A comprehensive synthetic genetic
interaction network governing yeast
histone acetylation and deacetylation
Yu-yi Lin,1,2Yan Qi,1,3Jin-ying Lu,1,4Xuewen Pan,1,2,6Daniel S. Yuan,1,2Yingming Zhao,5
Joel S. Bader,1,3and Jef D. Boeke1,2,7
1High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA;
2Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,
USA;3Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA;4Department
of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA;
5Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated
during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered
during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and
histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an
underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the
histone variant Htz1p at Lys 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of
H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of
NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found
that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an
elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These
new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic
interactions may help illuminate the mechanisms of intricate cellular processes.
[Keywords: Systems biology; histone; NuA4; acetylation; DNA repair]
Supplemental material is available at http://www.genesdev.org.
Received March 31, 2008; revised version accepted June 6, 2008.
Post-translational modifications of histones control many
DNA-related processes (Kouzarides 2007). Dynamic his-
tone (de)acetylation regulates gene transcription and si-
lencing, chromosome condensation, DNA replication, and
preservation of DNA integrity via DNA damage repair
(Millar and Grunstein 2006). There are over 20 known
histone acetyltransferases (HATs) and histone deacety-
lases (HDACs) in Saccharomyces cerevisiae; virtually all
function as protein complexes (Lee and Workman 2007;
Shahbazian and Grunstein 2007). These activities are co-
ordinated in the cell, and comprise a system that dy-
namically regulates chromatin state. Systems with this
many components are difficult to analyze using conven-
tional genetics and biochemical methods, although some
large-scale attempts have been made (Collins et al. 2007;
Mitchell et al. 2008). Comprehensive assessment of this
system is further complicated by the inclusion of essen-
tial genes (e.g., the essential acetyltransferase ESA1), re-
quiring suitable conditional or hypomorphic query al-
leles. Moreover, recent studies in higher organisms have
shown that HATs and HDACs have many substrates
apart from histones (Glozak and Seto 2007; Xu et al. 2007),
hinting that such substrates may exist in yeast as well.
Several recent studies have demonstrated that compre-
hensive genetic interaction profiling can effectively re-
solve complex pathways into conceptually and experi-
mentally tractable modules (Tong et al. 2004; Schuldiner
et al. 2005; Pan et al. 2006; Collins et al. 2007). Intergenic
interactions can be either aggravating (negative), such as
synthetic fitness or lethality defects (SFL), or alleviating
(positive) such as synthetic rescue (SR). The genes in-
volved can function either in a common essential path-
way or in distinct but compensatory pathways converg-
ing on the same essential function (Hartman et al. 2001).
Genetic interaction networks can be further organized
into interacting functional modules based on a statistical
analysis of the number of genetic interactions observed
between sets of genes (Hartman et al. 2001; Tong et al.
6Present address: Verna and Marrs McLean Department of Biochemistry
and Molecular Biology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA.
E-MAIL firstname.lastname@example.org; FAX (410) 502-1872.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1679508.
2062GENES & DEVELOPMENT 22:2062–2074 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org
2004; Schuldiner et al. 2005; Segre et al. 2005; Pan et al.
2006; Collins et al. 2007).
Here we present a comprehensive genetic interaction
network of HAT and HDAC protein complexes in yeast,
generated using “diploid-based Synthetic Lethality Analy-
sis on Microarray” (dSLAM) (Pan et al. 2006). The high
degree of connectivity in this network enabled classifi-
cation of interactions across protein complexes and iden-
tification of gene sets that function together as modules.
Analysis of these modules revealed that histone hyper-
acetylation is as deleterious as hypoacetylation, consis-
tent with previous studies showing that balanced acety-
lation status is crucial for cell viability (Vogelauer et al.
2000). Genetic interactions between HDAC complex
(Carmen et al. 1996) and complexes involved in acetyla-
tion or deposition of the histone H2A variant Htz1p led
us to demonstrate that HDA is the previously unidenti-
fied deacetylase for Htz1p. Other genetic interactions
revealed that Esa1p acetylates and stabilizes the nucleo-
some acetyltransferase of H4 (NuA4) subunit Yng2p, in-
sights that helped formulate a new model for how the
NuA4 complex undergoes rapid remodeling during the
repair of DNA double-stranded breaks (DSBs). These new
functions of the HDA and NuA4 complexes exemplify
the value of comprehensive surveys of genetic interac-
tions for exploring the roles of key protein complexes in
controlling the dynamic balance of acetylation and
deacetylation histones and other proteins.
