MOLECULAR AND CELLULAR BIOLOGY, Apr. 2008, p. 2244–2256
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 7
Functional Dissection of the NuA4 Histone Acetyltransferase Reveals
Its Role as a Genetic Hub and that Eaf1 Is Essential for
Leslie Mitchell,1,2Jean-Philippe Lambert,1,2Maria Gerdes,1,2Ashraf S. Al-Madhoun,2
Ilona S. Skerjanc,2Daniel Figeys,1,2and Kristin Baetz1,2*
Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada,1and Department of Biochemistry,
Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada2
Received 6 September 2007/Returned for modification 22 October 2007/Accepted 8 January 2008
The Saccharomyces cerevisiae NuA4 histone acetyltransferase complex catalyzes the acetylation of histone H4
and the histone variant Htz1 to regulate key cellular events, including transcription, DNA repair, and faithful
chromosome segregation. To further investigate the cellular processes impacted by NuA4, we exploited the
nonessential subunits of the complex to build an extensive NuA4 genetic-interaction network map. The map
reveals that NuA4 is a genetic hub whose function buffers a diverse range of cellular processes, many not
previously linked to the complex, including Golgi complex-to-vacuole vesicle-mediated transport. Further, we
probe the role that nonessential subunits play in NuA4 complex integrity. We find that most nonessential
subunits have little impact on NuA4 complex integrity and display between 12 and 42 genetic interactions. In
contrast, the deletion of EAF1 causes the collapse of the NuA4 complex and displays 148 genetic interactions.
Our study indicates that Eaf1 plays a crucial function in NuA4 complex integrity. Further, we determine that
Eaf5 and Eaf7 form a subcomplex, which reflects their similar genetic interaction profiles and phenotypes. Our
integrative study demonstrates that genetic interaction maps are valuable in dissecting complex structure and
provides insight into why the human NuA4 complex, Tip60, has been associated with a diverse range of
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 medi-
ated 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 di-
verse biological roles and a detailed understanding of the pro-
teins 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, 20, 58) and the histone H2A variant Htz1 (2,
28, 42). Similar to other HATs, NuA4 has been implicated in
numerous nuclear events, including regulation of gene expres-
sion (reviewed in reference 18), DNA repair (reviewed in ref-
erences 18 and 59), cell cycle progression (12, 13), and chro-
mosome 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, 51), 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, 7). As with other
HAT enzyme complexes, the ability of Esa1 to target nucleo-
somes 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, 55).
* Corresponding author. Mailing address: Ottawa Institute of Sys-
tems Biology, Department of Biochemistry, Microbiology and Immu-
nology, University of Ottawa, Roger Guidon Hall, 451 Smyth Rd.,
Ottawa, ONT K1H 8M5, Canada. Phone: (613) 562-5800, ext. 8592.
Fax: (613) 562-5840. E-mail: email@example.com.
?Published ahead of print on 22 January 2008.
at Univ of Ottawa on March 15, 2008
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 rec-
ognize and acetylate nucleosomes (7, 55). 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 remain-
ing 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 re-
mains 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, individ-
ual subunits or subcomplexes may be required for the targeted
recruitment of NuA4 to distinct chromatin loci. Indeed, tar-
geting 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, 17). Similarly, Tra1 directly interacts with the
acidic transcriptional activators Gcn4, Hap4, and Gal4 (8).
Further, Tra1 is required for both the acetylation of H4 sur-
rounding the promoters and the transcription of Gcn4-depen-
dent 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?, yng2?, and eaf1? cells display hypersensitivity to the
microtubule-destabilizing drug benomyl, eaf5? and eaf7? cells
display no sensitivity to this drug (30, 31, 36). Similarly, while
eaf1? and yaf9? cells display high rates of chromosome loss (41
and 23 times greater than that of wild-type [WT] cells, respec-
tively), eaf3?, eaf5?, eaf6?, and eaf7? cells display either no or
only modest increases in their rates of chromosome loss com-
pared to WT cells (31; L. Mitchell and K. Baetz, unpublished
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 syn-
thetically sick (SS) genetic interactions by double mutant anal-
ysis. 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 chromo-
some biology, no insights were gained into possible novel cel-
lular 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 ves-
icle-mediated transport. In addition, during our complex in-
tegrity 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 com-
plex integrity and discover that the Eaf1 subunit, whose dele-
tion is responsible for a large portion of the genetic interac-
tions 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.
MATERIALS AND METHODS
Yeast strains and plasmids. Yeast strains used in this study are listed in Table
1. The MATa deletion mutant array was purchased from Open Biosystems
(catalog no. YSC1053). The SGA starting strain Y7092 (62) and the media used
in the SL-SGA analysis have been described previously (63, 64). Genomic dele-
tion or epitope tag integrations made for this study were designed with PCR-
amplified cassettes as previously described (38, 47) and confirmed by PCR
analysis. Plasmid pGST-MSN4 (pKB1) was generated by PCR amplification of
MSN4 from genomic DNA using the primers 5?-ATCGGGATCCATGCTAGT
CTTCGGACCTAATAG (forward) and 5?-GCATGCTCGAGTCAAAAATCA
CCGTGCTTTTTG (reverse). The resulting PCR product was digested with
BamHI and XhoI and ligated into pGEX6p-2 (Amersham Bioscience) also
digested with BamHI and XhoI.
SL-SGA screens. Robotic manipulation of the deletion mutant array was
conducted using a Singer RoToR HDA (Singer Instruments), and SL screens
were performed as described previously (63). Genome-wide SL screens were
conducted three times at 30°C for the following query strains: the eaf1?, eaf3?,
eaf5?, eaf6?, and eaf7? strains. The resultant double mutants were scored for SL
or SS interactions by visual inspection. For the eaf1? screen, putative genetic
interactions, identified in a minimum of two out of three screens, were confirmed
by tetrad dissection on yeast-peptone-dextrose (YPD) medium at 25°C. For the
eaf3?, eaf5?, eaf6?, and eaf7? screens, only those putative interactions identified
in a minimum of two out of three screens that had not been previously published
(as listed at www.thebiogrid.org as of April 2007) were confirmed by tetrad
dissection, as described above. Any published interactions in the BioGrid data-
base that were not identified in our screens were incorporated into our data set.
The complete list of interactions and references are provided at www.oisb.ca
NuA4 PrA-tagged protein purification and identification. One-step affinity
purification of protein A (PrA; one epitope of the tandem affinity purification
[TAP] tag)-tagged NuA4 components was performed. Cells from 200 ml of
mid-log-phase culture (optical density at 600 nm [OD600], ?0.6 to 0.8) grown in
YPD at 25°C were collected by centrifugation, washed in 10 ml lysis buffer (20
mM HEPES, pH 7.4, 0.1% Tween 20, 2 mM MgCl2, 300 mM NaCl, protease
inhibitor cocktail [P-8215; Sigma]) and transferred to a 1.5-ml Eppendorf tube.
Cells were resuspended in 300 ?l lysis buffer plus an equal volume of acid-
washed glass beads (catalog no. 35-535; Fisher Scientific), and cells were lysed
through vortexing (six 1-min blasts with incubation on ice in between vortexing).
