Báo cáo khoa học: Site specificity of yeast histone acetyltransferase B complex in vivo pot

Site specificity of yeast histone acetyltransferase B
complex in vivo
Ana Poveda* and Ramon Sendra
Departament de Bioquı
´
mica i Biologı
´
a Molecular, Universitat de Vale
`
ncia, Spain
Histone acetylation is a highly dynamic post-transla-
tional modification involved in the regulation of chro-
matin activity in eukaryotic organisms [1,2]. Although
the mechanism is not completely understood, the long-
known link between histone acetylation and gene
expression was definitively settled by the identification
of a number of transcriptional regulators as histone
acetyltransferases (HATs) and histone deacetylases.
Acetylation influences transcription by facilitating the
access of the transcriptional machinery to the DNA
sequence and by creating specific recognition sites for
regulatory proteins that promote transcription [1].
Histone acetylation has also been proposed to be
involved in chromatin assembly during replication
[1,3]. This notion emerged from the finding in different
eukaryotic organisms that newly synthesized histones
are acetylated [4,5], and deacetylated shortly after their
incorporation into chromatin [6]. In many eukaryotes,
newly synthesized histone H4 assembled onto nascent
DNA is diacetylated on Lys5 and Lys12 [5,7]. The
N-terminus of newly synthesized histone H3 is also

acetylated, but in a more heterogeneous and less con-
served manner [5,8,9]. It is considered that the acetyla-
tion of histones may somehow favor their deposition
onto DNA mediated through specific interactions with
histone chaperones [1].
The enzyme that is assumed to catalyze the specific
acetylation of newly synthesized histone H4 on its
N-terminal tail is the type B HAT, HAT-B complex.
Enzymes operationally classified as type B, in contrast
Keywords
acetylation; acetyltransferase; chromatin;
histones; yeast
Correspondence
R. Sendra, Departament de Bioquı
´
mica i
Biologia Molecular, Facultat de Cie
`
ncies
Biolo
`
giques, C ⁄ Dr Moliner 50, 46100-
Burjassot, Vale
`
ncia, Spain
Fax: +34 96 354 4635
Tel: +34 96 354 3015
E-mail:
*Present address
IGH-Institute of Human Genetics, CNRS

Montpellier, France
(Received 25 January 2008, revised 25 Feb-
ruary 2008, accepted 28 February 2008)
doi:10.1111/j.1742-4658.2008.06367.x
Saccharomyces cerevisiae Hat1, together with Hat2 and Hif1, forms the his-
tone acetyltransferase B (HAT-B) complex. Previous studies performed
with synthetic N-terminal histone H4 peptides found that whereas the
HAT-B complex acetylates only Lys12, recombinant Hat1 is able to modify
Lys12 and Lys5. Here we demonstrate that both Lys12 and Lys5 of solu-
ble, non-chromatin-bound histone H4 are in vivo targets of acetylation for
the yeast HAT-B enzyme. Moreover, coimmunoprecipitation assays
revealed that Lys12 ⁄ Lys5-acetylated histone H4 is bound to the HAT-B
complex in the soluble cell fraction. Both Hat1 and Hat2, but not Hif1, are
required for the Lys12 ⁄ Lys5-specific acetylation and for histone H4 bind-
ing. HAT-B-dependent acetylation of histone H4 was detected in the solu-
ble fraction of cells at distinct cell cycle stages, and increased when cells
accumulated excess histones. Strikingly, histone H3 was not found in any
of the immunoprecipitates obtained with the different components of the
HAT-B enzyme, indicating the possibility that histone H3 is not together
with histone H4 in this complex. Finally, the exchange of Lys for Arg at
position 12 of histone H4 did not interfere with histone H4 association
with the complex, but prevented acetylation on Lys5 by the HAT-B
enzyme, in vivo as well as in vitro.
Abbreviations
FACS, fluorescence-activated cell sorting; H4K12ac, histone H4 isoform with acetylated Lys12; H4K12R, K12R substitution mutant of
histone H4; H4K5ac, histone H4 isoform with acetylated Lys5; HA, hemagglutinin; HAT, histone acetyltransferase; HAT-B, histone
acetyltransferase B; HU, hydroxyurea; WCE, whole cell extract.
2122 FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS
to type A, only acetylate histones not associated with
DNA, and are not involved in transcriptional regu-

lation. HAT-B enzymes were originally isolated from
cytosolic extracts [10–15], but several immunolocaliza-
tion analyses have indicated a mainly nuclear localiza-
tion [16–20]. In vitro, native HAT-B enzymes from
a wide variety of species establish the specific
Lys5 ⁄ Lys12 acetylation pattern characteristic of newly
synthesized histone H4 [10,13,15–17,21–23]. In the
yeast Saccharomyces cerevisiae, the HAT-B complex
consists of at least three protein subunits [19,20]: the
catalytic subunit, Hat1; the enzymatic activity stimula-
tory protein, Hat2 [13]; and Hif1, which, in vitro, has
histone chaperone and chromatin assembly activity
[20]. Recently, Hat1 and Hat2 have been found to
interact with the origin recognition complex, suggest-
ing a novel role for the Hat1–Hat2 subcomplex at the
replication fork [24]. It has been reported that
although recombinant Hat1 is able to modify Lys5
and Lys12 [13,25], the isolated HAT-B complex exclu-
sively acetylates Lys12 of histone H4 [13,22]. Deletions
of HAT1, HAT2 or HIF1 produce no apparent pheno-
type [13,19,20,22], but combined with specific muta-
tions in the N-terminus of histone H3, cause defects in
both telomeric gene silencing [19,20,26] and resistance
to DNA-damaging agents [20,27]. Such defects are
reproduced by the substitution of Lys for Arg at posi-
tion 12 of histone H4, but not at position 5 [26,27].
Moreover, Hat1 is recruited to the sites of DNA
double-strand breaks, where it is specifically required
for the histone H4 acetylation on Lys12, but appar-
ently not on Lys5 [28].

Despite many correlations linking the acetylation of
histone H4 with chromatin assembly, direct evidence
actually indicates that the specific histone H4
Lys5 ⁄ Lys12 diacetylation pattern, and also the HAT-B
enzymes that generate it, are dispensable for this pro-
cess. In yeast, the substitution mutation of Lys5 and
Lys12 of histone H4, in combination with deletion of
the histone H3 N-terminus, does not result in defective
chromatin assembly, either in vitro or in vivo [29],
although the acetylation state of newly synthesized
yeast histone H4 is not known. Likewise, in chicken
DT40 cells, it has been shown that HAT1 is not neces-
sary for replication-coupled chromatin assembly [30].
Thus, the biological role of the conserved Lys5 ⁄ Lys12
acetylation of histone H4 and hence the function of
the HAT-B enzymes found in all eukaryotes are elu-
sive.
Many reports have described the characterization
and the site specificity of type B enzymes from differ-
ent species in vitro [10,13,15–17,21–23,31], but analyses
of their in vivo specificity are few and not at all conclu-
sive [19]. Only recently has it been demonstrated in
chicken DT40 cells that the homozygous HAT1 dele-
tion results in a reduced diacetylation level on Lys5
and Lys12 of histone H4 in a cytosolic extract [30].
S. cerevisiae Hat1 was the first HAT to be identified
[22], and moreover its biochemical properties, both as
an isolated subunit and as part of the HAT-B complex
[13,19,20,25,31–33], have been studied. Despite all
these studies, its in vivo site specificity has not been