Features of the interaction network
We used dSLAM to determine genome-wide genetic in-
teraction profiles of 38 query genes involved in histone
(de)acetylation. Query genes included the HAT and
HDAC catalytic subunits (Lee and Workman 2007) and
associated protein complex subunits. Mutations used as
queries included knockout deletions (KO) of 32 nones-
sential genes plus seven temperature-sensitive (Ts) or
hypomorphic alleles of six essential genes (Fig. 1A; also
see Supplemental Table S1 for a list of point mutations
of the Ts and hypomorphic alleles). None of the essential
genes had been used previously as query genes in genome
wide studies. We validated 2823 unique pairwise genetic
interactions involving 763 genes (∼12.5% of yeast genes)
by tetrad dissection and/or random spore analysis. There
were comprise 105 (∼4.2%) SR interactions, and 2718
SFL defects (Fig. 1A; see also Supplemental Fig. S1 for a
high-resolution image of the entire clustogram; see
Supplemental Table S2 for a complete list of genetic in-
teraction pairs). Only 14% of the interactions identified
here were reported previously. Essential query genes had
188 genetic interaction partners on average, whereas
nonessential counterparts had 42, comparable with pre-
vious studies (Tong et al. 2004; Davierwala et al. 2005).
We arranged query genes by hierarchical clustering
based on genetic interaction pattern similarities (Eisen et
al. 1998). The essential query genes involved in histone
acetylation formed a compact cluster, indicating that
their patterns of genetic interaction were correlated (Fig.
1A). By contrast, essential genes involved in different
biological processes shared little correlation with each
other: Two examples are shown in Supplemental Figure
S2 (CDC20 is the essential coactivator of anaphase-pro-
moting complex, while CDC45 is an essential DNA rep-
lication initiation factor). The interaction profiles iden-
tified using Ts alleles did not appear to be dominated by
temperature effects, since the genetic interaction profile
of esa1-Hm1, a hypomorphic allele of ESA1, with a lower
growth rate than wild-type allele at 30°C, most closely
resembled that of the Ts allele esa1-531.
To discern relationships among genes at a higher level
of organization, we examined interactions among sets of
genes whose products might function together as a com-
plex. We inferred membership in a complex from the
subunit composition of known protein complexes or
from highly interconnected patterns of genetic interac-
tions indicating functional similarity. These complexes
were regarded as nodes in a network in which the edges
connected those pairs of nodes exhibiting more genetic
interactions than expected by chance. Figure 2 shows a
subnetwork highlighting HATs and HDACs (Supplemen-
tal Fig. S3 shows a comprehensive network; Supplemental
Table S3 shows the complete data set). The resulting com-
plex-to-complex network was highly connected. The close
interactions among HATs and HDACs suggest that cells
must maintain global acetylation levels within a certain
range for viability. There were many aggravating inter-
actions between different functional modules collec-
tively comprising the same protein complex. Prominent
SFL (blue) network hubs (see Supplemental Fig. S4 for
degree distribution of network and hub definition) in-
cluded the NuA4 and SAGA (Spt–Ada–Gcn5–acetyl-
transferase) complexes and ACS2 (encoding an essential
acetyl-CoA synthetase supplying the nucleocytosolic
acetyl-CoA pool) (Takahashi et al. 2006).
The HDA complex was the most prominent alleviat-
ing (SR, red) hub, showing strong alleviating interactions
with the NuA4, SAGA, and Elongator complexes. Not
all deacetylase complexes had alleviating interactions
with these complexes; for example, large and small
forms of the Rpd3 complex [Rpd3C(L) and Rpd3C(S), re-
spectively] had alleviating interactions only with NuA4
core acetylation machinery and aggravating interactions
with most other HAT complexes, despite a broader his-
tone substrate spectrum than the HDA complex. This
finding suggests an unexpectedly detrimental effect of
the HDA complex when the balance of chromatin acety-
lation and deacetylation is tilted. Moreover, the strong
aggravating interaction between the HDA complex and
Rpd3C mutants, and the fact that the growth defect and
histone hyperacetylation of a hda1? rpd3? double mu-
tant can be partially rescued by esa1-531 or gcn5? (Fig.
1B,C), supports the idea that these two complexes are
collectively (and redundantly) responsible for bulk cellu-
lar histone deacetylation, and suggests an overlooked
role of deacetylation in maintaining cell viability.
Finally, our genetic interaction maps were enriched with
genes annotated by Gene Ontology (GO) as having vacu-
Yeast histone (de)acetylation network
GENES & DEVELOPMENT2063
tal Table S4 for a full list of enriched GO annotations),
which is also revealed in the comprehensive complex-
to-complex network (Supplemental Fig. S3). Similar en-
richments have been observed in other studies (Takaha-
shi et al. 2006; Mitchell et al. 2008). Previous expression
microarray experiments of major HATs and HDACs
(Choy and Kron 2002; Robyr et al. 2002; Le Masson et al.