The soluble whole-cell extract (WCE) was isolated by centrifugation at 13,200
rpm for 20 min. Ten milligrams of the WCE was incubated with 25 ?l of magnetic
VOL. 28, 2008GENOMIC AND STRUCTURAL DISSECTION OF NuA42245
at Univ of Ottawa on March 15, 2008
Dynabeads (catalog no. 143-01; Dynal, Invitrogen) crossed-linked to rabbit im-
munoglobulin G (IgG) (catalog no. PP64; Chemicon) as per Invitrogen’s instruc-
tions. Following 2 hours of end-over-end incubation at 4°C, Dynabeads were
collected with a magnet and washed five times with 1 ml cold lysis buffer. The
Dynabeads were resuspended in 25 ?l of modified 1? loading buffer (50 mM
Tris, pH 6.8, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10%
glycerol), and PrA-tagged proteins and copurifying proteins were eluted from the
beads with moderate heat (65°C for 10 min). Loading buffer was transferred to
a new tube, and 2-?-mercaptoethanol was added to a final concentration of 200
mM. Samples were boiled for 5 min, and 20 ?l was resolved on 4 to 12%
polyacrylamide gradient gels (catalog no. NP0321BOX; Invitrogen) in 1? MES
(morpholineethanesulfonic acid) buffer. Proteins were visualized by silver
Immunoprecipitation and immunoblotting. PrA purification of Msn4-TAP
was performed as described above except 15 mg of WCE was incubated with the
IgG-coated Dynabeads overnight. Immunoprecipitations were separated by
7.5% SDS-polyacrylamide gel electrophoresis (PAGE). Standard Western blot-
ting procedures were performed using the following antibodies: anti-TAP
(CAB1001; Open Biosystems), anti-Myc (catalog no. 11667149; Roche), perox-
idase-conjugated goat anti-rabbit IgG (catalog no. AP307P; Chemicon), and
peroxidase-conjugated goat anti-mouse IgG (catalog no. 170-6516; Bio-Rad). In
cases where the anti-TAP antibody cross-reacted with the IgG eluted from the
magnetic beads, the anti-TAP antibody was conjugated to horseradish peroxi-
dase using the SureLINK horseradish peroxidase conjugation kit (catalog no.
84-00-01; KPL) as per the manufacturer’s instructions.
Mass spectrometric detection of proteins by liquid chromatography-MS/MS.
Gel bands were excised and subjected to in-gel tryptic digestion by following
standard protocols (56). For the knockout analysis, in cases where bands were
missing, areas corresponding to the missing bands were also excised to confirm
that the proteins were truly missing. Liquid chromatography-tandem mass spec-
trometry (MS/MS) was performed using the LTQ quadrupole ion trap mass
spectrometer (Thermo-Electron, Waltham, MA) as described previously (50).
MS/MS data were analyzed and matched to S. cerevisiae protein sequences in the
NCBI nonredundant database using the Mascot database search engine (Matrix
Science Inc., Boston, MA).
Fluorescence microscopy. Cells grown at 25°C in YPD medium were resus-
pended at 2 to 4 OD600U/ml and stained with FM4-64 (catalog no. T35356;
Molecular Probes) at a final concentration of 20 ?M for 25 min at 30°C. Cells
were resuspended in fresh medium and incubated at 30°C for a 2-h chase period.
The esa1(L254P) cells were incubated at 37°C for the chase period. Cells were
then resuspended at 4 to 8 OD600U/ml in fresh YPD medium. Slides were
analyzed with a Leica DM IRE2 microscope using a 625-nm filter. Images
were acquired using a Retiga 12-bit camera (Leica) and analyzed using Impro-
vision 3.1 software.
In vitro binding assay. Msn4 fused to glutathione S-transferase (GST) (pKB1)
as well as GST alone (pGEX-6P2; Amersham) was purified from Escherichia coli
on glutathione-Sepharose as recommended by Amersham. GST-Msn4 and GST
protein concentrations were normalized by Coomassie blue staining on 10%
SDS-PAGE gels. The NuA4 complex from yeast cells expressing Esa1-TAP or
background control (WT) yeast cells with no TAP-tagged proteins was purified
using IgG-coated magnetic Dynabeads as described above. Twenty-five microli-
ters of Dynabeads complexed with NuA4 (Esa1-TAP), the background (WT)
protein, or beads alone was equilibrated twice with 1-ml washes in cold binding
buffer (20 mM HEPES, pH 7.4, 0.001% Tween 20, 2 mM MgCl2, 100 mM NaCl).
Equivalent amounts of GST and GST-Msn4 were then incubated with NuA4, the
WT protein, or Dynabeads alone for 2 h at 4°C with end-over-end rotation in a
TABLE 1. Yeast strains used in this study
Strain Auxotrophies Reference or source
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1
MAT? can1?::STE2pr-Sp_his5 lyp1? his3?1 leu2?0 ura3?0 met15?0
MAT? can1?::STE2pr-Sp_his5 lyp1? his3?1 leu2?0 ura3?0 met15?0 eaf1?::NAT
MAT? can1?::STE2pr-Sp_his5 lyp1? his3?1 leu2?0 ura3?0 met15?0 eaf3?::NAT
MAT? can1?::STE2pr-Sp_his5 lyp1? his3?1 leu2?0 ura3?0 met15?0 eaf5?::NAT
MAT? can1?::STE2pr-Sp_his5 lyp1? his3?1 leu2?0 ura3?0 met15?0 eaf6?::NAT
MAT? can1?::STE2pr-Sp_his5 lyp1? his3?1 leu2?0 ura3?0 met15?0 eaf7?::NAT
MATa his3?200 leu2-3,112 trp1?1 ura3-52 esa1?::HIS3 esa1(L245P)::URA3
MAT? can1?::STE2pr-Sp_his5 lyp1? his3?1 leu2?0 ura3?0 met15?0 LYS2?
MAT? can1?::STE2pr-Sp_his5 lyp1? his3?1 leu2?0 ura3?0 met15?0 htz1?::NAT
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 EAF1-TAP::TRP
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 eaf1?::kanMX
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 eaf3?::TRP
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 eaf5?::TRP
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 eaf6?::TRP
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 yaf9?::kanMX
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 yng2?::kanMX
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 htz1?::kanMX
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 MSN4-TAP::TRP
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1EAF7-MYC::kanMX
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 msn4-TAP::TRP
MATa ?trp ura3-1 leu2-3,112 his3-11,15 ade2-1 can1-100 ESA1-TAP::TRP
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 msn4-TAP::TRP eaf1?::kanMX
MATa ESA1-TAP::TRP eaf1?::kanMX
MATa ?trp ura3-1 leu2-3,112 his3-11,15 ade2-1 can1-100 ESA1-TAP::TRP eaf3?::kanMX
MATa ?trp ura3-1 leu2-3,112 his3-11,15 ade2-1 can1-100 ESA1-TAP::TRP eaf5?::kanMX
MATa ?trp ura3-1 leu2-3,112 his3-11,15 ade2-1 can1-100 ESA1-TAP::TRP eaf6?::kanMX
MATa ?trp ura3-1 leu2-3,112 his3-11,15 ade2-1 can1-100 ESA1-TAP::TRP eaf7?::kanMX
MAT? ESA1-TAP::TRP yaf9?::kanMX
MAT? ESA1-TAP::TRP yng2?::kanMX
MATa esa1-TAP::TRP EAF7-MYC::kanMX
MATa esa1-TAP::TRP EAF7-MYC::kanMX eaf5?::kanMX
MATa his3?1 leu2?0 met15?0 ura3?0 EAF7-TAP::HIS
MATa EAF7-TAP::HIS eaf1?::kanMX
MATa ura3-52 lys2-801 ade2-101 trp1-?63 his3-?200 leu2-?1 msn2?::TRP msn4?::kanMX
Gift from J. Greenblatt
Gift from M. C. Keogh
Gift from N. Krogan
2246MITCHELL ET AL.MOL. CELL. BIOL.