directly ascertained.
In this article, we demonstrate that both Lys12 and
Lys5 of non-chromatin-bound histone H4 are authen-
tic targets of acetylation for the S. cerevisiae HAT-B
complex in vivo . Moreover, these positions are acety-
lated in histone H4 associated with the HAT-B enzyme
from the yeast soluble fraction. The requirements for
the distinct components of the complex for the acetyla-
tion and the association of histone H4 have also been
analyzed.
Results
Direct identification of Lys12 and Lys5 of soluble,
non-chromatin-bound histone H4 as in vivo
targets of acetylation by yeast Hat1
Previous work in our laboratory failed to detect any
defect in the in vivo steady-state level of acetylation on
Lys12 of histone H4 in hat1, hat2 or hif1 null mutant
strains as compared to the wild-type under normal
growth conditions [19]. The apparent independence of
histone H4 Lys12 acetylation from the HAT-B enzyme
in vivo was actually interpreted as a consequence of the
very short half-life of this modification, which would
make detection difficult.
We persisted in investigating the in vivo specificity of
the yeast HAT-B complex, and found that incubation
of cells with hydroxyurea (HU) resulted in an increase
of the histone H4 isoform with acetylated Lys12
(H4K12ac) in a HAT1-dependent manner (Fig. 1A).
HU is a ribonucleotide reductase inhibitor that causes
a depletion of deoxynucleotides, and thereby slows

down DNA synthesis. The acetylation analysis was
carried out by immunoblotting with a specific antibody
to H4K12ac. Cells were incubated in the presence of
200 mm HU (a concentration commonly used to syn-
chronize yeast cultures) for the indicated time periods.
In wild-type cells, HU promoted acetylation of his-
tone H4 Lys12, which is reflected by an increase in the
H4K12ac level 2 h after HU addition. In contrast, the
H4K12ac amount did not significantly change in hat1D
mutant cells, even after 12 h of incubation (Fig. 1A).
Importantly, an antibody against the C-terminus of
A. Poveda and R. Sendra Yeast histone acetyltransferase B complex
FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS 2123
histone H3 (anti-H3Ct), used as a control for histone
loading, did not detect differences in the amount of
histone H3 between the two strains, indicating that
cells lacking Hat1 display normal levels of histones
during the course of HU treatment.
To investigate whether HU induces an increase of
the Hat1 protein, we used a yeast strain expressing a
hemagglutinin (HA)-tagged version of Hat1. The
Hat1–HA protein level did not increase with HU incu-
bation time, but actually slightly diminished (Fig. 1B).
Apparently, HU treatment does not alters the enzy-
matic activity of the HAT-B complex, as the chro-
matographic HAT profiles and activity levels, in
particular that corresponding to the HAT-B peaks,
were very similar in HU-treated and untreated cells
(supplementary Fig. S1).
We investigated whether HAT1-dependent his-

tone H4 Lys12 acetylation was also increased in
response to other genotoxic agents, such as methyl-
methanesulfonate, phleomycin, and 4-nitroquinoline
n-oxide (4NQO). Like HU, these other agents
increased the amount of H4K12ac in wild-type but not
in hat1D cells (supplementary Fig. S2). Fluorescence-
activated cell sorting (FACS) analysis (supplementary
Fig. S2) revealed a certain degree of qualitative corre-
lation between the H4K12ac level and the enrichment
of the culture in S-phase cells. The most potent effect
on both was generated by HU.
In order to further examine the effect of HAT1 dele-
tion on acetylation of histone H4 Lys12, we purified
histones from wild-type and hat1D mutant yeast chro-
matin, before and after incubation with 200 mm HU
for 3 h. In agreement with our previous results [19],
immunoblotting analysis revealed no difference in his-
tone H4 Lys12 acetylation between purified histones
from wild-type and mutant cells left without HU treat-
ment. However, in striking contrast to the results
obtained with whole cell extract (WCE), we did not
observe a significant difference in histone H4 Lys12
acetylation between the two strains after HU incuba-
tion (Fig. 2A). As histones were obtained from isolated
chromatin, these results show that HU-induced, Hat1-
dependent histone H4 acetylation (Fig. 1A) is
restricted to non-chromatin-bound, soluble, ‘free’ his-
tone H4. To investigate this further in yeast, sphero-
plasts of wild-type and hat1D cells (HU-treated and
untreated) were lysed and fractionated by centrifuga-

tion into soluble and chromatin pellet fractions, as
shown in Fig. 2B. A significant amount of H4K12ac
was found in the soluble fraction of wild-type cells
after incubation with HU, but not in hat1D mutant
cells (Fig. 2C). H4K12ac was even detected in the solu-
ble fraction of untreated wild-type cells, although its
level increased substantially after treatment with HU.
In addition, antibodies against the recombinant yeast
full-length histone H4 (anti-ryH4) and the C-terminus
of histone H3 (anti-H3Ct), which recognize the corre-
sponding histones independently of the modification
state, revealed that HU treatment increased the
amount of soluble histone H4 and histone H3, as had
been previously described [4,15,34]. Such an accumula-
tion of histones was identical in wild-type and hat1D
mutant cells. With respect to the chromatin fractions,
histone H4 Lys12 acetylation was not significantly dif-
ferent between wild-type and hat1D cells, supporting
the results obtained with purified histones.
We investigated the requirement for Hat1 for acety-
lation of other acetylatable positions on histone H4
and histone H3 in the soluble fraction (Fig. 3). The
results clearly indicate that histone H4 Lys5 is an
authentic target for Hat1 in vivo. As shown in the
immunoblot in Fig. 3, like H4K12ac, the histone H4
isoform with acetylated Lys5 (H4K5ac) was detected
Fig. 1. Hydroxyurea (HU) treatment of yeast cells reveals the
involvement of Hat1 in the acetylation of histone H4 on Lys12
in vivo. (A) Solid HU was added to exponential-phase cultures of
strains W303-1a (wild-type; +) and RS1263 (hat14; )) to a final

concentration of 200 m
M and, at the indicated time points, equal
amounts of cells were collected, and used for preparation of
WCEs. Proteins were resolved by 15% SDS ⁄ PAGE, and transferred
to a nitrocellulose membrane. The membrane was stained with
Ponceau S (upper panel), and probed with the antiserum against
histone H4 acetylated on Lys12 (middle panel). As a specific load-
ing control for histones, a second immunoblot with an antibody
against the C-terminus of histone H3 (a-H3Ct, lower panel) was
carried out. (B) Strain BQS1154, expressing HA-tagged Hat1, was
incubated with 200 m
M HU, and, at different time points, cells from
identical volumes were processed to obtain WCEs. Hat1 protein
levels were revealed by immunoblotting with mouse 12CA5 anti-
body against the HA epitope. Mr, molecular mass markers.
Yeast histone acetyltransferase B complex A. Poveda and R. Sendra
2124 FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS
in the soluble fraction of wild-type cells, but not of
hat1D mutant cells. In addition, HU treatment also
increased the amount of Hat1-dependent H4K5ac. In
contrast, acetylation at the other potentially acetylat-
able sites within the histone H4 N-terminus, Lys8 and
Lys16, was hardly visible on soluble histone H4,
although strong bands on histone H4 in purified con-
trol histones were observed. In any case, their acetyla-
tion levels were independent of Hat1.
In budding yeast, there is evidence that the N-termi-
nal tail of newly synthesized histone H3 is monoacety-
lated preferentially on Lys9, but also on Lys14, Lys23,
or Lys27 [8]. Except for Gcn5, which is responsible for