2003; Huisinga and Pugh 2004; Zhang et al. 2004; Durant
and Pugh 2006) identified no significant change of tran-
scription of the key genes required for vacuolar/endo-
somal function. The genetic interaction and transcrip-
tional profiles suggest possible roles of these HATs and
HDACs in regulating extranuclear functions through
mechanisms other than regulation of transcription, re-
calling an apparent role of Elongator in regulating polar-
ized exocytosis (Rahl et al. 2005), and the importance of
the nuclear pore complex in controlling transcription
(Akhtar and Gasser 2007) and the targeting of DNA DSBs
to the nuclear periphery for efficient repair (N.J. Krogan,
and black boxes represent aggravating, alleviating, and no interaction, respectively. Only validated data were used in hierarchical
clustering and all subsequent computational analysis. The dendrogram of query genes was expanded for visualization, which indicated
the similarities of their interaction patterns. The cluster of essential query genes is highlighted inside the blue box. (B) esa1-531 and
gcn5? partially rescue the growth defect of hda1? rpd3? double mutants. Growth of each strain was assessed by plating four 10-fold
serial dilution on SC–Ura medium (for esa1-531 rescue experiment) or on YPD medium (for gcn5? rescue experiment) at 30°C, a
semipermissive temperature for esa1-531. (C) esa1-531 and gcn5? partially reversed hyperacetylation of histone H4 and H3, respec-
tively, in hda1? rpd3? double mutant at 30°C, a semipermissive temperature for esa1-531. The acetylation levels of H4 K5, H4 K8,
H4 K12, and H4 K16, and H3 K9 and H3 K14 were analyzed by immunoblot.
Global features of genetic interaction patterns. (A) Full hierarchical clustering of genetic interaction patterns. Blue, yellow,
Lin et al.
2064GENES & DEVELOPMENT
HDA deacetylates Htz1-K14Ac
Htz1p is an H2A variant isoform in yeast with important
roles in transcription, DNA replication, chromosome
segregation, and the delineation of heterochromatin
boundaries (Santisteban et al. 2000; Meneghini et al.
2003; Krogan et al. 2004a; Dhillon et al. 2006). Htz1p is
exchanged for H2A by the SWR-C chromatin remodeling
complex (Krogan et al. 2003; Kobor et al. 2004; Mizugu-
chi et al. 2004), and acetylation of Htz1p is required for
this process (Millar et al. 2006). Htz1p and SWR-C share
similar genetic interaction patterns, and thus have been
assigned to the same functional module (Krogan et al.
2003; Collins et al. 2007). It is known that NuA4 and
SAGA acetylate Htz1p at all four lysine residues in its
N-terminal tail (K3, K8, K10, K14), with K14 being the
most prominent acetylation site, but no Htz1p HDAC
has been identified previously (Keogh et al. 2006; Millar
et al. 2006). Htz1p-dependent genes tend to reside in
small clusters called Htz1-activated domains (HZADs),
and several of them overlap with Hda1-affected subtelo-
meric domains (HASTs) in subtelomeric chromatin (Me-
neghini et al. 2003). The two domains share the common
feature of Hda1p-directed hypoacetylation of histone H3,
which dampens the expression of their constituent
Previous studies revealed synthetic lethality between
HTZ1 and three genes of NuA4 (EAF1, EAF5, and EAF7),
as well as components of Rpd3C(L) (Krogan et al. 2003;
Kobor et al. 2004). Our dSLAM data demonstrated strong
aggravating genetic interactions between the SWR-C/
Htz1p module and all the functional modules of NuA4
and SAGA (Fig. 3A), as expected. We also observed a
significant alleviating genetic interaction between the
SWR-C/Htz1p module and the HDA complex (Fig. 3A).
Deletion of HDA1 rescued the slow growth phenotype of
the htz1? mutant and also reversed its sensitivity to the
genotoxic agents hydroxyurea (HU) and methyl meth-
anesulfonate (MMS) and to the microtubule-interfering
drug benomyl (Fig. 3B). Transcription of many Htz1p-
dependent genes was also restored in the hda1? htz1?
double mutant, including subtelomeric genes both in
and out of HASTs and genes far away from telomeres
(Supplemental Table S5). The ability of Htz1p to form a
boundary adjacent to the silent mating-type cassette
HMR was preserved in the hda1? strain (Supplemental
Fig. S6). In contrast to the other htz1? phenotypes, de-
letion of HDA1 could not rescue the boundary function
defect of htz1? that limits spread of silent chromatin from
HMR locus (Supplemental Fig. S7; Meneghini et al. 2003).
Based on these observations, we investigated whether
the HDA complex deacetylates Htz1p biochemically.
Acetylation level of Htz1p at Lys 14 dramatically in-
creased in vivo in the hda1? mutant, but not in any
other HDAC deletion strain (Fig. 3C). Addition of affin-
ity-purified Hda1p to Flag-tagged Htz1p purified from
the hda1? strain sharply diminished the increased level
of K14 acetylation (Fig. 3D). Deletion of HDA1 also re-
and functional modules. Pairs of nodes were linked by an edge if they had significantly more genetic interactions than expected by
chance. Node sizes proportional to the number of subunits in the corresponding complex or module; edge thicknesses signifies the
statistical significance of the enrichment in genetic interactions (thick and thin edges indicate P < 10−16and 10−16? P < 10−5,
The derived complex-to-complex genetic interaction network for HATs and HDACs. Nodes represent protein complexes
Yeast histone (de)acetylation network
GENES & DEVELOPMENT 2065
versed the sensitivity of htz1-K14R to benomyl (Fig. 3E;
Keogh et al. 2006), which suggests Hda1p may also
deacetylate other lysine residues. These results showed
that the HDA complex, previously regarded as an H3 and
H2B-specific HDAC (Wu et al. 2001), also counteracts
the effects of NuA4 and SAGA by removing a critical
acetyl group from Htz1p.