at Univ of Ottawa on March 15, 2008
volume of 500 ?l. Dynabeads were washed twice with 1 ml cold binding buffer
and then five times with 1 ml cold lysis buffer (see above). Proteins were eluted
as described above and resolved by 10% SDS-PAGE. Anti-GST Western blots
were carried out with anti-GST (catalog no. A5800; Invitrogen) and peroxidase-
conjugated goat anti-rabbit IgG (catalog no. AP307P; Chemicon) using standard
Modified ChIP. TAP-tagged and untagged strains, grown in YPD medium at
25°C to an OD600of 0.8, were collected by centrifugation, washed with 5 ml
chromatin immunoprecipitation (ChIP) lysis buffer (100 mM HEPES, pH 8.0, 20
mM magnesium acetate, 10% glycerol, 0.1 mM EDTA, protease inhibitor cock-
tail [catalog no. P-8215; Sigma]) and transferred to 1.5-ml Eppendorf tubes. Cells
were resuspended in 500 ?l lysis buffer plus an equal volume of acid-washed glass
beads (catalog no. 35–535; Fisher Scientific) and lysed by vortexing (six 1-min
blasts, with incubation on ice in between vortexing). The crude WCE was sep-
arated from the beads into a fresh Eppendorf tube by centrifugation at 1,000 rpm
for 1 min through a hole in the bottom of the Eppendorf tube created using a
red-hot 18-gauge needle. Samples were subjected to three rounds of sonication
(10 seconds each) (Sonic Dismembrator model 60, at setting 2; Fisher Scientific),
with a 30-second incubation on ice between each pulse. NP-40 was added to each
sample to a final concentration of 1%. Samples were clarified by centrifugation
at 3,000 rpm for 10 min at 4°C. One hundred micrograms of WCE was reserved
to serve as an “input” control, while 10 mg of WCE was incubated overnight at
4°C with 25 ?l of IgG-coated Dynabeads as described above. Beads were washed
three times with 1 ml cold ChIP wash buffer (ChIP lysis buffer plus 0.5% NP-40).
Twenty percent of the beads were reserved to test for immunopurification of the
TAP-tagged proteins by Western blotting as described above. The remainder of
the beads and the input WCE were treated with protease K (0.5 mg/ml in
Tris-EDTA) for 2 h at 37°C. Protein was extracted using phenol-chloroform, and
DNA was ethanol precipitated in the presence of 1 ?g of glycogen. DNA pellets
were washed with 70% ethanol, air dried, and resuspended in 50 ?l of Tris-
EDTA. Immunoprecipitated DNA was amplified using multiplex PCR with the
following primer pairs: HSP12 F (5? CGCAAGCATTAATACAACCC) and
HSP12 R (5? CGCAATTGAGGAAGTAGAAC) and Chr V no-ORF F (5?
GGCTGTCAGAATATGGGGCCGTAGTA) and Chr V no-ORF R (5? CCCC
GAAGCTGCTTTCACAATAC). PCR products were resolved on a 2% agarose
gel and visualized with ethidium bromide.
Northern blot analysis. Yeast strains were grown at 25°C in YPD medium to
an OD600of 0.6 to 0.9. Heat shock conditions were carried out at 39°C for 30
min. RNA was isolated using a hot-phenol extraction method (53), except that
TES acid buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, pH 8.0, 0.5% SDS)
was used, and samples were incubated at 65°C for 1 h. Northern blotting was
carried out as previously described (3). The probes used for the Northern blot
analysis were created by PCR amplification of the coding sequences of HSP12 (5?
GTCTGACGCAGGTAGAAAAGG [forward], 5? CGCAAGCATTAATACA
ACCC [reverse]) and ACT1 (5? GCATCATACCTTCTACAACG [forward], 5?
GTGATGACTTGACCATCTGG [reverse]). Probes were labeled using the
Megaprime DNA labeling system (catalog no. RPN1607; Amersham) in the
presence of [?-32P]dCTP (GE Healthcare).
An extensive NuA4 genetic-interaction map indicates that
NuA4 impacts a diverse range of cellular processes. In an
effort to comprehensively identify the cellular processes poten-
tially impacted by NuA4, we performed genome-wide SL
screens using query strains with deletions of all seven nones-
sential NuA4 genes (eaf1?, eaf3?, eaf5?, eaf6?, eaf7?, yaf9?,
and yng2?). All seven genome-wide SL screens were per-
formed in triplicate using SGA methodology by mating each
query strain to the yeast deletion mutant array and selecting
for double mutants (63). Any double mutant combinations that
resulted in inviability (SL) or in reduced fitness (SS) that were
identified in a minimum of two out of three screens were con-
firmed by tetrad analysis (genetic interaction data set available at
Despite multiple attempts with yng2? and yaf9? query strains,
reproducible genetic-interaction profiles were not obtained.
The resulting confirmed data set contains 172 genetic interac-
tions among 149 genes, of which 18% (31/172) were SL inter-
actions and the remainder were SS interactions. To increase
the coverage of our data set, we also incorporated eaf1?,
eaf3?, eaf5?, eaf6?, or eaf7? genetic interactions confirmed in
previously published SL-SGA analyses or direct testing (see
Materials and Methods for details). The combined data set
contains 268 genetic interactions among 204 genes, of which
38% (101/268) were SL interactions and the remainder were
Given that genetic interactions predict functional relation-
ships (63), the NuA4 genetic interaction map identified many
genes that encode proteins implicated in cellular processes
previously associated with the NuA4 complex, including chro-
matin structure, transcription, DNA repair, and chromosome
stability, as determined by their gene ontology (GO) annota-
tions (Fig. 1). Significantly, the NuA4 genetic map enriched for
genes encoding proteins within the same protein complexes,
suggesting that our screening method provided extensive cov-
erage of the genome and thus the ability to predict interactions
between NuA4 and protein complexes. Some examples of this
are the SAGA complex (GCN5, SGF9, SGF73, SGF11), the
SWR complex (ARP6, SWC3, SWC5, SWR1, VPS71, VPS72),
and the MRX complex (MRE11, RAD50, XRS2). This is the
first time that genetic interactions have been identified for any
NuA4 subunit on a genome-wide scale, so in addition to sup-
porting the well-characterized roles for NuA4, the genetic in-
teraction map enriched for numerous other genes implicated
in a wide variety of functions, including vesicle-mediated trans-
port, stress response, and arginine biosynthesis.
Eaf1 functions primarily as a component of NuA4. Although
it has been shown that genes that function within the same
protein complex have similar genetic interaction profiles (14),
we observed that the five NuA4 subunits have remarkably
different genetic interaction profiles, particularly with respect
to the total number of genetic interaction partners. While
eaf3?, eaf5?, eaf6?, and eaf7? mutants genetically interacted
with 31, 42, 12, and 35 genes, respectively, the eaf1? mutant
genetically interacted with 148 genes. Further, 75% of the
eaf1? genetic interactions were specific to the eaf1? mutant
alone (genetic interaction data set available at www.oisb.ca
/personal_web_site/Baetz_Lab/publicationsFS.html). This sug-
gests that either Eaf1 plays a crucial role in NuA4 that is not
shared by the other nonessential subunits tested or Eaf1 func-
tions on its own or as part of an additional protein com-
plex(es). Previous studies were not able to identify copurifying
proteins when tagged Eaf1 was used as bait (22, 32). There-
fore, to test the hypothesis that Eaf1 is found only in NuA4, we
immunopurified a TAP-tagged version of Eaf1 and identified
its interacting proteins using a modified procedure (Fig. 2).
One-step purification was performed using IgG-coated mag-
netic beads that interact with the PrA component of the TAP
tag. Proteins were eluted from the beads in sample buffer at
65°C, separated by SDS-PAGE, and silver stained. This pro-
tocol results in low-level background binding of nonspecific
proteins to IgG beads (Fig. 2, WT lane) and a high yield of
TAP purifications. Protein bands were individually cut, and MS
was performed to identify the Eaf1-TAP-interacting proteins.
We discovered that Eaf1-TAP copurifies only with subunits of
the NuA4 complex and Fks1, which was identified in all NuA4
purifications using this protocol (Fig. 2 and see Fig. 5 and 6).
VOL. 28, 2008GENOMIC AND STRUCTURAL DISSECTION OF NuA4 2247
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As Fks1, a subunit of the 1,3-beta-D-glucan synthase enzymatic
complex (29), localizes to the plasma membrane and has not
previously been reported to interact with NuA4, we suspect
that this interaction is an artifact of the purification procedure.