histone H3 Lys9 acetylation [9], the acetylation
enzymes for the other positions are unknown. We
detected histone H3 acetylated at these positions in the
soluble fraction, although with varying degrees of
intensity. Except for the histone H3 isoform with acet-
ylated Lys14, which apparently did not change, the
other three histone H3 isoforms increased after HU
treatment. In neither case did loss of Hat1 have any
effect on acetylation at these Lys residues (Fig. 3).
Recent evidence also indicates acetylation in the glob-
ular domains of histone H3 and histone H4. In yeast,
acetylation of histone H3 Lys56 and histone H4 Lys91
has been described, and both seem to be linked to
nucleosome assembly [35,36]. As shown in Fig. 3, the
histone H3 isoform with acetylated Lys56 and the his-
tone H4 isoform with acetylated Lys91 were detected in
the soluble fraction, and the levels of both were signifi-
cantly increased by HU treatment, but the amount of
neither of them was dependent on the presence of Hat1.
Although some caution must accompany the
interpretation of the immunoblotting assays, due to a
possible lack of reactivity or specificity of the anti-
bodies, our results indicate that Hat1 is apparently not
involved in the acetylation of any site on soluble
histone H4 and histone H3 except for Lys12 and Lys5
of histone H4.
Involvement of different components of the yeast
HAT-B complex in the acetylation of soluble
histone H4
We examined the presence of histone H4 acetylation

on Lys12 and Lys5 in soluble fractions obtained from
wild-type and hat1D, hat2D and hif1D deletion strains.
Deletion of the HAT2 gene caused a considerable
reduction in the acetylation of Lys12 and Lys5 of solu-
ble histone H4 (Fig. 4). Consistently, very low immu-
nosignals were also obtained in soluble fractions of
HU-treated hat1D and hat2D cells. In contrast, the
levels of Lys12 and Lys5 acetylation were equal in
wild-type and hif1D soluble fractions. On the other
Fig. 2. Hat1 acetylates Lys12 of soluble,
non-chromatin-bound histone H4 in vivo. (A)
Histones purified from wild-type (W303-1a)
and hat1D mutant (RS1263) cells, before
and after incubation with HU, were sepa-
rated by SDS ⁄ PAGE and immunoblotted
with antibody to H4K12ac (lower panel). His-
tone species are indicated on the Pon-
ceau S-stained membrane (upper panel). In
order to facilitate the detection of even
small differences in acetylation degree, two
distinct amounts of histones were loaded
(1.0 and 0.25 lg). (B) Schematic representa-
tion of the experimental procedure used to
fractionate yeast cells. (C) Equal numbers of
wild-type and hat1D mutant cells were har-
vested at 0 or 4 h after incubation with HU.
Soluble and chromatin-associated proteins
(pellet) were fractionated as illustrated in
(B), and histones in both fractions were ana-
lyzed by immunoblotting with antibody to

H4K12ac, anti-ryH4, and anti-H3Ct. Owing
to the low level of histones in the soluble
fraction, approximately 10 times more was
loaded as compared to the pellet fractions.
A. Poveda and R. Sendra Yeast histone acetyltransferase B complex
FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS 2125
hand, the amount of total soluble histone H4 was
similar in all four strains, and was equally increased by
HU treatment (as revealed with anti-ryH4). These data
indicate that Hat2, but not Hif1, participates in the
catalytic function of the HAT-B complex in vivo.
Hat1-dependent acetylation of soluble histone H4
throughout the cell cycle and in Rad53-deficient
cells
We previously observed fairly constant levels of yeast
Hat1 protein throughout the cell cycle [19]. We there-
fore checked the presence of soluble H4K12ac at
distinct cell cycle stages. Wild-type and hat1D mutant
cells growing asynchronously were left without treat-
ment or incubated with either a-factor, which arrests
cells in G
1
phase, or hydroxyurea or nocodazole,
which prevent the G
2
⁄ M transition, or transferred to
minimal medium without a nitrogen source, which
arrests cells in G
0
phase. The cell cycle phases of the

arrested cells were confirmed by DNA flow cytometry.
Soluble histone H4 Hat1-dependently acetylated on
Lys12 was present in cells arrested at all cell cycle
stages, G
1
,S,G
2
⁄ M and also G
0
(Fig. 5A). Similar
results were obtained with cells at different cell cycle
stages from synchronized cultures by release from a
a-factor block [19] (results not shown).
In S. cerevisiae, the checkpoint protein kinase Rad53
regulates histone protein levels, and thus Rad53-defi-
cient yeast cells exhibit abnormally high amounts of
soluble histones [34]. We therefore investigated whether
such an excess of soluble histone H4 is also acetylated
by Hat1. For this purpose, we deleted the HAT1 gene
in wild-type and rad53D mutant strains, and examined
the levels of H4K12ac in the corresponding soluble
fractions (Fig. 5B). As expected, asynchronously grow-
ing rad53D mutant cells displayed a higher amount of
soluble histone H4 than wild-type cells, but only the
excess soluble histone H4 from HAT1 cells was acety-
lated on Lys12. The accumulation of HAT-B-dependent
acetylation of Lys12 in Rad53-deficient cells was further
confirmed on yeast strains harboring the chromosomal
Fig. 3. Histone H4 Lys12 and histone H4 Lys5 are the only acetyla-
tion sites in soluble histone H4 and histone H3 that are dependent

on Hat1. Soluble histones from wild-type (W303-1a) or hat1D
(RS1263) cells, in the presence or absence of HU, were analyzed
by immunoblotting using antibodies against different acetylated iso-
forms of histone H4 and histone H3. A Ponceau S-stained mem-
brane (top panel) and an immunoblot with anti-H3Ct (lowest panel)
are shown as a loading control. Purified yeast histones (yhis) were
included to check antibody reactivity.
Fig. 4. Acetylation on Lys12 and Lys5 of soluble histone H4 by the
HAT-B complex in vivo. Soluble fractions were prepared from
wild-type (W303-1a), hat1D (RS1263), hat2D (YSTT11) and hif1D
(YSTT49) yeast cells, before and after incubation with 200 m
M HU
for 3 h. Histones in these extracts were analyzed by immunoblot-
ting using antibody to H4K12ac, antibody to H4K5ac, and anti-ryH4.
Yeast histone acetyltransferase B complex A. Poveda and R. Sendra
2126 FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS
RAD53 gene under the glucose-switched off GAL1 pro-
moter in wild-type, hat1D, hat2D or hif1 D strains
(supplementary Fig. S3). Results showed that Lys12
acetylation of excess soluble histone H4 present in
Rad53-deficient cells was absolutely dependent on Hat1
and Hat2, but not on Hif1.
Histone H4 acetylated on Lys12 and Lys5 is
associated with the HAT-B complex in the yeast
soluble fraction
To gain further insights into the organization and the
molecular determinants of the yeast HAT-B complex,
we attempted to determine: (a) whether HAT-B
enzyme, present in the soluble fraction, contains asso-
ciated histone H4; (b) the acetylated sites; and (c) the