The alleviating genetic interactions between hda1 and
htz1 suggested the existence of parallel substrates for
Hda1p, which would be predicted to display SFL inter-
actions with the htz1? mutant and SR interactions with
the hda1? mutant. Histone H3 is a well-known sub-
strate of HDA complex (Wu et al. 2001), and an N-tail
deletion strain [hht1 (?1-36)] shows the expected genetic
interaction pattern (Fig. 3F). Moreover, hht1 (?1-36) has
genetic interaction with 88 out of 129 known genetic in-
teraction partner genes of htz1 [see Supplemental Table S6
for a complete list of genetic interactions of hht1 (?1-36)
H2A variant, and histone H3 share redundant functions.
Functional organization of NuA4—a new look
NuA4, the only essential HAT in yeast, acetylates the
N-terminal tails of H2A, H4 and Htz1p. The processes
are critical for regulating gene transcription, limiting the
spread of silent heterochromatin and repairing DNA
DSBs (Doyon and Cote 2004). Yeast cells lacking normal
NuA4 arrest at the G2/M phase in a RAD9-dependent
manner (Choy and Kron 2002). Esa1p is the essential
catalytic subunit of the NuA4 complex. It forms the core
acetylation machinery with Epl1p and Yng2p, termed
Piccolo NuA4, to maintain global histone H4 and H2A
acetylation in vivo (Boudreault et al. 2003; Selleck et al.
2005). Impaired function of any of the three component
genes drastically decreases catalytic activity of the com-
plex (Smith et al. 1998; Allard et al. 1999; Clarke et al.
1999; Choy and Kron 2002; Boudreault et al. 2003).
We found that the lethality of the esa1-531 and epl1-1
Ts alleles and the growth defect of yng2? could each be
suppressed by deletion of either HDA1 or RPD3 (Fig. 2).
Htz1-K14Acin vitro and in vivo. (A) Over-
lap of the enzyme–substrate relationship
with genetic interactions. A schematic
model depicts genetic and enzyme–sub-
strate interactions between HATs (NuA4
and SAGA), HDACs (HDA complex), and
their substrates (Htz1p and histone H3).
(B) Deletion of HDA1 rescues the slow-
growth and drug-sensitivity phenotype of
htz1?. Four tetrads dissected from an
hda1?/HDA1 htz1?/HTZ1 diploid strain
on YPD medium are shown. Drug sensi-
tivities were assessed by plating 10-fold se-
rial dilution on YPD medium containing
HU (200 mM), MMS (0.03%), or benomyl
(15 µg/mL). (C,D) Hda1p deacetylates
Htz1-K14Ac in vivo and in vitro. The K14
acetylation level in wild-type and HDAC
mutant strains was analyzed by immuno-
blot. (D) In vitro deacetylation activity of
Hda1-TAP purified by tandem affinity pu-
rification was assessed by incubating it
with Flag-tagged Htz1p purified from
hda1? strain and then monitoring the K14
acetylation level by immunoblot. No pro-
tein was added to the control samples. (E)
Deletion of HDA1 rescues the sensitivity
of htz1-K14R to benomyl. The experimen-
tal conditions are described in B. (F) Ge-
netic interactions of an N-tail deletion of
histone H3 [hht1(?1–36)] with hda1? (al-
leviating) and htz1? (aggravating) mu-
tants, suggesting that histone H3 reside in
the same functional module as Htz1p.
Lin et al.
2066GENES & DEVELOPMENT
In other words, growth defects resulting from impaired
NuA4 acetylation were rescued by compensatory loss of
either of the corresponding HDACs. We expected Ts mu-
tants esa1-531 and epl1-1 to cluster most tightly but sur-
prisingly, epl1-1 clustered with yng2? (Fig. 4A). Also, to
our surprise, esa1-531 had a distinct response to the
genetic interaction patterns shows functional association. Font colors indicate distinct functional modules (see legend). Subsets of
genetic interactions between NuA4 and other complexes were organized for visualization. (B) Core acetylation machinery mutants are
differentially sensitive to TSA. Growth curves of esa1-531, epl1-1, and yng2? mutants in SC–Ura medium at 37°C (restrictive
temperature) with or without TSA (100 µM) are shown. Error bars indicate ±1 SEM from three biological replicates. (C) ESA1 stabilizes
and controls the protein abundance of Yng2p. Wild-type (WT) and esa1-531 strains stably expressing tagged Yng2p or Epl1p were grown
at 25°C (permissive temperature) to OD600∼0.3, then shifted to 37°C or kept for 4 h at 25°C; whole-cell extracts (WCEs) were then
collected and probed with anti-HA, and band intensities were quantified. (D) Yng2-3HA turnover kinetics in wild-type and esa1-531
strains investigated by cycloheximide turnover experiments. Cells were grown at 25°C to OD600∼0.3, then either shifted to 37°C or
kept at 25°C, and treated with 0.1 µg/mL cycloheximide simultaneously. Equal amount of cells were collected at indicated time points
and analyzed by immunoblot as shown in Supplemental Figure S9A. The fraction of Yng2-3HA remaining after cycloheximide addition
was plotted. Error bars indicate ±1 SEM. (E) Yng2p is stabilized by MG-132 in esa1-531 cells. Wild-type and esa1-531 cells stably
expressing tagged Yng2p were incubated with MG-132 (75 µM) for 4 h at 25°C (permissive temperature) or 37°C (restrictive tempera-
ture), after which WCEs were analyzed by immunoblot.