Since Eaf1-TAP copurified only with NuA4 proteins (and
Fks1), this suggests that the 148 eaf1? genetic interactions are
attributable to Eaf1’s role in NuA4. Hence, we hypothesized
that the eaf1? genetic interactions would be largely shared with
a strain with a mutation in the essential NuA4 catalytic subunit
Esa1. To test this, we directly mated the esa1(L245P) mutant
(13), a temperature-sensitive point mutant that displays acet-
ylation defects, with 148 deletion mutants that displayed ge-
netic interactions with the eaf1? strain. Diploids were sporu-
lated, and tetrads were dissected at 25°C. Of the 148 matings,
140 produced reliable tetrads. Dot assays were performed at
30°C to test for synthetic genetic interactions of these double
mutants, except for three double mutants that proved to be SL
or extremely SS on the dissection plates (the YKE2, TPM1, and
RIB4 mutants). Nineteen displayed no growth phenotype de-
fects in combination with the esa1(L245P) mutation at 30°C,
FIG. 1. Synthetic genetic-interaction map of five NuA4 subunits. Genome-wide SL-SGA screens were performed using query strains for five
nonessential NuA4 subunits: the eaf1? (YKB622), eaf3? (YKB993), eaf5? (YKB852), eaf6? (YKB623), and eaf7? (YKB853) strains. The 199
NuA4-interacting genes are represented by nodes that are color coded based on the GO process. Gray lines indicate genetic interactions (both SS
and SL interactions). The 139 interacting genes highlighted in bold text indicate those genes whose interaction with at least one NuA4 query strain
was confirmed by tetrad dissection in our lab. The remaining interacting genes in the map represent previously published genetic interactions that
were either identified in our SGA screens but were not confirmed by tetrad dissection or not identified in our screens. Genes encoding subunits
of a single protein complex are grouped together with black circles.
2248 MITCHELL ET AL.MOL. CELL. BIOL.
at Univ of Ottawa on March 15, 2008
while the remaining 118 deletion mutants were either SL or
SS in combination with the esa1(L245P) mutation (see Suple-
mental Table2at www.oisb.ca/personal_web_site/Baetz_Lab
/publicationsFS.html). The facts that 86% of eaf1? genetic inter-
actions are shared with the acetylation-deficient esa1(L245P)
mutant and that Eaf1-TAP copurifies only with NuA4 suggest
that Eaf1 functions primarily as a component of NuA4.
NuA4 function impacts vesicle-mediated transport. Surpris-
ingly, the NuA4 genetic map identified 15 genes that encode
proteins with well-established roles in vesicle-mediated trans-
port (Fig. 1 and Table 2). Remarkably, most of the vesicle-
mediated transport gene deletions displayed only genetic in-
teractions with eaf1? and the esa1(L245P) catalytically
deficient mutant (Table 2). Indeed, no genetic interactions
were identified between eaf3? or eaf6? mutants and this group
of genes. Further, of the 13 eaf1? mutant genetic interactions
with the vesicle-mediated transport genes, eight were SL, in-
cluding the interactions with all four nonessential subunits of
the conserved oligomeric Golgi complex (COG5, COG6,
COG7, COG8) (reviewed in reference 65), the soluble N-eth-
ylmaleimide-sensitive factor attachment protein receptors
(GOS1 and VAM7) (reviewed in reference 26), the Golgi com-
plex-associated retrograde protein complex subunit (VPS54)
(reviewed in reference 44), and the GTPase (YPT6) (reviewed
in reference 46). Many of the vesicle-mediated transport genes
identified have specific roles in Golgi complex-to-vacuole
transport, and deletion of most of these genes results in altered
vacuole morphology (54). The strong genetic interactions of
eaf1? and esa1(L245P) with these mutants suggest that NuA4
may impact vesicle-mediated transport and that NuA4 mutants
may also display aberrant vacuole morphology. To test this
hypothesis, we examined vacuolar morphology in all seven
nonessential NuA4 mutants and the esa1(L245P) mutant using
the fluorescent vacuolar vital stain FM4-64 (66). As expected
eaf3?, eaf5?, eaf6?, and eaf7? cells, which do not have any or
have only a few genetic interactions with vesicle-mediated
transport genes, displayed vacuole morphology similar to that
of the WT (Fig. 3A). In contrast, eaf1?, yaf9?, yng2?, and
esa1(L245P) cells displayed altered vacuole morphology, with
the majority of cells displaying one large vacuole (Fig. 3A).
This suggests that Eaf1, Yaf9, Yng2, and the acetyltransferase
activity of Esa1 are required for vacuole function. As NuA4
may mediate some of its cellular roles through Htz1 acetylation
(2, 28, 42) and htz1? mutants display some phenotypes similar
to those of NuA4 mutants (30, 31), we next tested whether
htz1? and htz1-ND cells, in which the N-terminal amino acids
3 to 14 (KAHGGKGKSGAK) are replaced with 24 amino
acids (CRSTTLNITSYNVCYTKLLGDIRT), also display de-
fects in vacuole morphology. While the majority of htz1? cells
displayed large vacuoles similar to those of eaf1?, yaf9?,
yng2?, and esa1(L245P) cells, the htz1-ND cells displayed WT
vacuole morphology (Fig. 3B). This suggests that the Htz1
globular core, but not the N terminus or N-terminal acetylation
sites, is required for vacuole function. We next directly tested
whether htz1? mutants display synthetic genetic interactions
with a subset of the identified deletion mutants which interact
with both eaf1? and esa1(L245P) mutant: the TVP23, VPS51,
GOS1, VPS8, and COG6 mutants. htz1? mutants shared all
these genetic interactions (Fig. 3C). This suggests that both
NuA4 and Htz1 have a role in vesicle-mediated transport,
likely through the transcriptional regulation of a key gene(s)
required for vesicle-mediated transport.
FIG. 2. Eaf1-TAP purifies the NuA4 complex. SDS-PAGE (gradi-
ent gel) and silver staining comparing NuA4 that was affinity purified
via Eaf1-TAP (YKB1007) and NuA4 from an untagged strain (WT;
YPH499), using IgG-coated magnetic beads that bind the PrA com-
ponent of the TAP tag. IgG heavy and light chains coelute upon
heating. Protein bands were identified by MS as indicated. Eaf6 was
not efficiently silver stained. The ? indicates a non-NuA4, copurifying
protein, Fks1. Anti-TAP Western blotting demonstrates the expression
of Eaf1-TAP in the WCE. This gel is representative of those from
three purification experiments. Numbers at the left indicate molecular
masses (in kDa).
TABLE 2. Genetic interactions of NuA4 mutants with
vesicle-mediated transport genes
Phenotype with indicated NuA4 genea
aEmpty spaces indicate that no genetic interaction was found by genome-wide
SL-SGA screens. ND indicates that no data were obtained from esa1(L245P)
mutant direct tests either because the gene was not identified in the eaf1? cell
screen (TFP1 and RIC1) or reliable tetrads were not obtained (TVP23, RMD7,
and MDM39). eaf3? and eaf6? mutants did not display genetic interactions with
any of the vesicle-mediated transport genes.
VOL. 28, 2008GENOMIC AND STRUCTURAL DISSECTION OF NuA4 2249
at Univ of Ottawa on March 15, 2008
NuA4 and the stress-responsive transcription factor Msn4.
The genetic interaction map also identified 10 genes that have
been implicated in the yeast stress response, thereby suggesting
a functional connection between NuA4 and the stress response
(Fig. 1). Additionally, during the course of our analysis of
NuA4 complex integrity (see below), we identified the general
stress-responsive transcription factor Msn4, which comigrates
with Esa1-TAP, in four separate Esa1-TAP purifications.