involvement of the different HAT-B components in
histone H4 binding. To address these questions, we
performed immunoprecipitation experiments with solu-
ble extracts from yeast strains that express tagged
forms of each of the three components of the HAT-B
complex (Hat1–HA, Hat2–HA, and Hif1–Myc). Im-
munoprecipitates (bound fractions), input materials
and unbound materials were examined by immuno-
blotting with antibodies against histone H4, his-
tone H3, and acetylated isoforms. All three HAT-B
components, Hat1, Hat2 (Fig. 6A, lanes 6 and 15,
respectively) and Hif1 (Fig. 6B, lane 27) coimmunopre-
cipitated H4K12ac. Furthermore, histone H4 present
in the soluble extracts from yeast cells lacking Hat1 or
Hat2 was not coprecipitated with any of the other
complex components (Fig. 6A, lanes 9 and 21; and
Fig. 6B, lanes 30 and 33), indicating that both Hat1
and Hat2 are necessary for histone H4 binding. In
contrast, both Hat1 and Hat2 were still able to copre-
cipitate H4K12ac in the absence of Hif1 (Fig. 6A,
lanes 12 and 18), indicating that Hif1 is dispensable
for the interaction of histone H4 with Hat1 ⁄ Hat2.
Results corresponding to Fig. 6A,B were entirely
reproduced when blots were probed with the antibody
to H4K5ac (results not shown).
When the blots were probed with anti-H3Ct, an im-
munosignal was not obtained in any of the immuno-
precipitates of Hat1, Hat2, or Hif1 (Fig. 6A, lanes 6,
Fig. 5. Soluble histone H4 at different cell
cycle stages and excess histone H4 in

Rad53-deficient cells is acetylated by the
HAT-B complex. (A) Wild-type and hat1D
mutant cells were grown asynchronously to
exponential phase, and either harvested
(asy.) or arrested in G
1
phase (by incubation
with 4.5 lgÆmL
)1
a-factor for 3 h; a), in
S phase (200 m
M HU for 3 h), in
G
2
⁄ M phase (15 lgÆmL
)1
nocodazole, 3 h);
NZ and in G
0
phase by nitrogen deprivation
for 14 h (DN). Cells were fractionated, and
the soluble fractions were analyzed by
immunoblotting with antibody to H4K12ac
and anti-ryH4. Cell cycle stages were
monitored by FACS (bottom). (B) Soluble
fractions from the yeast strains YAV49
(sml1D), BQS1386 (hat1D, sml1D), YAG101
(rad53D, sml1-1) and BQS1358 (hat1D,
rad53D, sml1-1) were analyzed with
antibody to H4K12ac and anti-ryH4. These

strains also bear an sml1 mutation to
suppress the lethality due to rad53 deletion
[34].
A. Poveda and R. Sendra Yeast histone acetyltransferase B complex
FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS 2127
12, 15 and 18; and Fig. 6B, lane 27). These results are
disturbing, because it is assumed that histone H3 and
histone H4 form tetramers [37] or dimers [38], with an
equal stoichiometry. It is well known that histone H3
is particularly susceptible to proteolytic degradation.
We cannot completely rule out the possibility that his-
tone H3 proteolysis is also occurring in yeast soluble
extracts in our experiments, but its presence in input
and unbound fractions argues against this possibility.
Moreover, intact histone H3, and also H4K12ac, were
detected in immunoprecipitates from soluble extracts
of cells expressing Flag-tagged Cac1 or Asf1, two his-
tone H3 ⁄ H4 chaperones (Fig. 6C). These additional
controls also indicate the absence of specific his-
tone H3 degradation under our immunoprecipitation
assay conditions. Likewise, none of the specific anti-
bodies to acetylated histone H3 used in Fig. 3 gener-
ated immunosignals corresponding to histone H3 on
the HAT-B complex immunoprecipitates (results not
shown). Altogether, our data suggest that histone H3
is not part of the HAT-B complex in the soluble frac-
tion of yeast cells.
In vivo, the HAT-B complex requires an
acetylatable Lys at position 12 for acetylation
on Lys5, but not for binding histone H4

Recombinant yeast Hat1, as well as native HAT-B
enzymes from various species, modify histone H4
in vitro on Lys12 preferentially over Lys5
[13,17,22,23,25,31]. Thus, HAT-B complex acetyltrans-
ferase activity results in an ordered acetylation, with
Lys12 being acetylated before Lys5 [23,31]. To investi-
gate the in vivo requirement for histone H4 Lys12 on
Fig. 6. Histone H4 acetylated on Lys12 and Lys5 is bound to the HAT-B complex in the soluble cell fraction. Soluble extracts of yeast cells
expressing Hat1–HA, Hat2–HA (A) or Hif1–Myc (B) were used for immunoprecipitation with rat monoclonal antibody to HA (3F10) and mouse
monoclonal antibody to Myc (9E10), respectively. Yeast strains expressing a tagged HAT-B component, but with a deletion of any other
companion protein (hat1D, hat2D,orhif1D), were also examined. The input (I), unbound (U) and bound (B) fractions were analyzed by immu-
noblotting with antibody to H4K12ac, anti-ryH4, and anti-H3Ct. Untagged wild-type strain (wt, no tag) W303-1a was used as a negative con-
trol. (C) Extracts of exponentially growing cells expressing Cac1–Flag or Asf1–Flag were used for immunoprecipitation with Flag M2 antibody
agarose beads. I, U and B fractions were probed by immunoblotting with anti-H3Ct or antibody to H4K12ac. Purified yeast histones (yhis)
were used as a control. Yeast strains (with the relevant gene modifications in parentheses) used in these experiments were: (A) BQS1154
(HAT1-HA); BQS1189 (HAT2-HA); BQS1172 (HAT1-HA, hat2D); BQS1184 (HAT1-HA, hif1D); BQS1309 (HAT2-HA, hat1 D) and BQS1304
(HAT2-HA, hif1D); (B) BQS1187 (HIF1-MYC); BQS1202 (HIF1-MYC, hat1D); and BQS1225 (HIF1-MYC, hat2D); (C) YAV49 (CAC1-FLAG); and
YAV52 (ASF1-FLAG).
Yeast histone acetyltransferase B complex A. Poveda and R. Sendra
2128 FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS
the acetylation of Lys5 and also on the histone
H4–HAT-B complex association, we made use of yeast
strains expressing wild-type or a K12R substitution
mutant histone H4 (H4K12R) from a centromeric
plasmid as the only source of histone H4. In addition,
these strains contained Hat1 or Hif1 tagged with the
HA epitope. First, we checked that K12R substitution
does not interfere with the recognition of H4K5ac by
the antibody to H4K5ac (supplementary Fig. S4). In
agreement with this, antibody to H4K5ac yielded