Functional dissection of the NuA4 complex. (A) Hierarchical clustering of NuA4 component genes based on genome-wide
Yeast histone (de)acetylation network
GENES & DEVELOPMENT2067
HDAC inhibitor trichostatin A (TSA) to that of epl1-1
and yng2?. TSA at 100 µM, which inhibits most HDAC
activity of Hda1p and Rpd3p in vitro (Carmen et al.
1999), restored growth of epl1-1 and yng2? to that of
wild type, whereas esa1-531 lethality was only partially
rescued (Fig. 4B). This suggested a broader substrate spec-
trum for Esa1p than for NuA4 per se; thus, Esa1p might
act independently of other NuA4 components. Con-
versely, nicotinamide (a chemical that inhibits another
major type of HDAC named sirtuin) (Denu 2005) had
little effect on the three mutants (Supplemental Fig. S8),
and there were few SR interactions between sirtuin and
NuA4 genes (Fig. 2).
Based on the distinct behavior of ESA1 relative to
other genes encoding core NuA4 acetylation machinery,
we hypothesized that Esa1p performs functions beyond
its well-established global chromatin acetylation activ-
ity in the context of Piccolo NuA4. We discovered that
the abundance of Yng2-3HA (but not Epl1-3HA) depended
on normal ESA1 function (Fig. 4C). We also showed that
protein degradation (Fig. 4D; Supplemental Fig. S9A) and
not transcriptional activation (Supplemental Fig. S9B),
since Yng2p is stabilized by the proteasome inhibitor
MG-132 (Fig. 4E).
To further investigate the mechanism of Esa1p on
regulating the protein stability of Yng2p, we examined
the acetylation status of Yng2-Myc by immunoprecipi-
tation with mouse monoclonal anti-acetylated lysine.
To our surprise, a large proportion of Yng2p was acety-
lated in vivo (Fig. 5A). The signal of Yng2p acetylation
could be efficiently competed away with acetylated BSA
(Supplemental Fig. S10A), and mouse monoclonal anti-
HA could not pull down acetylated Yng2p to any detect-
able level (Supplemental Fig. S10B), indicating the speci-
ficity of the detected acetylation signals. The acetylation
level of Yng2p diminished dramatically in an esa1-531
mutant at restrictive temperature (Fig. 5A), suggesting
its acetylation depends on normal ESA1 function. A can-
didate lysine residue (K170) of Yng2p for acetylation was
identified by tandem mass spectrometry (Supplemental
Figure S11), which was confirmed by the loss of acetyla-
tion of Yng2p when substituting K170 with arginine
(K170R), a mutation blocking acetylation (Fig. 5B). A
whereas substituting K170 with glutamine (K170Q, a mu-
tation mimicking constitutive acetylation) did not cause
detectable change of protein abundance relative to the
wild type, but rendered it insensitive to the effects of an
esa1-531 Ts mutation (Fig. 5C). The K170R and K170Q
mutants were each hypersensitive to benomyl and MMS
(Fig. 5D), consistent with dynamic acetylation and
deacetylation of Yng2p affecting its normal function. A
screen of several known HDAC mutants identified in-
of Yng2p controls its protein stability and
function. (A) Yng2p is acetylated in vivo
through an Esa1p-dependent mechanism.
Wild-type (WT) and esa1-531 strains sta-
bly expressing Myc-tagged Yng2p were
grown at 25°C (permissive temperature) to
OD600∼0.3, then shifted to 37°C or kept
for 4 h at 25°C; WCEs were then collected,
immunoprecipitated with anti-Ac-K and
probed with anti-Myc. (B) K170 is the ma-
jor acetylated lysine residue of Yng2p in
vivo. K170 was identified by tandem mass
spectrometry. Substitution of K170 with
arginine (K170R) diminishes acetylation of
Yng2p. (C) Effects of K170 substitutions
on Yng2p stability. Cells were grown in
conditions describes in Figure 3A. WCEs
were collected and probed with anti-HA,
and band intensities were quantified.
K170R mutation causes decreased protein
abundance of Yng2p, while K170Q causes
no detectable change. (D) Effects of K170
substitutions on Yng2p function. Drug
sensitivities were assessed by plating 10-
fold serial dilution on YPD medium con-
taining MMS (0.03%) or benomyl (15 µg/
mL). (E) Yng2p is deacetylated through an
Cells were grown at 30°C to OD600∼0.6,
then WCEs were collected, immunopre-
cipitated with anti-Ac-K and probed with
Acetylation and deacetylation
Lin et al.