Msn4, along with its functionally related homolog Msn2, are
required for the transcriptional induction of numerous stress
response genes upon various environmental and metabolic
cues (39). We were also interested in the physical interaction
between NuA4 and Msn4 because microarray analysis of sev-
eral NuA4, SWR, and Isw1 mutants previously demonstrated
that the complex plays a role in the transcriptional repression
of a large number of Msn2/4-dependent stress response genes
(25, 37). Further, it was demonstrated that the derepression of
a subset of Msn2/4 target genes in NuA4, SWR, or Isw1 mu-
tants in the absence of environmental stress requires Msn2
and/or Msn4 (37). Having initially identified the physical in-
teraction between NuA4 and Msn4 by MS, we sought to test
the validity of the interaction in vivo by reciprocal immunopu-
rification of NuA4 using a tagged version of Msn4. To accom-
plish this, we generated a strain expressing both Msn4-TAP
and Eaf7-Myc from their endogenous promoters. IgG-coated
magnetic beads were used to immunopurify Msn4-TAP, and
Western blot analysis confirmed the presence of Eaf7-Myc
(Fig. 4A). Given that Eaf7 has been identified only within the
NuA4 complex, we conclude that Msn4 physically interacts
directly or indirectly with the NuA4 enzyme complex. Though
Msn4 and Msn2 migrate at the same rate on SDS-PAGE gels,
we never identified Msn2 by MS in our Esa1-TAP purification.
However, as Msn2 and Msn4 are largely redundant, we also
asked whether NuA4 and Msn2 interact. Experiments using
Msn2-TAP showed that Msn2-TAP coimmunoprecipitates
Eaf7-Myc (data not shown), indicating the NuA4 interacts with
both Msn2 and Msn4. While this experiment verified the in-
teraction between NuA4 and both Msn4 and Msn2, it told us
little about whether the interaction is mediated by other, non-
NuA4 proteins. As Msn2 and Msn4 likely act in the same
manner, the remaining experiments were performed with
Msn4 alone. To test whether the physical interaction between
NuA4 and Msn4 is direct, we performed an in vitro binding
experiment using recombinant GST-Msn4 purified from E. coli
and NuA4 isolated from yeast using IgG-coated magnetic
beads after extensive washes. We found that while GST alone
does not bind to NuA4, GST-Msn4 specifically interacts with
the NuA4 enzyme complex (Fig. 4B), suggesting that the in-
teraction between Msn4 and the NuA4 enzyme complex is
likely direct. However, although we believe our NuA4 immu-
nopurification to be extremely clean (Fig. 5A), given that
NuA4 was purified from yeast, we cannot rule out the possi-
bility that non-NuA4 copurifying proteins mediated the inter-
action with Msn4.
In light of the physical interaction between NuA4 and Msn4/
Msn2 and the derepression of a subset of Msn2/4 genes in
NuA4 mutants, we next decided to further explore the molec-
ular mechanism connecting Msn2/4 and NuA4. Under non-
stress conditions, the majority of Msn2 and Msn4 proteins are
localized to the cytoplasm, and upon environmental stress,
they enter the nucleus and bind to stress response elements
upstream of their target genes (24). However, strains with
mutations in NuA4 do not display increased Msn2 or Msn4
nuclear localization, nor is the H4 acetylation of the promoters
of derepressed Msn4/2 target genes significantly changed from
that of control promoters in yaf9? cells (37). This suggests that
FIG. 3. NuA4functionimpactsvesicle-mediatedtransport.(A)eaf1?,
yaf9?, yng2?, and esa1(L245P) cells display vacuolar-morphology de-
fects. Vacuole morphology was examined in WT (YPH499) cells, in
strains deficient in each of the seven nonessential NuA4 subunits
(eaf1? [YKB44], eaf3? [YKB654], eaf5? [YKB658], eaf6? [YKB662],
eaf7? [YKB853], yaf9? [YKB464], and yng2? [YKB494]), and in a
temperature-sensitive point mutant
[esa1(L245P) (LPY3500)] using the dye FM4-64. The WT and deletion
mutants were grown at 30°C, while esa1(L245P) mutants were grown at
37°C for 2 h after FM4-64 treatment. Images shown were taken after
the merger of fluorescent light and transmitted light. (B) htz1-ND cells
(YKL35) do not show vacuolar-morphology defects, while htz1?
(YKB625) cells display defects similar to those of the eaf1?, yaf9?,
yng2?, and esa1(L245P) mutants. Vacuole morphology was examined
in mutant cells with FM4-64 as described for panel A. (C) htz1?
(YKB500), esa1(L245P) (LPY3500), and eaf1? (YKB622) mutants
(indicated by the red dots) genetically interact with a subset of vesicle-
mediated transport genes (indicated by green dots) identified in the
NuA4 SGA screens. Lines connect genes with synthetic genetic
ofthe catalytic subunit
2250MITCHELL ET AL.MOL. CELL. BIOL.
at Univ of Ottawa on March 15, 2008
Msn4 and/or Msn2 may bind stress response element promot-
ers in the absence of stress and that NuA4-dependent modifi-
cation of the chromatin structure may not regulate Msn2 or
Msn4 binding to promoters. To test this, we examined the
occupancy of Msn4 on the promoter of the HSP12 gene by
ChIP. The transcriptional induction of HSP12 is dependent
largely on Msn2/4 under stress conditions (39, 52) and is highly
derepressed in NuA4 mutants in a manner dependent on
Msn2/4 (37), and Esa1 has been shown to localize to the
HSP12 promoter (49). Using Msn4-TAP, we attempted a tra-
ditional ChIP experiment but found that due to the reduced
accessibility of the epitope tag upon cross-linking, we were
unable to immunoprecipitate Msn4-TAP. We therefore devel-
oped a modified ChIP protocol, utilizing a gentle clarification
that circumvents the use of formaldehyde cross-linking, and
tested for the presence of Msn4-TAP on the promoter of
HSP12. Our modified ChIP analysis demonstrated that under
normal growth conditions, Msn4-TAP associates specifically
with the promoter region of HSP12 but not with an untran-
scribed control region on chromosome V (Fig. 4C, row no
ORF) (27). Through Northern blot analysis, we determined
that HSP12 is derepressed at 25°C in log-phase eaf1?cells and
that derepression is largely dependent on Msn2 and Msn4 (Fig.
4D). Therefore, we also performed the modified Msn4-TAP
ChIP protocol with eaf1? cells and determined that the dele-
tion of EAF1 does not alter Msn4 localization to HSP12 pro-
moters (Fig. 4C). As NuA4 has been localized to the HSP12
promoter in the absence of stress (49) and the deletion of
EAF1 causes the collapse of the NuA4 complex, the ChIP
results suggest that Msn4 localization to the HSP12 promoter
does not require NuA4. We were next interested in determin-
ing whether NuA4 played a significant role in the activation of
HSP12 under heat shock conditions. To do this, we performed
HSP12 Northern blot analysis and compared WT cells to eaf1?
FIG. 4. NuA4 physically interacts with Msn4 but does not regulate Msn4 binding to the HSP12 promoter or heat shock induction of HSP12.
