Eaf1 is the platform for NuA4 molecular assembly that evolutionarily links chromatin acetylation to ATP-dependent exchange of histone H2A variants

Abstract

Eaf1 (for Esa1-associated factor 1) and Eaf2 have been identified as stable subunits of NuA4, a yeast histone H4/H2A acetyltransferase complex implicated in gene regulation and DNA repair. While both SWI3-ADA2-N-CoR-TF IIIB domain-containing proteins are required for normal cell cycle progression, their depletion does not affect the global Esa1-dependent acetylation of histones. In contrast to all other subunits, Eaf1 is found exclusively associated with the NuA4 complex in vivo. It serves as a platform that coordinates the assembly of functional groups of subunits into the native NuA4 complex. Eaf1 shows structural similarities with human p400/Domino, a subunit of the NuA4-related TIP60 complex. On the other hand, p400 also possesses an SWI2/SNF2 family ATPase domain that is absent from the yeast NuA4 complex. This domain is highly related to the yeast Swr1 protein, which is responsible for the incorporation of histone variant H2AZ in chromatin. Since all of the components of the TIP60 complex are homologous to SWR1 or NuA4 subunits, we proposed that the human complex corresponds to a physical merge of two yeast complexes. p400 function in TIP60 then would be accomplished in yeast by cooperation between SWR1 and NuA4. In agreement with such a model, NuA4 and SWR1 mutants show strong genetic interactions, NuA4 affects histone H2AZ incorporation/acetylation in vivo, and both preset the PHO5 promoter for activation. Interestingly, the expression of a chimeric Eaf1-Swr1 protein recreates a single human-like complex in yeast cells. Our results identified the key central subunit for the structure and functions of the NuA4 histone acetyltransferase complex and functionally linked this activity with the histone variant H2AZ from yeast to human cells.

FIG. 1.

Eaf1 is found exclusively in the NuA4 HAT complex and is required for its functions but not for global histone H4/H2A acetylation. (A) Eaf1 coelutes with the HAT activity of NuA4 as well as with subunits of the NuA4 HAT complex. An extract from Δeaf1 cells harboring an episomal HA-EAF1 gene was fractionated on nickel and Mono Q columns. The desired fractions (fns) were pooled and loaded on a Superose-6 gel filtration column. Western blots show Arp4, Tra1, and Esa1 coelution with NuA4 HAT activity (fractions 19 to 23). α, anti. (B) Eaf1 is specific to the NuA4 complex. The TAP of Eaf1 shows the association with NuA4 subunits. Purified material from untagged and EAF1-TAP strains was loaded onto gradient gels and visualized by silver staining. Bands corresponding to the NuA4 subunits are indicated on the right. The asterisk corresponds to a nonspecific band known to purify with TAP-tagged protein. (C) Schematic representation of Eaf1. Eaf1 contains an HSA domain,a charged domain (gray) partially overlapping the HSA domain, and a SANT domain at the positions indicated. The C terminus of Eaf1 is enriched in glutamine (Q) (black). (D) Full-length Eaf1 is required for growth in the presence of DNA damage or crippled ribosome biogenesis. Serial 10-fold dilutions of Δeaf1, eaf1ΔN, eaf1ΔSANT, and isogenic wild-type (WT) strains were grown in the presence or absence of 0.03% MMS or 25 nM rapamycin. (E) Deletion of Eaf1 results in a slow G₂/M passage. Liquid cultures of wild-type and Δeaf1 cells were blocked in G₁ by the addition of α-factor. DNA content was quantified by fluorescent-activated cell sorter analysis. The 1n peak represents cells in the G₁/S stage, whereas the 2n peak represents cells in the G₂/M stage. (F) Deletion of Eaf1 does not affect global acetylation by NuA4. Nucleosomal histones from isogenic wild-type and Δeaf1 strains were purified and analyzed by Western blotting using antibodies indicated on the left (the top panel shows Coomassie-stained histones). (G) Eaf1 is implicated in gene-specific regulation. RNAs from isogenic wild-type, Δeaf1, eaf1ΔSANT, eaf1ΔN, and EAF1 cells were isolated, and Northern blot analyses were performed using the probes indicated on the left.

FIG. 2.