2068GENES & DEVELOPMENT
creased acetylation of Yng2p in rpd3? mutant (Fig. 5E),
suggesting that deacetylation of Yng2p in vivo depends
on the balance of normal ESA1 and RPD3 activities.
The genetic interaction profile of other NuA4 compo-
nents provided additional information on the functional
organization of the NuA4 complex (Fig. 4A). Arp4p is an
actin-related gene shared among the NuA4, Ino80, and
SWR-C chromatin remodeling complexes, and Arp4p is
important for recruiting NuA4 to DSBs and specifically
regulating the transcription of ESA1-dependent genes
(Galarneau et al. 2000; Downs et al. 2004). ARP4 was
previously assigned to the same module with three other
genes (ACT1, SWC4, and YAF9) also shared between
NuA4 and SWR-C (Doyon and Cote 2004; Auger et al.
2008). However, our genetic interaction data indicated
that ARP4 has a closer functional relationship to EAF1
than to SWC4 and YAF9 (Fig. 4A). This finding agrees
with previous chemical genomics surveys implicating
EAF1 in DNA damage repair (Parsons et al. 2004). Based
on this, we assign ARP4 and EAF1 to the DNA damage
repair and transcription regulation module, keeping
SWC4 and YAF9 in the SWR-C module.
Molecular choreography at DSBs
Given that Esa1p-dependent acetylation and Rpd3p-de-
pendent deacetylation controls the protein stability of
Yng2p, which is essential for the enzymatic activity of
Piccolo NuA4 on acetylating nucleosomal histones (Bou-
dreault et al. 2003; Selleck et al. 2005), we investigated
the influence of these post-translational modifications
on DSB chromatin dynamics. We used a galactose-induc-
ible DSB induction system to monitor protein species
and chromatin state at the DSB. The DSB, introduced by
HO endonuclease at the mating type locus (MAT), can
only be repaired by nonhomologous end joining in this
strain (Lee et al. 1998). In agreement with a previous study
(Downs et al. 2004), we found transient hyperacetylation of
histone H4 followed by a distinct hypoacetylation phase
near the DSB (Fig. 6A). This change and others reported
below were observed near the break and 2 kb distal to it
but not 10 kb away. The recruitment of NuA4 to DNA
DSBs is important for local acetylation of histone H4
(Bird et al. 2002; Downs et al. 2004; Tamburini and Tyler
2005), but the recruitment kinetics of Esa1p, Epl1p, and
Yng2p were distinct from that of bulk NuA4 (Fig. 6B;
Supplemental Fig. S12). Whereas Eaf1p, the only NuA4-
specific subunit protein not shared with any other pro-
tein complex (Auger et al. 2008), remained steadily
enriched near the DSB, Esa1p, Epl1p, and Yng2p were
initially recruited with kinetics similar to Eaf1p but
were evicted from the broken chromatin region at dis-
tinct rates thereafter. Addition of MG-132, a proteasome
inhibitor that prevents Yng2p degradation (Fig. 4E), greatly
delayed eviction of Yng2p from the DSB, suggesting an
ubiquitin based displacement mechanism (Fig. 6B). Con-
sistent with altered Yng2p recruitment kinetics, MG-132
also inhibited Ac-H4 depletion (Fig. 6A). In contrast,
MG-132 did not affect histone H3 eviction kinetics
(Supplemental Fig. S13). Rpn11p, a metalloprotease sub-
unit of the 19S regulatory particle of the 26S proteasome
lid (Verma et al. 2002), is concomitantly recruited to the
DSB, suggesting that the proteasome is responsible for
degrading Yng2p locally at the DSB (Fig. 6C).
A dynamic system with H4 acetylation followed by
deacetylation might be crucial for efficient DSB repair is
suggested by the hypersensitivity of GAL-HO strains to
MG-132 upon DSB induction by galactose (Supplemental
Fig. S14). Rpd3p was recruited to DSBs preceding
deacetylation of histone H4 and eviction of Yng2p (Fig.
6C), and addition of MG-132 did not change the kinetics
of Rpd3p recruitment (Supplemental Fig. S15). By con-
trast, Hda1p and Hos2p were not enriched at the DSB
(Supplemental Fig. S16). These data suggest that in addi-
tion to conducting global nucleosomal acetylation, Pic-
colo NuA4 is elaborately remodeled independently of
the rest of NuA4 locally at DSBs and that this molecular
choreography is governed by dynamic post-translational
modification occurring specifically at DSBs (Fig. 7).
