Collectively, the data presented here using three different human cell types clearly demonstrate the presence of NuA4 HAT complexes in higher eukaryotes. The subunit identifications indicate a strikingly high level of structural conservation, i.e., 11 of 12 subunits conserved from yeast to humans. We also showed that chromatin modification by the complex functions through a highly conserved enzymatic core complex, i.e., Piccolo NuA4. Finally, we presented evidence supporting a role for the human NuA4 complex in the control of cell proliferation in part by cooperation with p53 in transcription regulation of cell cycle control genes. Interestingly, the previously proposed role of Tip60 in gamma irradiation induced DNA damage response is also conserved since Esa1 is required for DNA double-strand break repair in yeast (6
Human NuA4 subunit identifications highlighted multiple protein domains that have been linked to chromatin function, all encompassed in the same multiprotein complex. Besides the MYST HAT domain of Tip60, hNuA4 contains chromodomains that have been proposed to be methylated-histone or RNA-binding modules, bromodomains that can be acetylated-histone binding modules (reviewed in reference 17
), SANT domains that have been proposed to bind histone tails (8
), an ATPase domain of the SWI2 family of chromatin remodelers (58
), an actin-related globular domain that may have histone chaperone activity (20
), a PHD finger domain which is commonly found in chromatin modifying complexes and a phosphatidylinositol 3-kinase domain related to ATM/ATR, which phosphorylate histone H2A.X in response to DNA damage (46
). The presence of the essential GAS41 protein, a member of the AF9/ENL-related (YEATS) family, also confirmed the exclusive association of these proteins to transcription/chromatin-modifying complexes, including yeast NuA4, NuA3, Sas2, SWI/SNF, TFIID/mediator/TFIIF, and human SWI/SNF complexes (40
). We showed that the Enhancer of Polycomb homology domain of human EPC1, like Epl1 in yeast (7
), is a conserved functional key for histone acetylation since it bridges the MYST HAT with the ING protein to enable potent nucleosome histone acetyltransferase activity (Fig. ). Interestingly, a splice variant and a distinct protein highly related to EPC1 were also found in the human NuA4 complexes, suggesting the possibility of functionally different complexes within the same cell or specific to cell types, development stages.
Identification of a bromodomain-containing subunit in human NuA4 distinguished it from its yeast counterpart. Brd8 links NuA4 to ligand-dependent transcription regulation by the thyroid hormone receptor (35
) and could be implicated in local retention on chromatin after initial recruitment of NuA4 (24
). Interestingly, Brd8 is related to yeast Bdf1, which has been shown to functionally interact with Esa1 and preferentially bind acetylated histone H4 (32
). Genetic interactions have also been detected between yeast NuA4 and other bromodomain-containing proteins (N. Bouchard and J. Côté, unpublished). Another protein that initially appeared specific to the human complex is p400/hDomino. We propose that in fact yeast NuA4 subunit Eaf1 is the functional homolog of p400/hDomino since they share four regions of homology, including SANT and HSA domains (A. Auger, D. Cronier, L. Galarneau, A. Nourani, R. T. Utley, and J. Côté, unpublished data). However, Eaf1 lacks the SWI2-related ATPase domain of p400/hDomino. Interestingly, the remaining proteins specific to the human NuA4 complex are the ruvB-like helicases (RUVBL1/2), which are likely implicated in the DNA repair function of the complex. We speculate that their absence in the yeast complex is explained by the lack of the SWI2 domain in Eaf1 since they were shown to depend on this domain for interaction with p400/hDomino (19
). In fact, we recently characterized a separate non-HAT yeast complex that shares four subunits with NuA4 and contains a SWI2-related protein, Swr1, and ruvB-like helicases, Rvb1/2 (A. Auger, D. Cronier, L. Galarneau, A. Nourani, R. T. Utley, and J. Côté, unpublished data). This raises the possibility that the human NuA4 complex is functionally equivalent to a fusion of two distinct complexes in yeast, one harboring HAT activity and the other involved in ATP-dependent chromatin remodeling.
Our finding of DMAP1 in human NuA4 implicates the complex in DNA replication since this protein has been found associated to the major DNA methyltransferase at replication foci (48
). On the other hand, we did not detect DNMT1 signals in our NuA4 purification (data not shown). Thus, the interaction could be transient or DMAP1 may have other roles outside of NuA4. Indeed, we found that DMAP1 is also present in one or more distinct protein complexes (Fig. ). The complex or complexes lack HAT activity but contain ruvB-like helicases and SWI2-related ATPase subunits. p400/hDomino was previously shown to be part of a non-HAT complex containing other proteins that are also found with Tip60 (19
). We now show that one of these proteins is DMAP1. Another SWI2-related ATPase was also identified in DMAP1 purification, SRCAP. This large protein is related to p400/hDomino over the SWI2 domain but lacks the SANT and HSA domains. It was shown to be involved in transcription regulation by CBP (27
The presence of MRG15 in human NuA4 confirms its importance in the control of cell proliferation. Mortality factor-4 is a truncated version of MRG15 lacking the N-terminal chromodomain that induces senescence in a number of cell lines (5
). We show that, like homologs in lower eukaryotes (14
), MRG15 is present in both NuA4 HAT and Sin3/HDAC complexes. This certainly reflects its key role in the interaction of these complexes with chromatin, most likely through the chromodomain. Implication of human NuA4 in the control of cell division was recently highlighted by a study showing recruitment of Tip60 and p400/hDomino to promoter regions by Myc in vivo (18
). Our identification of candidate tumor suppressor ING3 further supports an important role for NuA4 in cell cycle control. Furthermore, ING3 also links hNuA4 to p53 function in transcription and apoptosis (15
). Accordingly, we show that Tip60 can affect p53-dependent transcription in vivo, a result reminiscent of our study with the yeast complex (41
). It will be interesting to continue the structural and functional characterization of human NuA4 complexes since they are poised to play essential roles in such diverse nuclear functions as gene regulation, DNA repair, and cell cycle control.