Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans - PubMed (original) (raw)
Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans
Yannick Doyon et al. Mol Cell Biol. 2004 Mar.
Abstract
The NuA4 histone acetyltransferase (HAT) multisubunit complex is responsible for acetylation of histone H4 and H2A N-terminal tails in yeast. Its catalytic component, Esa1, is essential for cell cycle progression, gene-specific regulation and has been implicated in DNA repair. Almost all NuA4 subunits have clear homologues in higher eukaryotes, suggesting that the complex is conserved throughout evolution to metazoans. We demonstrate here that NuA4 complexes are indeed present in human cells. Tip60 and its splice variant Tip60b/PLIP were purified as stable HAT complexes associated with identical polypeptides, with 11 of the 12 proteins being homologs of yeast NuA4 subunits. This indicates a highly conserved subunit composition and the identified human proteins underline the role of NuA4 in the control of mammalian cell proliferation. ING3, a member of the ING family of growth regulators, links NuA4 to p53 function which we confirmed in vivo. Proteins specific to the human NuA4 complexes include ruvB-like helicases and a bromodomain-containing subunit linked to ligand-dependent transcription activation by the thyroid hormone receptor. We also demonstrate that subunits MRG15 and DMAP1 are present in distinct protein complexes harboring histone deacetylase and SWI2-related ATPase activities, respectively. Finally, analogous to yeast, a recombinant trimeric complex formed by Tip60, EPC1, and ING3 is sufficient to reconstitute robust nucleosomal HAT activity in vitro. In conclusion, the NuA4 HAT complex is highly conserved in eukaryotes, in which it plays primary roles in transcription, cellular response to DNA damage, and cell cycle control.
Figures
FIG. 1.
Tip60 and Tip60b are associated with homologs of yeast NuA4 complex subunits. (A) Amino acid sequence alignment between the yeast Esa1, human Tip60, and Tip60b/PLIP histone acetyltransferases. The chromodomain and MYST HAT family homology regions are indicated. (B) Nuclear extracts from mock, FLAG-Tip60, and FLAG-Tip60b-transduced MCF7 cells were immunoprecipitated with α-FLAG resin, eluted with FLAG peptide, and analyzed by Western blotting with antibodies against protein homologous to yeast NuA4 subunits. Native FLAG eluates were also tested in HAT assays on free histones and chromatin substrates. Specific association of actin with Tip60(b) cannot be concluded from this experiment since it is also detected in the control lane. Proteins previously shown to be associated with Tip60 are marked with an asterisk (26). (C) Tip60b is part of a large HAT protein complex. The Tip60b FLAG eluate from panel B was purified over a calibrated Superose 6HR gel filtration column, and fractions were analyzed by Western blotting and HAT assay. Tip60b, EPC1, and H4/H2A HAT activity coelute as a single high-molecular-mass complex of ∼1.8 MDa. Molecular mass standards eluted as follows: void volume, fraction 19; 670 kDa, fraction 28; 158 kDa, fraction 32; 44 kDa, fraction 35; and 17 kDa, fraction 37.
FIG. 2.
Purification of the Tip60-associated proteins identifies the human NuA4 HAT complex. (A) Scheme of the protocol for triple-affinity (using TAP [protein A/calmodulin binding peptide] and FLAG tags) purification of the Tip60-containing complex. (B) Silver-stained gel of mock-treated and triple-affinity-purified material. The Tip60-containing complex was purified from N-terminal FLAG- and C-terminal TAP-tagged Tip60-transduced HeLa cells and then analyzed by silver staining. An extract from nontransduced cells was used as control. Specific bands not present in the control and identified by mass spectrometry and/or Western analysis are labeled on the right. Nonspecific bands are labeled with asterisks. (C) Tip60-associated proteins were identified by tandem mass spectrometry. Tryptic digestion of gel slices from a sample as in panel B were analyzed by tandem mass spectrometry. The proteins identified are listed with the number of different peptide sequences detected for each of them (their lengths in amino acids are indicated in parentheses). Each protein is homologous to a yeast NuA4 subunit, with the exception of Brd8 and RUVBL1/2. MRG15, did not produce peptide hits but was identified by Western analysis. Proteins with asterisks indicate the detection of protein products from splice variants. One mass spectrometry hit was specific for the shorter isoforms of both Brd8 (also known as p120) and EPC1 (also known as EPC2). p400 is named hDomino because of the close homology with mouse and Drosophila Domino proteins (43). Identified human protein FLJ11730 is named hEaf6 because of the homology with the yeast NuA4 subunit Eaf6. The accession number for the EPC1 paralog named EPC-like is NP_056445.
FIG. 3.
SANT domain-containing protein DMAP1 is a stable subunit of the human NuA4 complex. (A) Amino acid sequence alignment between yeast NuA4 subunit Eaf2, human DMAP1, and a Drosophila homologous protein (dEaf2, accession number AAF57436). The conserved SANT domain region is indicated. (B) DMAP1 associates with the same set of proteins as Tip60. Nuclear extracts from FLAG-Tip60-TAP, FLAG-DMAP1-TAP, or mock-transduced HeLa S3 cells were partially purified over IgG-Sepharose beads, and the TEV eluate was analyzed by Western blotting as in Fig. 1B. (C) Triple affinity purification of DMAP1 identifies distinct non-HAT complex(es). The DMAP1-containing complexes were purified as in Fig. 2 and analyzed on a gel by quantitative Sypro Ruby staining. Proteins identified by Western analysis or tandem mass spectrometry are labeled on the right. New proteins not identified in the Tip60 purification are in italics. The positions of MRG15 and Tip60 based on Western analysis of the previous fraction are given in parentheses. The asterisk indicates a nonspecific band. A significant amount of GAS41 protein was also detected but was run out of the gel presented here (see Western signal in panel B). The numbers of specific peptide sequences obtained by tandem mass spectrometry analysis performed as described for Fig. 2 are as follows: 6 for TRRAP, 8 for p400/hDomino, 7 for SRCAP (2,971 aa), 4 for Brd8, 13 for DMAP1, 4 for YL-1 (364 aa), 10 for RUVBL1, 14 for RUVBL2, 5 for BAF53a, and 5 for actin.