In this study we systematically surveyed the functional
associations among genes that dynamically regulate his-
tone acetylation and deacetylation in yeast, generating a
comprehensive network of genetic interactions. The in-
clusion of six essential query genes significantly en-
hanced our ability to identify functionally important tar-
Our analysis focused on the abstracted network of
functional modules and protein complexes rather than
those of individual genes. The network was highly con-
nected, and revealed a close functional relationship be-
tween HAT and HDAC complexes, indicating that these
complexes share common essential cellular functions
despite the fact that most of them modify distinct spec-
tra of histone lysine residues. The HDA complex was the
most distinct SR hub, pointing to major counterbalanc-
ing effects on the NuA4, SAGA, and Elongator HAT
complex, which indicates that the HDA complex re-
moved the largest amount of acetyl groups. By contrast,
the Rpd3C had aggravating interactions with most of the
HAT complexes, except Piccolo NuA4; these interac-
tions are consistent with the cooperation between
Rpd3C(S) and various HATs in regulating transcriptional
elongation (Carrozza et al. 2005; Keogh et al. 2005; Li et
al. 2007), and also the role of Rpd3C in governing histone
H4 deacetylation following an acetylation conducted by
NuA4 in the vicinity of DNA DSBs.
Our results also revealed the general finding that the
HDA and Rpd3 complexes together define the major
HDAC activities that counteract the HAT activity of the
NuA4 and SAGA complexes, and that these complexes
provide the bulk control of the dynamic balance of global
histone acetylation and deacetylation essential to cell
viability. Requirement of histone acetylation by various
HATs for maintaining cell viability have been well stud-
ied (Smith et al. 1998; Zhang et al. 1998; Allard et al. 1999;
Clarke et al. 1999; Howe et al. 2001). Our data suggest that
Yeast histone (de)acetylation network
GENES & DEVELOPMENT2069
hyperacetylation of histone H3 and H4 in the hda1? rpd3?
double mutant is as detrimental to cell viability as hypo-
acetylation, and can be rescued when the responsible
acetylase activity is repressed.
In addition to its effect on many aspects of chromo-
some biology, a relationship between global histone
acetylation/deacetylation and vacuolar function is also
revealed by these studies. This relationship raises the
possible existence of nonhistone substrates of HATs and
HDACs in yeast.
The genetic interaction profile of htz1? and follow-up
experiments led us to conclude that the HDA complex,
previously known to acetylate histones H3 and H2B, re-
moves the acetyl group from K14 and possibly also other
lysine residues of the N-tail of Htz1p. However, lack of
reliable antibodies limited our ability to test the acety-
lation level of lysine residues other than K14. We also
propose that histone H3 and Htz1p reside in the same
functional module based on their similar genetic inter-
action profiles. However, although being less enriched in
the promoter region and defective in blocking telomeric
heterochromatin spreading, an unacetylatable Htz1p
alters the kinetics of histone H4 acetylation near an HO-induced DNA DSB. Chromatin immunoprecipitation (ChIP) assays with
anti-H4-K8Acwere used to assess kinetics of histone H4 acetylation. Enrichment of histone H4-K8Acwas quantified by RT–PCR and
normalized to the GEA2 internal control and also the local abundance of histone H3 (Supplemental Fig. S11). (B) NuA4 core acetylation
machinery components show distinctive kinetics of recruitment to an HO-induced DNA DSB. The enrichment of tagged Esa1p,
Yng2p, and Eaf1p after HO induction was assessed by ChIP assays with anti-HA. GAL-HO pdr5? strains were used in MG-132
experiments. (C) Proteasome and Rpd3C are recruited to an HO-induced DSB. Enrichment of tagged Rpn11p and Rpd3p after HO
induction was assessed by ChIP with anti-HA.
Post-translational protein modification of the NuA4 core acetylation machinery choreographs DSB repair. (A) MG-132
Lin et al.
2070 GENES & DEVELOPMENT
mutant (htz1-K3,8,10,14R) is insensitive to genotoxic
agents lethal to htz1?, suggesting that acetylation is im-
portant in some but not all aspects of Htz1p function
(Babiarz et al. 2006; Millar et al. 2006). The genetic in-
teractions between HTZ1 and its corresponding HATs
(ESA1 and GCN5) and HDAC (HDA1) suggest that these
complexes not only affect Htz1p modification but also
modulate the essential pathway through additional un-
In addition to examining functional relationships
among protein complexes, synthetic genetic interaction
profiles can be used for dissecting more elaborate protein
complexes like NuA4 into separate functional modules.
A recent genetic interaction survey of a subset of nones-
sential NuA4 subunits revealed that Eaf1p is important
for maintaining the integrity of NuA4 complex (Mitchell
et al. 2008). Here, comprehensive incorporation of most
essential and nonessential NuA4 subunit genes led to
many new findings. The genetic interaction profile of
esa1-531 revealed new functions of Esa1p beyond its
well-known nucleosomal acetylation activity. For ex-
ample, we found that the enzymatic activity of Piccolo
NuA4, the core acetylation machinery of NuA4, was
maintained by control of Esa1p on the protein turnover
of Yng2p through an acetylation-dependent mechanism.