Eaf2, another SANT-containing subunit, is essential for cell viability and affects a subset of NuA4 functions. (A) Schematic representation of Eaf2 (Swc4) and human DMAP1. (B) The SANT-containing N-terminal portion of Eaf2 is essential for viability, and human DMAP1 does not complement eaf2 mutants in yeast. Strains in which the EAF2 gene has been deleted but that express an episomal version of EAF2, the truncated EAF2(1-285) or EAF2(285-476), DMAP1, or an empty vector were streaked on solid YPD medium. (C) Deletion of the C-terminal portion of Eaf2 causes sensitivity to MMS but not to rapamycin. Serial 10-fold dilutions of the indicated strains were incubated on solid YPD medium containing either 0.03% MMS or 25 nM rapamycin. (D) Episomal full-length and truncated Eaf2 are expressed at similar levels in vivo. Whole-cell extracts (WCE) from the indicated strains were analyzed by Western blotting with anti-HA (α HA). (E) Eaf2 is essential for cell cycle progression. The depletion of Eaf2 leads to cells being blocked at G₂/M. Cells in which the endogenous EAF2 promoter has been replaced by the inducible GAL1 promoter were incubated in liquid medium in the presence or absence of galactose (GAL), and the DNA content was analyzed by flow cytometry. (F) Eaf2 is not essential for the global acetylation of chromatin by NuA4. Cells harboring EAF2 under the control of the GAL1 promoter as well as wild-type (WT) cells were incubated in medium containing galactose or glucose for 12 h at 30°C. Western blot analyses were performed with histones purified from those strains using the indicated antibodies.

FIG. 3.

Eaf1 serves as a platform for the assembly of four different functional modules into the NuA4 complex. (A) Epl1 interacts with Eaf1 and bridges picNuA4 with the remaining complex, while the association with an Eaf5/7/3 trimer also is dependent on Eaf1. Purified material from untagged strains and the indicated TAP-tagged strains was loaded onto gradient gels and visualized by silver staining. The asterisk corresponds to a nonspecific band known to purify with TAP-tagged proteins. (B) Western blots using the final elution of the TAP purifications shown in panel A were probed with antibodies α against the indicated proteins. (C) The HSA domain region of Eaf1 interacts with the subunits shared with the SWR1/INO80 ATP-dependent chromatin remodeling complexes. Purified material from the indicated TAP-tagged strains supplemented with an episomal version of either EAF1, Eaf1ΔHAS, or Eaf1ΔSANT was loaded onto a gradient gel and analyzed by Western blotting using the antibodies indicated on the right. In lane 7, there is no Esa1 signal but rather a nonspecific keratin band (asterisk). (D) Tra1 requires the SANT domain region of Eaf1 for its association with NuA4. Purified material from strains indicated in the legend to panel C was loaded onto low-percentage gels and visualized by silver staining. The 400-kDa band visible on this part of the gel represents Tra1. (E) Requirement of the HSA and SANT regions of Eaf1 for proper growth. Tenfold dilutions of mid-log-phase Δeaf1, EAF1, Eaf1ΔHSA, and Eaf1ΔSANT yeast cultures were grown in the presence or absence of 0.03% MMS or 25 nM rapamycin. (F) Expression levels of episomal wild-type (WT) and mutant Eaf1 are similar to that of endogenous Eaf1. Whole-cell extracts (WCE) from the strains used for panel E were analyzed by Western blotting with anti-Eaf1. (G) Schematic representation of Eaf1 as the platform for the assembly of the different functional modules of NuA4 and the subunits shared with other chromatin modifying/remodeling complexes.

FIG. 4.

Structural and functional relationships between NuA4 and SWR1 complexes. (A) The SWR1 complex shares subunits with NuA4. Purified material from untagged strains and Swr1-TAP strains was loaded onto gradient gels and visualized by silver staining. Bands corresponding to the SWR1 subunits are indicated on the right. (B) Yaf9 is a subunit common to the NuA4 and Swr1 complexes. Shown is a Western blot analysis using the final elution of YAF9-TAP and EPL1-TAP purifications using the indicated antibodies (α). (C) Yaf9 requires the C-terminal portion of Eaf2 for its association with NuA4 or SWR1. Purified material from untagged strains and EAF2-TAP and Eaf2(1-285)-TAP strains was loaded onto gradient gels and analyzed by Western blotting using the indicated antibodies. (D) The Δyaf9 and Δeaf1 mutants show a defect in telomere silencing. Wild-type (WT), Δyaf9, and Δeaf1 strains were plated on 5-FOA to verify the expression of the URA3 marker positioned in a silent telomeric region. The absence of growth on 5-FOA indicates URA3 marker expression. (E) Deletion of EAF1 cripples H4 acetylation (AcH4) at the PHO5 promoter, while SWR1 deletion has no effect. Shown is a ChIP analysis with anti-H3 (C terminus) and anti-H4AcK8 in wild-type, Δeaf1, and Δswr1 strains. Precipitated DNA was analyzed by PCR with primers corresponding to chromosomal regions at the PHO5 promoter (UAS2 region). Data are presented as an IP ratio of the amount of AcK8 H4 on total H3 (to correct for a change in nucleosome occupancy) relative to that of the wild-type strain. The values are based on two independent experiments. (F) Incorporation of Htz1 at the PHO5 promoter depends on SWR1 but also is affected by Eaf1. ChIP analysis was performed as described for panel E with anti-H3 and anti-Htz1 in wild-type, Δeaf1, Δyaf9, and Δswr1 strains. (G) SWR1/Htz1 do not influence H4 acetylation at the PHO5 promoter. ChIP analysis was done as described for panel E with wild-type, Δhtz1, and Δyaf9 strains. (H) NuA4 and SWR1 mutants show common and distinct phenotypes. Serial 10-fold dilutions of Δeaf1, Δswr1, Δarp8 (specific to the INO80 complex), and wild-type mid-log-phase cultures were grown in the presence or absence of 0.03% MMS, 25 nM rapamycin, or 130 mM hydroxyurea (HU). (I) swr1/htz1 mutants show synthetic lethality with mutant cells lacking normal NuA4 complex but containing picNuA4 global HAT activity. Wild-type, Δswr1, epl1(1-380), and epl1(1-380)Δswr1 cells were plated onto solid medium containing 5-FOA (to lose an episomal copy of wild-type EPL1 on a URA3 vector). (J) Serial 10-fold dilutions of wild-type, epl1(1-380), and epl1(1-380)Δhtz1 strains carrying an episomal copy of wild-type EPL1 on a URA3 vector were plated onto YPD with or without 5-FOA.

