complexes: the complete 13-subunit NuA4 complex and a trimer subcomplex composed of Esa1, Yng2, and Epl1 called Piccolo NuA4 (7
). Studies suggest that Piccolo NuA4 cannot be recruited by transcription factors to specific locations, leading to the hypothesis that Piccolo NuA4 is responsible for nontargeted global histone acetylation, whereas the full NuA4 complex is responsible for targeted NuA4-dependent histone acetylation (7
Esa1 and Yng2 interact independently with the N-terminal portion of Epl1, and it has been demonstrated that the N-terminal Enhancer of Polycomb A (EPcA) region of Epl1 and the N-terminal region of Yng2 are required for Esa1 to recognize and acetylate nucleosomes (7
). Though it is known that the C-terminal half of Epl1 is required to bridge Esa1 and Yng2 with the remaining 10 NuA4 subunits (7
), very little is known regarding the physical interactions between the remaining NuA4 subunits. Studies have found that Yaf9 interacts with NuA4 through Swc4 in vivo (5
), and both Eaf3 and Arp4 interact directly with Esa1 in vitro (21
). As seen in other chromatin modification complexes, such as the SWR1 complex (67
) and SWI/SNF (68
), it is likely that the non-Piccolo NuA4 subunits may form multiple and distinct subcomplexes that perform a subset of the diverse NuA4 cellular functions.
The functional role of each individual NuA4 subunit remains to be fully understood, particularly with respect to the 10 non-Piccolo NuA4 subunits. One possibility is that some NuA4 subunits may be required for complex integrity, acting as a scaffold upon which other subunits bind. Alternatively, individual subunits or subcomplexes may be required for the targeted recruitment of NuA4 to distinct chromatin loci. Indeed, targeting roles have been characterized for some subunits. For example, upon DNA damage, Arp4 recognizes and interacts with histone H2A phosphorylated at serine 129. This action recruits NuA4 to regions of DNA double-strand breaks where histone H4 acetylation is required for DNA double-strand break repair (4
). Similarly, Tra1 directly interacts with the acidic transcriptional activators Gcn4, Hap4, and Gal4 (8
). Further, Tra1 is required for both the acetylation of H4 surrounding the promoters and the transcription of Gcn4-dependent genes, suggesting that Tra1 may mediate the recruitment of NuA4 to certain promoters.
While specific targeting roles for the other subunits are undefined, the different phenotypes of the nonessential NuA4 subunits strongly support the hypothesis that different subunits are required for distinct NuA4 functions. For instance, while yaf9
Δ, and eaf1
Δ cells display hypersensitivity to the microtubule-destabilizing drug benomyl, eaf5
Δ and eaf7
Δ cells display no sensitivity to this drug (30
). Similarly, while eaf1
Δ and yaf9
Δ cells display high rates of chromosome loss (41 and 23 times greater than that of wild-type [WT] cells, respectively), eaf3
Δ, and eaf7
Δ cells display either no or only modest increases in their rates of chromosome loss compared to WT cells (31
; L. Mitchell and K. Baetz, unpublished data).
Defining the roles of the individual subunits will provide critical insight into the NuA4 enzyme complex as a whole. In S. cerevisiae
, a genetic method for exploring gene function is through the identification of synthetically lethal (SL) or synthetically sick (SS) genetic interactions by double mutant analysis. Mutants that are defective in the same essential or parallel nonessential pathways often display SL or SS interactions. The development of synthetic genetic array (SGA) analysis has enabled genetic screens to be performed systematically with yeast and has proven to be a powerful tool for predicting the cellular functions of a protein (63
). Recently, subunits of NuA4 were analyzed in an epistatic miniarray profile (E-MAP) screen that identified pairwise genetic interaction between 743 genes implicated in various aspects of chromosome biology (14
). This study solidified the role of NuA4 in its previously characterized functions, such as chromosome stability, DNA repair, transcription, and chromatin remodeling. However, given that the study was limited to genes involved in chromosome biology, no insights were gained into possible novel cellular functions of NuA4.
To comprehensively explore the global cellular functions of NuA4, we performed genome-wide SL-SGA analysis for five nonessential subunits. Our genetic interaction map reveals over 200 genetic interactions for the NuA4 subunits tested, dramatically expanding our knowledge of the potential cellular functions of the complex. Using the genetic interaction map, we identify a role for NuA4 in Golgi complex-to-vacuole vesicle-mediated transport. In addition, during our complex integrity studies, we discovered that NuA4 physically interacts with the stress response transcription factor Msn4. We also examine the effect of each nonessential subunit on NuA4 complex integrity and discover that the Eaf1 subunit, whose deletion is responsible for a large portion of the genetic interactions in our map, is essential for maintaining the complex integrity of NuA4. Moreover, Eaf5 and Eaf7, which display similar genetic interaction profiles and phenotypes, are found in a subcomplex. This integrative study provides novel insights into the pathways and processes impacted by NuA4 and sheds light on the roles of subunits within the complex.