Deacetylation of Yng2p was dependent on Rpd3p, which
potentially precedes the degradation of Yng2p by protea-
some. Tandem mass spectrometry and further biochemi-
cal experiments confirmed that K170 is the major acety-
lated lysine residue of Yng2p. The hypersensitivity of
both yng2-K170R and yng2-K170Q mutants (mutations
mimicking constitutive deacetylation and acetylation,
respectively) to benomyl and MMS suggests that dynamic
acetylation and deacetylation is important to its normal
function in DSB repair. Moreover, the recruitment kinet-
ics of the Piccolo NuA4 subunits to an HO-induced DSB
is distinct from the rest of NuA4. NuA4 is actively re-
cruited focally and rapidly hyperacetylates nearby his-
tone H4 at a DSB. We propose that the dynamic protein
turnover of Yng2p mediated by an acetylation–deacety-
lation cycle with cooperative recruitment of Rpd3C and
proteasome at DSBs disrupts the enzymatic activity of
Piccolo NuA4, which stops ongoing acetylation. This
finding is consistent with previous findings showing that
proteasome is involved in the repair of DSBs (Krogan et
al. 2004b). The recruited Rpd3C further removes acetyl
groups from histone H4, and ATP-dependent chromatin
remodeling complexes actively conduct nucleosome dis-
placement (Tsukuda et al. 2005). These three mecha-
nisms allow for dynamic hyperacetylation and subse-
quent hypoacetylation of histone H4 nearby facilitating
DSB repair (Fig. 7).
In this study, we applied genetic interaction analysis
on a large scale as a general approach for analyzing the
complexities of histone (de)acetylation in yeast. New
functions of the NuA4 and HDA complexes were iden-
tified, and a potential nonhistone substrate of Esa1p and
Rpd3p was found. Extensions of this powerful strategy to
mammalian systems will certainly be of interest in light
of recent advances in high-throughput methodologies
based on RNA interference (Silva et al. 2008).
Materials and methods
Full experimental details and data analysis methods
are provided in the Supplemental Material.
acetylation status near DSBs. Upon introduction of a DSB, such
as that caused by HO endonuclease at the HO recognition site,
NuA4 is actively recruited focally and rapidly hyperacetylates
nearby histone H4. The function of the core acetylation ma-
chinery of NuA4 is disrupted when Yng2p is deacetylated
through an Rpd3C-depedent mechanism followed by degrada-
tion by proteasome, followed by eviction of Esa1p and Epl1p.
Three distinct processes appear to be at play to facilitate tran-
sition to the hypoacetylation phase crucial for proper DSB re-
pair: (1) The breakdown of Piccolo NuA4 stops ongoing acety-
lation, (2) Rpd3C is recruited and could actively remove acetyl
groups, and (3) ATP-dependent chromatin remodeling com-
plexes, which conduct active nucleosome displacement.
Schematic model for the dynamic regulation of
Yeast histone (de)acetylation network
GENES & DEVELOPMENT 2071
Ts alleles creation
The method to generate Ts alleles was performed as described
(Huang et al. 2008). To construct a cloning vector, the 5? pro-
moter and 3? terminator regions of an essential gene were cloned
into a CEN plasmid with URA3 marker. A pool of mutations was
generated in a cassette containing the promoter, the open reading
frame, and the terminator regions by manganese-driven error-
prone PCR reaction. The mutagenized PCR products and the
digested cloning vector (to create a gap between the promoter
and the terminator) were then cotransformed into a haploid-
convertible heterozygous YKO strain of the corresponding es-
sential gene. Vectors harboring mutated cassettes were gener-
ated by recombinational gap repair. Cells containing a library of
mutations were sporulated and then subjected to selection for
Ura+-G418Rphenotype in magic medium without uracil (MM–Ura;
SC–LeuHis–Arg–Ura + canavanine + G418). These spores were
then incubated at 25°C or 37°C to screen for a Ts phenotype.
dSLAM and genetic interaction target gene validation
The synthetic lethality screen was performed as described previ-
ously (Pan et al. 2006). A pool of haploid-convertible heterozygous
diploid YKO library was transformed either once with a URA3
knockout cassette for nonessential query genes, or sequentially
with a natMX knockout cassette followed by YCplac33 harbor-
ing a Ts allele for essential query genes. The resulting hetero-
zygous double-mutant pool was then subjected to selection for
a mixed population of single and double mutants as the control
pool and a pure population of double mutants as the experimen-
tal pool in magic medium with the appropriate combination of
selecting drugs. Three different semipermissive temperatures
were tested for optimizing the screen conditions for essential
query genes. Strains with a control/experiment ratio ?2 in ei-
ther UPTAG or DNTAG were selected for validation either
with random spore analysis or tetrad dissection.
Microarray data were submitted to GEO with accession code
BioGRID database: http://www.thebiogrid.org; MIPS database:
http://mips.gsf.de; Saccharomyces genome database: http://www.
We thank W. Zachariae for providing the cdc20-1 Ts allele; E.M.
Cooper and J. Dai for gifts of reagents; members of the Boeke
laboratory for valuable discussions throughout the course of the
work; and P.B. Meluh, A. Norris, K.A. O’Donnell, and O.J. Rando
for their critical comments on the manuscript. Y.Q. is an IBM
predoctoral fellow. X.P. was a fellow of the Leukemia and Lym-
phoma Society. This work was supported by NIH Roadmap
grant “Technology Center for Networks and Pathways” (U54
RR 020839) and grant R01 HG 02432 to J.D.B.
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