FIG. 5.

Fusion of Eaf1 and Swr1 proteins reconstitutes a human TIP60-like complex in yeast cells. (A) Construction scheme of yDomino. The SWI2 domain of Swr1 was introduced by restriction endonucleases into Eaf1 to produce a yeast version of human p400/Domino (p400/hDomino). (B) Synthetic construct of a yeast Domino-like protein is expressed at levels similar to that of Eaf1 in vivo. The Western blot analysis of whole-cell extracts (WCE) from strains carrying the indicated expression vectors using anti-Eaf1 is shown. (C) The Eaf1-Swr1 fusion protein suppresses the MMS and rapamycin sensitivity of eaf1 mutant cells. Serial 10-fold dilutions of mid-log-phase Δeaf1, Δeaf1+Eaf1, and Δeaf1+yDomino cultures were spotted on medium containing 0.03% MMS or 25 nM rapamycin. (D) Reconstitution of the human NuA4/TIP60-like complex in yeast. TAP-purified material from strains EPL1-TAPΔeaf1 and EPL1-TAPΔeaf1 expressing Eaf1 or yDomino from a vector was visualized on a gel after silver staining. The asterisk corresponds to nonspecific bands known to purify with TAP-tagged protein. (E) Rvb1/2 helicases associate with NuA4 harboring the yDomino fusion protein. Shown is a Western blot analysis of purified material described for panel C using the indicated antibodies (α), which reveals normal NuA4 assembly in the presence of yDomino and the association of helicases Rvb1/2 with the complex. (F) yDomino-containing complex increases the amount of Htz1 at the PHO5 promoter in the absence of Swr1. ChIP analysis was performed as described in the legend to Fig. 4E with anti-H3 and anti-Htz1 in wild-type (WT), Δswr1, and Δswr1+yDomino strains. Data are presented as IP ratios of the amount of Htz1 on total H3 relative to that of the wild-type strain. (G) yDomino-containing complex allows the acetylation of Htz1 (AcHtz1) at the PHO5 promoter in the absence of Eaf1. ChIP analysis was performed as described in the legend to Fig. 4E with anti-AcK14 Htz1 and anti-Htz1 (C terminus) in wild-type, Δeaf1, and Δeaf1+yDomino strains. Data are presented as IP ratios of the amount of AcK14 Htz1 on total Htz1 (to correct for the effect of Eaf1 on Htz1 incorporation [Fig. 4F]) relative to that of the wild-type strain.

FIG. 6.

Model for the evolution of NuA4 HAT and SWR1 ATP-dependent remodeling complexes from yeast to human cells. The yeast NuA4 and SWR1 complexes are depicted. SWR1 could be present in two forms, with or without a bromodomain-containing Bdf1 subunit. These distinct complexes regulating histone H2AZ incorporation in chromatin would differentially reflect the link to NuA4-dependent chromatin acetylation. In human cells, SWR1 plus a Bdf1 equivalent is physically merged with NuA4 into the TIP60/hNuA4 complex (through the fusion of yeast Eaf1 and Swr1 platform proteins into human p400/Domino). The yeast SWR1 lacking Bdf1 corresponds to the human SRCAP complex, which should not be linked to chromatin acetylation.