bands with similar intensity on WCEs prepared from
cells containing wild-type histone H4 or H4K12R
(Fig. 7A). However, when soluble fractions were ana-
lyzed with the same antibody, cells expressing wild-
type histone H4 were characterized by a well-defined
band, whereas in cells expressing H4K12R, only a very
weak signal was detected (Fig. 7A). These results
imply that an acetylatable Lys at position 12 is
essential for the efficient acetylation of Lys5 of soluble
histone H4.
Analyses of histone H4 association with the HAT-B
complex were performed by coimmunoprecipitation
and subsequent immunoblotting. As expected, the
antibody to H4K12ac (control) generated a signal in
immunoprecipitates from soluble extracts containing
wild-type histone H4 (Fig. 7B, lanes 3 and 9) but not
in those containing H4K12R (Fig. 7B, lanes 6 and 12).
Remarkably, anti-ryH4 revealed that as much Hat1–
HA as Hif1–HA coimmunoprecipitated both wild-type
and K12R mutant histone H4 (Fig. 7B, lanes 3, 6, 9
and 12). This finding demonstrates that Lys12 and its
acetylation are not involved in the binding of his-
tone H4 to the HAT-B complex. In addition, the anti-
body to H4K5ac revealed that H4K12R coprecipitated
with Hat1 was acetylated very weakly on Lys5
(Fig. 7B, lane 6).
Finally, we investigated the in vitro activity of yeast
Hat1 and Hat1-dependent type B complex towards
wild-type or K12R mutant histone H4 in purified yeast
core histones. A HAT-B complex, partially purified by

anion exchange chromatography of soluble extracts
from wild-type cells, was used in the enzymatic assays
(Fig. 8A). As a control, equivalent chromatographic
fractions from a hat1D mutant strain were also
assayed. A recombinant yeast Hat1 (ryHat1) was also
included in the assays (Fig. 8C). Whereas wild-type
histone H4 was efficiently acetylated by native HAT-B
enzyme, H4K12R was modified very weakly, if at all
(Fig. 8A). Thus, in vitro as well as in vivo, Lys5 in
H4K12R represents only a very poor substrate for the
yeast HAT-B complex. Importantly, this finding sup-
ports the in vivo results that an acetylatable Lys at
position 12 of soluble histone H4 is required for fur-
ther modification on Lys5. Moreover, immunoblotting
with antibody to H4K5ac, after HAT assays, revealed
that the yeast HAT-B complex indeed acetylates Lys5
on wild-type histone H4. Figure 8B shows that incuba-
tion of yeast or chicken histones with acetyl-CoA and
chromatographic fractions containing the HAT-B
enzyme increased the H4K5ac immunosignal, which
was not the case when hat1D fractions (or buffer solu-
tion) were used. Thus, these results demonstrate that
the yeast HAT-B complex acetylates Lys5 in the con-
text of intact histone H4 in vitro.
In contrast to Hat1 as part of the HAT-B complex,
recombinant Hat1 was able to acetylate H4K12R,
although to a lesser extent than wild-type histone H4
(Fig. 8C).
Fig. 7. Exchange of Lys for Arg at position 12 of histone H4 pre-
vents acetylation of Lys5 in the soluble cell fraction. (A) H4K5ac in

WCEs and soluble fractions from yeast strains expressing either
wild-type (PKY501, wt H4) or the K12R mutated version (LDY105,
K12R H4) of histone H4 was analyzed by immunoblotting. (B)
Hat1–HA and Hif1–HA were immunoprecipitated from soluble
extracts of cells that express wild-type histone H4 or H4K12R.
Input (I), unbound (U) and bound (B) fractions were analyzed for the
presence of associated histone H4. Yeast strains: BQS1399
(expressing Hat1–HA, wild-type histone H4); BQS1401 (Hat1–HA,
K12RH4); BQS1403 (Hif1–HA, wild-type histone H4); and BQS1405
(Hif1–HA, K12RH4). Blots were probed with antibody to H4K12ac,
anti-ryH4, and antibody to H4K5ac.
A. Poveda and R. Sendra Yeast histone acetyltransferase B complex
FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS 2129
Discussion
The main result of this study has been the demonstra-
tion that the S. cerevisiae HAT-B complex is involved
in the acetylation of both Lys12 and Lys5 of soluble
histone H4 in vivo. We showed that both Hat1 and
Hat2 are essential for this specific histone H4
Lys12 ⁄ Lys5 acetylation, whereas the third component
of the complex, Hif1, is not. These results are in agree-
ment with in vitro data indicating that the absence of
Hif1 alters neither the activity nor the specificity of the
rest of the HAT-B enzyme [19,20], whereas Hat2 has
the ability to enhance the catalytic potential of the
Hat1 subunit [13]. Therefore, the functional role of
Hif1 in the HAT-B complex is downstream of the acet-
ylation of histone H4.
It had been previously determined that, in vitro, the
yeast HAT-B complex exclusively acetylates Lys12

on histone H4 N-terminal synthetic peptides [13,22],
whereas recombinant Hat1 modifies Lys12 and Lys5
[13,25]. Moreover, in yeast cells, indirect evidence has
shown the involvement of Hat1 in the modification of
Lys12, although not of Lys5, of histone H4 [26–28].
However, we have demonstrated that Lys5 is a bona
fide target for acetylation by the yeast HAT-B complex
in vivo. Remarkably, the inability of the HAT-B
complex to acetylate histone H4 containing the K12R
substitution, both in vivo and in vitro, indicates a
sequential order of acetylation, with Lys12 being modi-
fied before Lys5. As Arg mimics unacetylated Lys, this
inability to use Lys5 as a target on H4K12R strongly
suggests that acetylation of Lys12 is a prerequisite for
the subsequent acetylation of Lys5. An identical
sequential order of site usage has been found for
HAT-B enzymes isolated from maize and rat liver [23],
and also from human cells [31], in vitro. Yeast recom-
binant Hat1 is able to modify H4K12R, which comple-
ments previous findings showing less stringent site
specificity for Hat1 alone [13,25], and suggests the
involvement of the other complex components in the
site selection mechanism. In contrast to previous stud-
ies [13,22], we have found that, even in vitro, the yeast
HAT-B complex modifies Lys5 as well as Lys12, just
like other type B enzymes from diverse species
[10,15,16,23,31]. The reason for this discrepancy may
be the different substrates used. Earlier experiments,
indicating Lys12 as the only acetylation site, were car-
ried out with histone H4 N-terminal peptides [13,22].

We have used whole histone H4 of yeast or chicken
erythrocytes, as in numerous other studies
[10,15,16,23,31]. We suggest that an interaction of his-
tone H4, beyond its N-terminus, with the HAT-B com-
plex is needed in order to establish a physiological
acetylation pattern. Interestingly, in line with this idea,
in vitro, the human HAT-B enzyme acetylates the his-
tone H4(1–41) N-terminal fragment more efficiently
than the shorter histone H4(1–34) fragment [16]. It
seems reasonable that at least part of the differential
Fig. 8. Effect of K12R substitution on histone H4 acetylation by
the yeast HAT-B complex and ryHat1 in vitro. (A) Soluble extracts
from wild-type and hat1D strains were subjected to Q-Sepharose
HP chromatography, and fractions containing HAT-B enzyme
(HAT1) were used for HAT activity assays. Equivalent fractions
from the deletion mutant strain were also tested as negative con-
trols (hat1D). These acetyltransferase assays were carried out by
mixing 2 lg of purified whole yeast histones, containing wild-type
histone H4 (wt H4) or K12R mutant histone H4 (K12R H4),
[1-
14
C]acetyl-CoA, and aliquots of the chromatographic fractions (2
and 8 lL). After incubation, histones were separated by 15%
SDS ⁄ PAGE, and gels were stained with Coomassie brilliant blue
(upper panel), and subsequently subjected to fluorography (lower
panel). Histones are indicated on the left. (B) Yeast histones with
wild-type histone H4 and chicken erythrocyte core histones were
assayed with chromatographic fractions containing (HAT1) or lack-
ing (hat1D, or buffer) HAT-B enzyme, and subsequently analyzed by
immunoblotting with antibody to H4K5ac. (C) Recombinant yeast