(A) NuA4 interacts with Msn4 in vivo. Protein extracts expressing the indicated tagged proteins (Msn4-TAP [YKB1035], Eaf7-MYC [YKB518],
Msn4-TAP/Eaf7-MYC [YKB1069]) or an untagged WT control (YPH499) were immunoprecipitated with magnetic beads coated with IgG
antibodies that bind the PrA component of the TAP tag. Total protein extracts (WCE) and immunoprecipitates (?-TAP IP) were resolved by 7.5%
SDS-PAGE and subjected to Western blot analysis with anti-Myc and anti-TAP antibodies (?-MYC and ?-TAP, respectively), as indicated at the
right side of the panels. (B) NuA4 and Msn4 interact in vitro. WCE from cells expressing Esa1-TAP (YKB440) were used to purify the NuA4
complex using IgG-coated magnetic beads, and results were compared to those for an untagged strain (WT; YPH499). IgG-coated magnetic beads
complexed with NuA4 (lane Esa1-TAP), background yeast proteins (lane WT), or beads alone (lane Beads) were incubated with equivalent
amounts of either full-length GST-Msn4 or GST. Resulting immunocomplexes were eluted with heat, resolved by 10% SDS-PAGE, and subjected
to Western blotting with anti-TAP and anti-GST antibodies, as indicated. Twenty-five percent of the input GST or GST-Msn4 fusion protein was
run alongside the pull-down experiments as an input control (lane Input). (C) Msn4 occupancy at the HSP12 promoter is independent of NuA4.
Modified ChIP (see Materials and Methods) was performed using an untagged strain (WT; YPH499) and the Msn4-TAP (YKB1035) and
Msn4-TAP eaf1? (YKB1091) strains. Immunoprecipitated (ChIP lanes) or WCE (Input lanes) DNA was subjected to multiplex PCR amplification
using primers specific to the promoter region of HSP12 and an intergenic region on chromosome V (no ORF). The results of this ChIP was
representative of three experiments. (D) Deletion of EAF1 causes derepression of HSP12 but does not inhibit the heat shock induction of HSP12.
WT (YPH499), eaf1? (YKB44), and eaf1? msn2? msn4 ? (YKB1097) cells were grown in YPD at 25°C to mid-log phase, and samples were
collected at 25°C and after 30 min of heat shock treatment at 39°C. Northern blots were probed with labeled DNA fragments of the HSP12 gene.
The signal was quantitated using AlphaEase FC (Alpha Innotech) and normalized to the ACT1 signal. The values are averages from three
independent RNA preparations, and error bars indicate standard deviations.
VOL. 28, 2008GENOMIC AND STRUCTURAL DISSECTION OF NuA4 2251
at Univ of Ottawa on March 15, 2008
cells after 30 min of heat shock at 39°C (Fig. 4D). The deletion
of EAF1 did not inhibit the heat shock induction of HSP12.
This suggests that though NuA4 is important for maintaining
the repression of the HSP12 promoter, NuA4 does not play a
significant role in the induction of HSP12. Surprisingly, we also
determined that in the absence of Eaf1, the heat shock induc-
tion of HSP12 is independent of Msn2 and Msn4 (Fig. 4D),
suggesting that other transcription factors are compensating.
Eaf1 is required for NuA4 complex integrity. The large size
of the eaf1? genetic-interaction profile relative to those of the
other nonessential NuA4 subunits suggested that Eaf1 might
have a unique and critical role in complex integrity. To test this
hypothesis, we examined protein association with TAP-tagged
Esa1 in cells deficient for one of the seven nonessential sub-
units (Fig. 5). Protein bands in the Esa1-TAP lane were iden-
tified through MS. Eaf3 comigrated with the IgG heavy chain,
and Eaf7 comigrated with Esa1-TAP, so while we detected
these subunits by MS in the Esa1-TAP purification, we cannot
make reliable predictions as to their interactions with the com-
plex in Esa1-TAP purifications in the various deletion mutant
backgrounds. Regardless, we found that the elimination of
Eaf3, Eaf5, Eaf6, Eaf7, and Yaf9 had no detectable effect on
the association of the remaining visible NuA4 subunits (Fig. 5),
indicating that the overall integrity of the NuA4 complex is not
dependent on these subunits. Removal of Yng2 resulted in the
loss of Eaf6 (Fig. 5, compare lane Esa1-TAP to lane Esa1-TAP
yng2?), indicating that Eaf6 interacts with the NuA4 complex
Remarkably, the removal of Eaf1 resulted in a dramatic loss
of NuA4 complex integrity (Fig. 5, compare lane Esa1-TAP to
lane Esa1-TAP eaf1?). Though faint bands corresponding to
the size of Epl1 and Yng2 were detected on the silver-stained
gel as interacting with Esa1-TAP in the absence of Eaf1, no
other subunits of NuA4 were detected in multiple purifica-
tions. This implies that in the absence of Eaf1, Piccolo NuA4,
which is sufficient for cellular viability (7), is still present. These
results suggest that Eaf1 is required to maintain the integrity of
the NuA4 complex and may link the Piccolo NuA4 subcomplex
to the remaining NuA4 components.
Eaf5 and Eaf7 form a subcomplex. We were surprised that
the deletion of most nonessential subunits revealed little about
the physical interdependencies of the NuA4 subunits within the
complex. As genes that function within the same complex or
subcomplex tend to have similar genetic-interaction profiles, to
gain further insight into NuA4 complex integrity, we per-
formed two-dimensional hierarchical clustering of our NuA4
genetic interactions (Fig. 6A). Our analysis revealed that
EAF5, EAF7, and EAF3 components cluster together. We also
performed a two-dimensional hierarchical clustering analysis of
our NuA4 genetic interactions in the context of a large combined
data set consisting of 12,954 previously published SL or SS inter-
actions that were identified using genome-wide SL-SGA screens
(supplementary cluster Treeview files are available at www.oisb
.ca/personal_web_site/Baetz_Lab/publicationsFS.html) (40, 45,
48, 64). This analysis further confirmed the idea that EAF3,
EAF5, and EAF7 cluster together. Similar clustering of EAF5
and EAF7 was seen in the recently published chromatin E-
MAPs (14), and eaf5? and eaf7? strains display similarity in
microarray transcriptional profiles (31). The repeated cluster-
ing of EAF5, EAF7, and EAF3 genetic profiles strongly sug-
gests that Eaf5, Eaf7, and potentially Eaf3 may form a sub-
complex that is responsible for mediating only a subset of the
cellular functions of NuA4. We have shown that the interaction
of Eaf5 with the NuA4 complex is not dependent on Eaf7 (Fig.
5); however, as Eaf7 was not visible on the silver-stained gel,
we could not visually determine whether Eaf5 is required for
Eaf7’s interaction with NuA4. Therefore, to further investigate
the possible existence of this subcomplex, we chose to integrate
a C-terminal Myc tag at the EAF7 chromosomal locus in order
to determine the effect of EAF5 gene deletion on the Eaf7-Myc
interaction with the NuA4 complex by Western blotting. We
purified the NuA4 complex using Esa1-TAP and found that
the eaf5? mutant disrupted the interaction of Eaf7-Myc with
the NuA4 complex (Fig. 6B). We were next interested in de-
termining whether Eaf7 and Eaf5 form a detectable subcom-
plex in the absence of NuA4. To explore this, we TAP tagged
Eaf7 and studied whether the Eaf5-Eaf7 subcomplex would
still form in the absence of Eaf1, which eliminates the full
NuA4 complex. We found that while all of the other NuA4
subunits were lost as a result of the Eaf1 deletion, the physical
interaction of Eaf7 with Eaf5 remained intact (Fig. 6C). Taken
together, these results indicate the presence of a novel sub-
complex forming between Eaf5 and Eaf7 within the NuA4
complex and, moreover, that Eaf7 interacts with NuA4
FIG. 5. Eaf1 is required for NuA4 complex integrity. SDS-PAGE
(gradient gel) and silver staining of NuA4 affinity purified via Esa1-
TAP (YKB440) compared to an untagged strain (WT; YPH499) and
Esa1-TAP purified from mutant strain backgrounds of without all
seven nonessential subunits (eaf1? [YKB855], eaf3? [YKB765], eaf5?