FIG. 4.
The chromodomain-containing MRG15 protein is present in both hNuA4 HAT and Sin3A/HDAC complexes. (A) MRG15 copurifies with specific subunits of the hNuA4 complex and nucleosomal H4/H2A HAT activity. Nuclear extracts from 293T cells transiently transfected with MRG15-TAP or mock transfected with pcDNA3-TAP were sequentially purified over IgG-Sepharose and calmodulin beads, analyzed by Western blotting, and nucleosomal HAT assay. Some nonspecific HAT activity was detected in the mock purification of this experiment, presumably due to high-copy transfected plasmid, but specific H4/H2A HAT activity is clearly seen in the tagged MRG15 fraction. (B) MRG15 coimmunoprecipitates with a Sin3A/HDAC complex. Nuclear extracts from 293T cells transiently transfected with FLAG-MRG15 or mock transfected with pcDNA3-FLAG were immunoprecipitated with α-FLAG resin, eluted with FLAG peptides, and analyzed by Western blot. (C) Increasing amounts of the native FLAG eluates from the experiment in panel B (1 and 2 μl) were tested for HAT activity with oligonucleosomes as the substrate. (D) The same eluates were also tested for HDAC activity by using in vivo-labeled purified free core histones. The specificity of the detected activity was verified by incubation with the HDAC inhibitor sodium butyrate (25 mM).
FIG. 5.
The PHD finger protein ING3 implicates human NuA4 in p53-dependent transcription activation and growth control. (A) Diagram of homology regions between yeast Yng2 and human ING3 (>30% identity, 48% similarity). (B) ING3 coimmunoprecipitates with human NuA4 subunits and histone H4/H2A HAT activity. Nuclear extracts from 293T cells transiently transfected with FLAG-ING3 or mock transfected with pcDNA3-FLAG were immunoprecipitated with α-FLAG resin, eluted with FLAG peptides, and analyzed by Western blot and HAT assay. Note that both Tip60 HAT isoforms are detected. (C) Chronic overexpression of ING3 suppresses cell growth. NIH 3T3 cells were transfected with FLAG-ING3 or pcDNA3-FLAG, diluted serially, and selected for 2 weeks with G418. Resistant colonies were stained with Giemsa and analyzed for ING3 expression by Western blot on protein extracts. G418-resistant clones have lost ING3 expression (BAF53a signal is used as internal control). (D) Tip60 stimulates p53-dependent transcription after gamma irradiation. RT-PCR analysis of the endogenous p21/WAF1, MDM2, and GADD45 mRNAs in 293T cells transfected with different combinations of CMV-driven p53, wild-type Tip60 (wt) and Tip60 HAT-dead mutant (mut.) expression plasmids. All cells were gamma irradiated with 15 Gy. p53 and GAPDH mRNA signals are used as internal controls. Protein extracts were made in the same conditions and analyzed by Western blotting with α-p21 and α-p53 antibodies to show the significant increase of p21/WAF1 protein in the presence of wild-type Tip60 but not with the mutant, even though p53 was highly expressed (lower panel).
FIG. 6.
Conservation of the core nucleosomal acetyltransferase trimeric complex, Piccolo NuA4, from yeast to humans. (A) Coexpression of EPC1 and ING3 with His-tagged Tip60 and Tip60b in bacteria enable their HAT activity on chromatin substrates. Tip60(b) were affinity purified from bacteria expressing each protein alone or in conjunction with EPC1 conserved N-terminal domain [EPC1(1-400)] and ING3 with or without its C-terminal PHD finger [ING3(1-300)]. Relative nucleosomal HAT activity of affinity-purified HIS-tagged Tip60 and Tip60b were measured by liquid scintillation counting of HAT assays on oligonucleosomes while the amount of Tip60(b) was kept constant (evaluated by Western blot). Note that Tip60 and Tip60b are not able to acetylate chromatin by themselves and that ING3 PHD finger is not required for nucleosomal HAT activity. Average counts per minute (cpm) from four different HAT assays are shown (ctl, 276 ± 74; Tip60, 425 ± 162; Tip60b, 273 ± 25; Tip60/EPC1(1-400)/ING3, 4,872 ± 271; Tip60b/EPC1(1-400)/ING3, 9,714 ± 531; Tip60/EPC1(1-400)/ING3(1-300), 7,876 ± 873; Tip60b/EPC1(1-400)/ING3(1-300), 11,522 ± 1,417). (B) Tip60, EPC1 and ING3 form a stable trimeric complex when coexpressed in bacteria. Protein extract from bacteria coexpressing EPC1(1-400) with a C-terminal His6 tag, ING3 (1-300) with a C-terminal Strep-II tag and untagged full-length Tip60b was successively affinity purified on Talon cobalt and Strep-Tactin resins and analyzed on gel after Coomassie blue staining. The presence of all three proteins was confirmed by Western blot and HAT activity (not shown).
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