Hat1 (ryHat1; approximately 0.01 and 0.04 lg) was used in HAT
assays carried out as in (A).
Yeast histone acetyltransferase B complex A. Poveda and R. Sendra
2130 FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS
potential as substrates of the two peptides is due to
different positions being modified by the enzyme. Our
data indicate a site specificity of the yeast HAT-B
complex that exactly matches the specificity of other
type B HATs [10,15,16,23,31], thus pinpointing a much
higher degree of conservation of these enzymes than
previously assumed.
The levels of acetylation on Lys16 and Lys8 are
extremely low in the soluble histone H4 of wild-type
cells, and also nearly undetectable on Lys12 and Lys5
in hat1D cells. These observations strongly suggest
that, in yeast, Lys12 and Lys5 are the only N-terminal
positions that are acetylated in soluble histone H4, and
that the HAT-B complex must be the only enzyme
involved in this specific modification. However, in con-
trast with these results, histone H4 acetylated on
Lys16 and, to a greater degree, on Lys8 has been
detected in the cytoplasmic fraction of chicken DT40
cells. In addition, chicken cells lacking Hat1 retain a
significant level of histone H4 Lys12 and Lys5 acetyla-
tion in the soluble fraction [30]. Although the acetyla-
tion pattern of the soluble H4 histones of yeast and
chicken could be different, contamination with chro-
matin could also explain the presence of Hat1-indepen-
dent histone H4 Lys12 and Lys5 acetylations and
other acetylated positions in the soluble fraction.

Hat1-dependent acetylated histone H4 is present in
the soluble fraction in different cell cycle stages, which
shows that, in addition to DNA replication, it may
also participate in other processes outside of S phase.
A dynamic nucleosome disassembly ⁄ reassembly pro-
cess is a well-established feature of sites undergoing
transcription [39,40], but a global histone H4 exchange
independent of replication and transcription has also
been described in yeast [41]. Reassembly makes use of
histones from the soluble pool [40,41], in which his-
tone H4, as our results indicate, must be acetylated by
the HAT-B complex.
In addition, we have found that excess histone H4
accumulating in the soluble fraction in cells treated
with HU or in cells deficient in the Rad53-dependent
histone degradation pathway [34] is acetylated by
Hat1. It therefore seems that all new histone H4 mole-
cules appearing in the soluble fraction contain the spe-
cific Lys12 ⁄ Lys5 acetylation pattern generated by the
type B enzyme.
In contrast to studies on different species where
newly synthesized histone H4 in a diacetylated form
was obtained from chromatin [4,5,42,43], we did not
detect HAT-B-dependent acetylation on chromatin
histone H4. In yeast, the Hat1-dependent acetylation
could be eliminated immediately upon the deposition
of histone H4 into chromatin. It cannot be ruled
out that this deacetylation occurs either during, or
even prior to, histone H4 deposition. Mutational
analysis has shown that specific Lys residues in the

N-termini of histone H3 and histone H4 play critical
roles in nuclear import, suggesting that acetylation
could serve to release histones from nuclear transport
factors [44]. Formally, for such a role, the deacety-
lation would not necessarily have to be post-
deposition.
Although, in vitro, Hat1 and Hat2 [44] and also
Hif1 [20] bind H4 ⁄ H3 histones, in vivo, both Hat1
and Hat2, together, are involved in the physical
interaction with histone H4, whereas Hif1 is not.
Furthermore, both targets of acetylation, Lys12 and
Lys5, are found to be acetylated in the histone H4
bound to the HAT-B complex. Current models
propose that the acetylated state at the his-
tone H4 N-terminus is involved in the stable binding
of histone H4 to the HAT-B complex [1,20,24]. How-
ever, this is not consistent with the ability of
H4K12R, which also lacks acetylation at Lys5, to be
bound by the HAT-B complex. Even Hif1, in the
context of the HAT-B complex, is associated with
histone H4 that lacks acetylation at the N-terminal
tail. As Hif1 exhibits chromatin assembly activity
in vitro [20], we must not rule out completely its par-
ticipation in chromatin assembly independently of
histone H4 acetylation. Our data also suggest that
the N-terminus (at least segment 1–12) is not
involved in the stable association of histone H4 with
the complex. Our previous two-hybrid assays indi-
cated an in vivo interaction between Hif1 and frag-
ment 1–59 of histone H4 that was dependent on

Hat2 [19]; thus, the portion of histone H4 involved
in the interaction with the HAT-B complex must be
located between residues 13 and 59. Verreault et al.
[16] found that helix 1 (residues 31–40) of his-
tone H4, situated in the histone-fold domain, is criti-
cal for binding to the Hat2 human homolog p46.
Reasonably, the yeast HAT-B complex could use the
same determinants to bind histone H4, although
additional contacts with Hat1 seem to be necessary
for efficient and stable binding of histone H4. It is
possible that all or some of these interactions are
also responsible for the acetylation specificity of the
HAT complex discussed above.
Although histone H3 has always been found with
histone H4 [37], and they are usually obtained from
the cells in a 1 : 1 ratio, we have not detected his-
tone H3 in the HAT-B complex from the yeast soluble
fraction. The controls carried out argue against the
specific proteolytic degradation of histone H3 in the
yeast fractions obtained and processed by our experi-
A. Poveda and R. Sendra Yeast histone acetyltransferase B complex
FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS 2131
mental procedures. Although these results must be
interpreted with caution, overall they suggest either
that histone H3 does not form part of the yeast HAT-
B complex in the soluble fraction, or that its binding
to histone H4 in the complex is weaker than in the
nucleosomal tetramer or in predeposition histone H3 ⁄
H4–chaperone complexes [34,38]. The possibility that
Hat1 takes part in alternative complexes with distinct

histone contents deserves to be taken into account.
Different complexes containing Hat1 as a catalytic
subunit have been described in yeast [20,24,33],
although the presence of histones in all of them has
not been analyzed. In any case, the absence of his-
tone H3 in the HAT-B complex is not a reason to
change significantly the widely assumed perception that
this enzyme plays a role in deposition of histones dur-
ing nucleosome assembly [45,46]. The recruitment of
histone H3 could occur in a subsequent step, resulting
in a transient, and less abundant, new HAT-B complex
containing histone H3, or simultaneously to its trans-
fer, together with acetylated histone H4, to a distinct
predeposition complex. In any case, the presence of
the HAT-B complex lacking histone H3 in the soluble
cell fraction would indicate that the interaction of
histone H4 with the complex and its acetylation by
Hat1 must be events that occur very early after the
biosynthesis of the histone H4 molecule.
Experimental procedures
Strains, media and culture conditions
The yeast strains used in this study are listed in Table 1.
All of the chromosomal integrated-tagged or deleted strains
were generated by a one-step PCR-based strategy [47,48] as
described previously [19]. Growth and manipulation of
yeast cells, and preparation of media, were performed
according to standard procedures. Strains BQS1391 to
BQS1394 were generated by replacing, also through homol-
ogous recombination with the appropriate PCR fragment,
the natural promoter of the essential RAD53 gene by the