[YKB854], eaf6? [YKB766], eaf7? [YKB964], yaf9? [YKB966], and
yng2? [YKB967] strains). Eaf6, as it does not stain well with silver, was
subjected to a longer exposure (middle panel). Proteins bands in the
Esa1-TAP lane were identified by MS as indicated. Anti-TAP Western
blotting demonstrated the expression of Esa1-TAP in the WCE of all
strains tested. Arrowheads point to proteins eliminated by subunit
deletion, while squares specify additional subunits lost. The * indicates
a non-NuA4, copurifying protein, Fks1. Numbers at the left are mo-
lecular masses (in kDa). The gel is representative of three purification
2252 MITCHELL ET AL.MOL. CELL. BIOL.
at Univ of Ottawa on March 15, 2008
NuA4 is a genetic hub. In an effort to further define the
cellular functions of NuA4 and provide insight into the func-
tion of individual NuA4 subunits, we performed genome-wide
SL-SGA analysis with five nonessential subunits of NuA4.
With the inclusion of previously confirmed genetic interac-
tions, the combined data set contains 268 genetic interactions
among 204 genes (Fig. 1). The most remarkable feature of the
genetic-interaction map is that the eaf1? query mutant ac-
counted for 148 genetic interactions alone, which is more than
four times greater than the average number of interactions per
query mutant in other genome-wide screens (64). Further, the
genes identified by the eaf1? mutant were not limited to genes
with roles in chromatin biology previously linked to NuA4;
rather, the genetic interaction profile for the eaf1? mutant
indicates new and diverse functional relationships for NuA4,
such as protein transport, arginine biosynthesis, stress re-
sponse, and ubiquitination (Fig. 1A). Though it is possible that
Eaf1 has cellular functions outside NuA4, the lack of addi-
tional protein interactions (Fig. 2) and the high degree of
overlap in genetic-interaction profiles between eaf1? and
suggestthatthesolefunctionofEaf1isasacomponent of NuA4.
Of the few eaf1? mutant genetic interactions that were not
shared with esa1(L245P), it is possible that the interactions
occur at higher temperatures or with other esa1 alleles. As
Esa1 is essential and part of Piccolo NuA4, esa1 mutants will
likely display numerous genetic interactions distinct from that
of the eaf1? mutant. Regardless, the large number of interac-
tions identified for both eaf1 and esa1(L245P) indicates that
these are “hub genes” (64).
It has been hypothesized that highly connected genetic-hub
genes are more important for cellular fitness, are more likely to
encode essential genes, and are highly conserved across species
(15, 35, 64). Genetic-hub genes may act as genetic buffers
because the loss of hub genes causes in enhancement of mu-
tant phenotypes in otherwise unconnected, diverse cellular
functions. This may explain the identification of seemingly
disparate genes in the eaf1? SL-SGA screen. Recently, system-
atic mapping of genetic interactions in C. elegans identified
numerous genes encoding chromatin-modifying proteins as ge-
netic hubs, including mys-1 and trr-1, the orthologs of ESA1
and TRA1, respectively (35). This indicates that NuA4/Tip60
mutants likely act as genetic hubs across species.
How might NuA4 and other chromatin modifiers act as
global genetic buffers? The most likely scenario is that a loss of
function of NuA4 leads to global changes in acetylation pat-
terns of histones, which subsequently affects transcription.
NuA4 mutants alone display relatively minor effects on global
transcriptional profiles, as detected in microarray studies (11,
19, 27, 31, 36, 69). However, these transcriptional deficiencies
in combination with other mutant genes may result in a dra-
matic enhancement of phenotypes or fitness defects. For ex-
ample, we determined that nonessential NuA4 mutants display
SS or SL interactions with mutant genes encoding proteins
required for arginine biosynthesis (Fig. 1). Microarray studies
have shown that NuA4 mutants display modest two- to three-
fold decreases in the transcription of numerous genes in the
FIG. 6. Eaf5 and Eaf7 form a subcomplex within NuA4.
(A) Two-dimensional, hierarchical clustering of the NuA4 synthetic
genetic interactions. Rows display NuA4 query genes, columns in-
dicate the interacting deletion mutant array genes, and a red box
indicates genetic interaction. The cluster includes genetic interac-
tions for the esa1(L245P) mutant that were directly tested against
all eaf1? genetic interactions. (B) Eaf7 interacts with NuA4 through
Eaf5. Shown are results with protein extracts prepared from strains
expressing the indicated tagged proteins (Esa1-TAP [YKB440],
Eaf7-Myc [YKB518], Esa1-TAP Eaf7-Myc [YKB1054], and Esa1-
TAP Eaf7-Myc eaf5? [YKB1043]) or an untagged WT (YPH499);
immunoprecipitations were performed using magnetic beads coated
with IgG antibodies that bind the PrA component of the TAP tag.
Total protein extracts (WCE) and anti-TAP immunoprecipitates
(IP) were then resolved by 7.5% SDS-PAGE and subjected to
Western blot analysis with anti-Myc and anti-TAP antibodies, as
indicated on the right side of the panels. ?, anti. (C) Eaf7 and Eaf5
form a subcomplex in the absence of Eaf1. SDS-PAGE (gradient
gel) and silver staining of NuA4 affinity purified with Eaf7-TAP in
the presence (YKB442) and absence (eaf1?) of Eaf1 (YKB1034).
Anti-TAP Western blotting was carried out on WCE as well as on
10% of the IP eluate. An anti-histone H3 (?-H3) Western blot
analysis of WCE demonstrates equal protein loadings. The results
are representative of three purification experiments.
VOL. 28, 2008GENOMIC AND STRUCTURAL DISSECTION OF NuA4 2253
at Univ of Ottawa on March 15, 2008
arginine biosynthesis pathway (11, 19, 31, 36). While the dele-
tion of single genes of this pathway does not result in fitness
defects, we suspect that this deletion in combination with de-
creases in the NuA4-dependent transcription of arginine path-
way genes results in fitness defects. Moreover, we hypothesize
that NuA4 buffers the effects of the vesicle-mediated transport
mutants in a similar manner. Microarray studies of NuA4 or
htz1? mutants (11, 19, 27, 30, 31, 36, 41, 42, 69) provide no
insights into the transcriptional misregulation of the key
gene(s) required for vesicle-mediated transport. However, as
htz1? cells display genetic interactions and vacuolar morpholog-
ical defects similar to those of eaf1? and esa1(L245P) cells (Fig.
3), we favor a model where NuA4’s and Htz1’s roles as genetic
not display vacuolar morphological defects, it is likely that
NuA4’s transcriptional effects are not mediated through Htz1
N-terminal tail acetylation but rather through H4 acetylation
or alternative targets. Further, numerous strains containing
deletions of NuA4, SWR complex, and HTZ1 genes were iden-
tified in a high-throughput screen aimed at identifying diploid,
homozygous deletion mutants with carboxypeptidase Y vacu-
olar sorting defects (6).
Genetic hubs are thought to be major contributors to com-
plex genetic diseases arising from multiple mutations (re-
viewed in reference 9). Mutations in genetic hubs may modify
the effects of numerous mutations, resulting in the implication
of genetic hub genes in a diverse range of apparently unrelated
diseases. As mutant components of NuA4/Tip60s in both yeast
and C. elegans act as genetic hubs, it is likely that this trait is
shared with human Tip60. If Tip60 is a genetic hub, it would
explain the connection of Tip60 with pleiotropic roles in a
diverse range of cancers, neurodegenerative diseases, and viral
infections (51). Insights derived from the NuA4 genetic map
presented in this paper, along with extension of the map using
mutant alleles of essential NuA4 genes, will provide an excel-
lent source of testable hypotheses for determining the exact
molecular mechanisms through which Tip60 may contribute to
NuA4 as a transcriptional repressor of stress response
genes. Traditionally, it has been proposed that HAT acetyla-
tion of histones is correlated with transcriptional activation but
that HDAC deacetylation of histones is correlated with tran-
scriptional repression (reviewed in reference 33). While NuA4
is required for transcriptional activation, growing evidence in-
dicates that NuA4 also has a role in the transcriptional repres-
sion of a subset of Msn2/4-dependent stress-responsive genes,
including HSP12 (Fig. 4D) (25, 37). In our study, the identifi-
cation of 10 genes involved in stress response in the NuA4
SL-SGA screen (Fig. 1) and the identification of a physical
interaction between Msn4 and NuA4 (Fig. 4A and B) further
support the role of NuA4 in stress response.