regulatable GAL1 promoter [47]. These strains were grown
in rich media containing 2% galactose (YPGAL). The
RAD53 expression was repressed by switching to a 2% glu-
cose medium (YPD).
Arrest of yeast cells at different cell cycle stages was
achieved by addition of a-factor (Sigma-Aldrich, Madrid,
Spain) to 4.5 lgÆmL
)1
, HU (Sigma-Aldrich) to 200 mm,
and nocodazole (Calbiochem, San Diego, CA, USA) to
15 lgÆmL
)1
, and a subsequent incubation for 3 h. For the
arrest caused by nitrogen deprivation, a minimal medium
was prepared with yeast nitrogen base without ammonium
sulfate (Pronadisa, Madrid, Spain). The cell cycle stages
were assessed by microscopic inspection and by FACS anal-
ysis of propidium iodide-stained cells with an Epics XL
(Coulter, Fullerton, CA, USA) flow cytometer. For the
analysis of the effect of DNA-damage-inducing agents,
YPD liquid exponentially growing yeast cultures were trea-
ted with HU, methylmethanesulfonate, phleomycin, and
4NQO, all of them purchased from Sigma-Aldrich, at the
final concentrations and for the time periods indicated in
the figure legends.
Preparation of cell extracts and cellular
fractionation
WCEs for SDS ⁄ PAGE were prepared using an alkali
method [49].
For the separation of the cellular content into a soluble

fraction and a precipitate containing the chromatin, yeast
cells were harvested by centrifugation (500 g, 5 min),
washed in distilled water, and resuspended in 10 mLÆg
)1
of
cells (fresh weight) of pretreatment medium (50 mm
Tris ⁄ HCl, pH 7.5, 5 mm MgCl
2
,1m sorbitol, 75 mm
2-mercaptoethanol). After incubation for 10 min, cells were
collected by centrifugation (500 g, 5 min), and dispersed in
4mLÆg
)1
of digestion buffer (pretreatment medium but
only 5 mm 2-mercaptoethanol). Spheroplasts were produced
by incubation with zymolyase (40 UÆg
)1
; Seikagaku, Tokyo,
Japan) at 30 °C for 20–30 min with gentle agitation. Upon
incubation, cell suspensions were diluted with 10 volumes
of cold wash buffer (50 mm Mes ⁄ NaOH, pH 6.0, 10 mm
MgCl
2
,1m sorbitol, 1 mm phenylmethanesulfonyl fluoride,
2 lm E64, 1 mm 2-iodoacetamide), and the spheroplasts
were collected by centrifugation at 1000 g for 5 min.
This and all subsequent steps were performed at 4 °C.
Sedimented spheroplasts were washed once with the
same buffer. Spheroplasts were lysed by adding 4 mLÆg
)1

of
fractionation buffer [50 mm Tris ⁄ HCl, pH 7.6, 75 mm
NaCl, 0.5 mm CaCl
2
, 0.1% (v ⁄ v) Tween-20, and the
protease inhibitors phenylmethanesulfonyl fluoride 1 mm,
3,4-dichloroisocoumarin 25 lm, and the complete inhibitor
cocktail (Roche, Basel, Switzerland)], and incubating for
10 min at 4 °C with orbital agitation. Soluble and pellet
fractions were obtained by centrifugation at 16 000 g for
5 min. The examination of the enzymatic activity of the
HAT-B complex from the soluble fraction was achieved
upon its partial purification by chromatography onto
Q-Sepharose HP (GE Healthcare, Little Chalfont, UK) (see
below).
Yeast histones were purified by acid extraction of iso-
lated chromatin as described previously [19].
Immunoblotting and immunoprecipitation assays
For western blotting of histones, WCEs or pellet fractions,
from 1 · 10
6
cells, or soluble fractions, from 1 · 10
7
cells,
Yeast histone acetyltransferase B complex A. Poveda and R. Sendra
2132 FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS
were resolved by 15% SDS ⁄ PAGE. For analysis of HA-
tagged, Myc-tagged or Flag-tagged proteins, the extracts
and cellular fractions were electrophoresed on 8% SDS
polyacrylamide gels. All protein gels were transferred

to 0.2 lm pore nitrocellulose membranes as previously
described [50]. Membranes were routinely stained with
Ponceau S as a loading control, and processed as described
by the manufacturer of the ECL Advance Western blotting
detection system (GE Healthcare). Blots were probed over-
night at 4 °C with specific antibodies against different iso-
forms of histone H4 and histone H3, and against the
specific tags. The primary antibodies used in immunoblot-
ting were as follows. From Upstate Biotechnology (Lake
Placid, NY, USA): anti-H4 acetylated at position 5, 12, or
16; and anti-H3 acetylated at position 9, 14, 27, or 56.
From Abcam (Cambridge, UK): anti-H4 acetylated at resi-
due 8 or 91, and also a-ryH4, anti-H3 acetylated at posi-
tion 18 or 23, and a-H3Ct. The anti-HA clone 12CA5, and
the anti-Myc clone 9E10 mouse monoclonal sera were
from Roche. All primary antibodies were utilized at a dilu-
tion approximately 10 times higher than suggested by
the manufacturer. The secondary antibodies were horse-
radish peroxidase-linked anti-rabbit or anti-mouse IgG
(GE Healthcare), and were employed at a dilution of
1 : 50 000.
For immunoprecipitation experiments, soluble cell
fractions were incubated with rat anti-HA (3F10;
Roche) or mouse anti-Myc (9E10) monoclonal sera to
a final concentration of approximately 2.5 lgÆmL
)1
, and
incubated for 4 h at 4 °C. Forty microliters of pre-
equilibrated protein G–Sepharose FF (GE Healthcare)
was then added and incubated for 4 h on a rotating

wheel. For pulling down Flag-tagged proteins (Cac1
and Asf1) from soluble fractions, mouse anti-Flag M2-
agarose beads were utilized. After centrifugation (500 g
for 1 min), supernatants were saved, and the beads
were washed five times with 0.5 mL of washing buf-
fer B [15 mm Tris ⁄ HCl, pH 7.6, 150 mm NaCl, 0.5 mm
EDTA, 0.1% (v ⁄ v) Tween-20, 10% (v ⁄ v) glycerol, plus
the protease inhibitors indicated above]. Input materi-
Table 1. Yeast strains used in this study.
Strain Relevant genotype Source
W303-1a MATa ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100 R. Rothstein
RS1263 As W303-1a, but hat1D::TRP1 R. Sternglanz
BQS1154 As W303-1a, but HAT1-HA6-TRP1 [19]
MCY730 MATa ura3-52 lys2-801 ade2-101 trp1-D1 his3-D200 leu2-3,112 can1-100 M. Carlson
BQS179 As MCY730, but hat1D::KanMX3 J. E. Pe
´
rez-Ortı
´
n
YSTT11 As W303-1a, but hat2D::HIS3 [19]
YSTT49 As W303-1a, but hif1D::his5
+
[19]
YAV49 MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11 ura3-1 sml1D::URA3 CAC1-FLAG3::TRP1 A. Verreault
YAV52 MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11 ura3-1 sml1D::URA3 ASF1-FLAG3::TRP1 A. Verreault
BQS1386 As YAV49, but hat1D::KanMX4 This work
YAG101 MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11 ura3-1 sml1-1
rad53D::HIS3 CAC1-FLAG3::TRP1 RAD5
+
A. Verreault

BQS1358 As YAG101, but hat1D::KanMX4 This study
BQS1189 As W303-1a, but HAT2-HA6-TRP1 [19]
BQS1172 As W303-1a, but HAT1-HA6-TRP1, and hat2D::KanMX4 [19]
BQS1184 As W303-1a, but HAT1-HA6-TRP1, and hif1D::natMX4 [19]
BQS1304 As W303-1a, but HAT2-HA6-TRP1, and hif1D::KanMX4 This study
BQS1309 As W303-1a, but HAT2-HA6-TRP1, and hat1D::KanMX4 This study
BQS1187 As W303-1a, but HIF1-Myc9-TRP1 [19]
BQS1202 As W303-1a, but HIF1-Myc9-TRP1, and hat1D::KanMX4 [19]
BQS1225 As W303-1a, but HIF1-Myc9-TRP1, and hat2D::KanMX4 [19]
PKY501 MATa ade2-101 arg4-1 his3-201 leu2-3,112 lys2-801 trp1-901 ura3-52 thr
)
tyr
)
hhf1::HIS3 hhf2::LEU2 pPK626(URA3-HHF2)
M. Grunstein
LDY105 MATa ade2-101 arg4-1 his3-201 leu2-3,112 lys2-801 trp1-901 ura3-52 thr
)
tyr
)
hhf1::HIS3 hhf2::LEU2 pPK626(URA3-HHF2-K12R)
M. Grunstein
BQS1399 As PKY501, but HAT1-HA6-TRP1 This study
BQS1401 As LDY105, but HAT1-HA6-TRP1 This study
BQS1403 As PKY501, but HIF1-HA6-TRP1 This study
BQS1405 As LDY105, but HIF1-HA6-TRP1 This study
BQS1391 As W303-1a, plus pRAD53::pGAL1-HA3(KanMX4) This study
BQS1392 As W303-1a, plus pRAD53::pGAL1-HA3(KanMX4), hat1 D ::TRP1 This study
BQS1393 As W303-1a, plus pRAD53::pGAL1-HA3(KanMX4), hat2 D ::HIS3 This study
BQS1394 As W303-1a, plus pRAD53::pGAL1-HA3(KanMX4), hif1D::his5
+

This study
A. Poveda and R. Sendra Yeast histone acetyltransferase B complex
FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS 2133
als, first supernatants (unbound materials) and immuno-
precipitates (bound materials) were resolved by SDS ⁄
PAGE and probed for the presence of tagged proteins
and for coimmunoprecipitated histone H4 and his-
tone H3 by immunoblotting.
Extraction of HAT enzymes and anion exchange
chromatography
Extracts for fractionation of HAT enzymes were obtained
by lysis of yeast spheroplasts in a low-salt medium. Briefly,
yeast cells growing exponentially, or after 3 h of incubation
with 200 mm HU, were harvested by centrifugation, and
washed in distilled water. Spheroplasts, prepared as
described above, were lysed in buffer containing 75 mm
Tris ⁄ HCl (pH 7.9), 0.25 mm EDTA, 5 mm MgCl
2
,10mm
2-mercaptoethanol, 0.1% (v ⁄ v) Tween-20, and the protease
inhibitors phenylmethanesulfonyl fluoride 1 mm, 3,4-dichlo-
roisocoumarin 25 lm, and the complete inhibitor cocktail
(Roche), and stirred for 30 min. The homogenates were ul-
tracentrifuged for 1 h at 100 000 g, and the resulting super-
natants, containing those ‘free’ or soluble HATs, such as
the HAT-B complex, and devoid of the chromatin-associ-
ated HAT enzymes, were saved and dialyzed against
buffer B [15 mm Tris ⁄ HCl, pH 7.9, 0.25 mm EDTA, 5 mm
2-mercaptoethanol, 0.05% (v ⁄ v) Tween-20, 10% (v ⁄ v) glyc-
erol, 10 mm NaCl]. The dialyzed extracts were loaded onto

Q-Sepharose HP columns, and after washing, bound pro-
teins were eluted with a linear 80–400 mm NaCl gradient in
buffer B. Fractions were collected and assayed for protein
content (A
280 nm
) and HAT activity. ryHat1 was generated
in bacteria as previously described [22], purified partially by
anion exchange chromatography on Sep-Pak Accell plus
QMA cartridges (Waters, Milford, MA, USA), and
employed for the in vitro HAT specificity assays.
HAT assays
For determination of enzymatic activity in chromatographic
fractions, a new assay method was used. Briefly, 12 lLof
chromatographic fractions was mixed with 4 lg of chicken
erythrocyte core histones and 0.005 lCi of [1-
14
C]acetyl-
CoA (50 mCiÆmmol
)1
; Moravek, Brea, CA, USA) in a final
volume of 16 lL, and incubated for 20 min at 30 °C. The
reaction was terminated by addition of 8 lL of SDS ⁄ PAGE
sample solution [3·; 0.187 m Tris ⁄ HCl, pH 6.8, 6% (w ⁄ v)
SDS, 1.5 m 2-mercaptoethanol, 30% (v ⁄ v) glycerol, 0.005%
(w ⁄ v) bromophenol blue]. The amount of [
14
C]acetate
incorporated into the protein substrates was quantified with
an FLA-3000 phosphoimager (Fujifilm, Tokyo, Japan) or
an InstantImager (Packard Meriden, CT, USA), and the

relative radioactivity values were expressed as arbitrary
units (a.u.). Full details of this method for assaying HAT
activity will be given elsewhere.
In vitro HAT specificity assay
The histone specificity of partially purified HAT-B complex
from the soluble cellular fraction was determined by HAT
assays as previously described [19] (see also above), using
chicken erythrocyte or yeast core histones and [1-
14
C]ace-
tyl-CoA as substrates. Acetylated histone products were
resolved by 15% PAGE in the presence of SDS, and subse-
quently the gels were stained with Coomassie brilliant blue,
destained, dried, and exposed on phosphor-record image
plates. Screens were scanned in an FLA-3000 fluorescent
imager analyzer. Immunoblotting of the resulting acetylated
histone products was also carried out, in some case, to
examine the site specificity on histone H4.
Acknowledgements
We wish to thank Dr M. R. Parthun and A. Verreault
for their generous gift of several yeast strains and anti-
bodies. We also acknowledge Drs P. Loidl, M. Pam-
blanco and E. Ralph for their useful comments on the
work and critical reading of the manuscript. This work
has been supported by Grant BFU2005-02603 from
Ministerio de Educacio
´
n y Ciencia, Spain.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. HU treatment of yeast cells does not change
the enzymatic activity of the HAT-B complex.
Fig. S2. HU and different genotoxic agents increase
the in vivo Hat1-dependent Lys12 acetylation of his-
tone H4.
Fig. S3. Hat1 and Hat2, but not Hif1, are implicated
in the acetylation of soluble histone H4 accumulated
in Rad53-deficient cells.
Fig. S4. K12R substitution in histone H4 does not
interfere with the recognition of Lys5 acetylation by
the antibody to H4K5ac.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Yeast histone acetyltransferase B complex A. Poveda and R. Sendra
2136 FEBS Journal 275 (2008) 2122–2136 ª 2008 The Authors Journal compilation ª 2008 FEBS