How might NuA4 repress the Msn2/4-dependent transcrip-
tion of stress response genes? Microarray studies indicate that
derepression of the subset of Msn2/4-dependent genes also
occurs in HTZ1 deletion mutants (41, 43) and histone H4
mutants defective in N-terminal acetylation (16), suggesting
that NuA4 could mediate the repression of these genes
through the acetylation of these two histones. We do not be-
lieve that NuA4 acetylation of histones inhibits the access of
Msn2 and/or Msn4 to stress-dependent promoters, as we re-
producibly detect Msn4 occupancy at the HSP12 promoter
under nonstress conditions and moreover show that the dele-
tion of EAF1 does not result in increased Msn4 localization to
this promoter (Fig. 4C). Though it is possible that the local-
ization of Msn4 to HSP12 promoters is dependent on Piccolo
NuA4, which appears to be present in an eaf1? strain, we
believe that this is unlikely, as there is presently no evidence of
targeted Piccolo NuA4 activity. Alternatively, the physical in-
teraction between Msn2/4 and NuA4 may target NuA4 histone
acetylation to promoters to promote a repressed chromatin
state through the recruitment of chromatin remodelers or re-
pressors. Despite multiple ChIP attempts, we were not able to
reproducibly determine whether or not Esa1-TAP localization
to the HSP12 promoter is changed in msn2? msn4? strains.
NuA4 may also directly acetylate Msn4 or Msn2, thereby
masking activation domains or regulating additional protein
interactions required for the activation or repression of Msn2/
4-dependent genes. This may explain the role of the physical
interactions seen between NuA4 and Msn2 and/or Msn4. Sur-
prisingly, though HSP12 heat shock induction has been shown
to be largely dependent on Msn2/4, in eaf1? msn2? msn4?
cells, HSP12 is still induced upon heat shock (Fig. 4D). This
suggests that alternative transcription factors, such as Hsf1
(49), may play a significant role in the absence of NuA4. Un-
raveling the mysteries of the molecular function of NuA4 in
Msn2/4-dependent gene repression will require further in-
NuA4 complex structure and function. Though it has been
proposed that genes encoding proteins in the same pathway or
complex should have similar synthetic genetic interactions, a
surprising feature of our NuA4 genetic map was the dramatic
difference in the numbers of interactions for components and
in some cases the lack of overlap between the queries. As
genetic interactions that were identified in one screen were not
directly tested against all the queries, some of the lack of
overlap may be explained by the inherent rate of false-negative
results of SL-SGA screening. In the case of the eaf3? and
eaf6? screens, some of the unique genetic interactions may be
the result of the Eaf3 and Eaf6 proteins also being part of the
Rpd3S and NuA3 complexes, respectively (10, 27, 60). How-
ever, in the case of the eaf1? mutant, the striking number of
genetic interactions displayed is likely reflective of the role that
Eaf1 plays in NuA4 complex integrity. We determined that in
the absence of Eaf1, the full NuA4 complex collapses (Fig. 5
and 6). Despite the dramatic effect on NuA4 complex integrity,
EAF1 is not an essential gene. This is likely a reflection of the
fact that in the absence of Eaf1, Piccolo NuA4 is still detected
(Fig. 5). Piccolo NuA4 contains the only two essential NuA4
proteins, Esa1 and Epl1, that are exclusively found in NuA4,
and as previously suggested, Piccolo NuA4 likely mediates the
essential nontargeted global acetylation of the histones of
NuA4 (7). However, with Eaf1 being a scaffold protein, the
diverse genetic interactions identified in the eaf1? screen may
reflect the sum of the proposed recruitment roles of the re-
maining 10 non-Piccolo NuA4 subunits. Alternatively, Eaf1
may perform unique recruitment or regulatory roles that are
not shared with the other nine subunits.
The clustering of our genome-wide NuA4 genetic interac-
tions (Fig. 6A; supplementary cluster Treeview files are avail-
able at www.oisb.ca/personal_web_site/Baetz_Lab/publicationsFS
2254MITCHELL ET AL.MOL. CELL. BIOL.
at Univ of Ottawa on March 15, 2008
.html) and the chromatin E-MAP (14) allowed us to make
predictions regarding a possible subcomplex involving Eaf5
and Eaf7. We confirmed that Eaf5 and Eaf7 form a
subcomplex and that Eaf7 interacts with the NuA4 complex
through Eaf5 (Fig. 6B and C). The genetic network also
predicts that Eaf3 may be a component of this subcomplex.
Work from Jacques Cote ´’s laboratory has confirmed an Eaf3
-Eaf5-Eaf7 subcomplex (personal communication). Though we
presently do not know if the subcomplex is purely structural in
nature, it is likely that it mediates a distinct set of NuA4
cellular functions. As the majority of the genetic interactions
shared between the eaf3?, eaf5?, and eaf7? mutants are with
SWR complex mutants or the htz1? mutant, this suggests that
the subcomplex performs a function distinct from NuA4’s role
in Htz1 acetylation. This idea is further solidified by the fact
that eaf3?, eaf5?, and eaf7? mutants do not display mutant
phenotypes that are shared with SWR complex, HTZ1, and
other NuA4 mutants implicated in Htz1 acetylation. For
example, eaf3?, eaf5?, and eaf7? mutants display either no or
only modest increases in chromosome segregation defects (31)
(data not shown), are not sensitive to benomyl (31), are not
defective in Htz1 K14 acetylation (28), and do not display
defects in vacuole morphology as detected with FM4-64 (Fig.
3A). As eaf5? and eaf7? mutants have similar effects on gene
expression (31), it is likely that the Eaf3-Eaf5-Eaf7 subcomplex
is required for a distinct subset of NuA4 cellular functions,
possibly through the recruitment of NuA4 to distinct
Our work indicates that genome-wide genetic interaction
maps not only provide valuable insights into query gene func-
tion and complex composition but also may be extremely use-
ful in discerning structural integrity and subcomplexes of large,
multisubunit protein complexes. It will be interesting to see
whether additional SGA screens using mutants with point or
domain mutations in NuA4 genes that specifically abolish pro-
tein interactions will provide greater insight into the function
of each NuA4 subunit.
We thank M. C. Keogh, J. Dillingham, and J. Greenblatt for pro-
viding yeast strains; M. C. Keogh, V. Measday, and members of the
Baetz laboratory for helpful discussion and critical reading of the
manuscript; A. Rudner and M. C. Keogh for technical support; and J.
Cote ´ for communicating results prior to publication.
This work was supported by operating grants from the National
Cancer Institute of Canada through funds raised by the Terry Fox
Research Foundation and an Early Research Award from the Ontario
Government (to K.B.). Funding was also received from the Canada
Foundation for Innovation, Natural Sciences and Engineering Re-
search Council of Canada; the Canadian Institute of Health Research
(CIHR); the Heart and Stroke Foundation of Ontario Centre for
Stroke Recovery; La Fondation Jean-Louis Le ´vesque; and the Ontario
Government (to D.F.). K.B. is a Canada Research Chair in Chemical
and Functional Genomics. D.F. is a Canada Research Chair in Pro-
teomics and Systems Biology. L.M was supported by an Ontario Grad-
uate Student Award and a CIHR Doctoral Award. J.-P.L was sup-
ported by an Ontario Graduate Student Award. I.S.S. was supported
by a Canadian Institute of Aging Investigator Award. A.S.A.-M. was
supported by an Ontario Women’s Health Council/CIHR Institute of
Gender and Health Fellowship.
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