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The Histone Chaperone Asf1 Increases the Rate of Histone
Eviction at the Yeast PHO5 and PHO8 Promoters
*
Received for publication, December 15, 2005 Published, JBC Papers in Press, January 4, 2006, DOI 10.1074/jbc.M513340200
Philipp Korber
‡1
, Slobodan Barbaric
§
, Tim Luckenbach
‡
, Andrea Schmid
‡
, Ulrike J. Schermer
‡
, Dorothea Blaschke
‡
,
and Wolfram Ho¨rz
‡†
From the
‡
Adolf-Butenandt-Institut, Universita¨tMu¨nchen, 80336 Mu¨ nchen, Germany and the
§
Laboratory of Biochemistry,
Faculty of Food Technology and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia
Eukaryotic gene expression starts off from a largely obstructive
chromatin substrate that has to be rendered accessible by regulated
mechanisms of chromatin remodeling. The yeast PHO5 promoter is
a well known example for the contribution of positioned nucleo-
somes to gene repression and for extensive chromatin remodeling
in the course of gene induction. Recently, the mechanism of this
remodeling process was shown to lead to the disassembly of pro-
moter nucleosomes and the eviction of the constituent histones in
trans. This finding called for a histone acceptor in trans and thus
made histone chaperones likely to be involved in this process. In this
study we have shown that the histone chaperone Asf1 increases the
rate of histone eviction at the PHO5 promoter. In the absence of
Asf1 histone eviction is delayed, but the final outcome of the chro-
matin transition is not affected. The same is true for the coregulated
PHO8 promoter where induction also leads to histone eviction and
where the rate of histone loss is reduced in asf1 strains as well,
although less severely. Importantly, the final extent of chromatin
remodeling is not affected. We have also presented evidence that
Asf1 and the SWI/SNF chromatin remodeling complex work in dis-
tinct parallel but functionally overlapping pathways, i.e. they both
contribute toward the same outcome without being mutually
strictly dependent.
The DNA of eukaryotic cells is compacted in the nucleus into a com-
plex structure called chromatin. The first level of chromatin organiza-
tion is formed by the nucleosome, which consists of a histone octamer
core organizing ⬃1.7 turns of double-stranded DNA around its surface
(1). DNA that is wound around a histone octamer in a canonical nucleo-
some is much less accessible for most DNA-interacting factors than
DNA in the linker regions between nucleosomes.
It is now widely accepted not only that nucleosomes serve a structural
role for the compaction of eukaryotic DNA but also that the obstructive
nature of the nucleosomal histone-DNA interactions is a means to reg-
ulate the expression of genetic information (2–4). This mode of regu-
lation involves changes in chromatin structure at, for example, pro-
moter or enhancer regions. A hallmark of such regulatory changes is the
switch of DNA regions from a state that is protected from nucleases to
a state that is sensitive, or even hypersensitive, to nucleases.
To understand the process of regulation through chromatin struc-
ture it is therefore crucial to study the molecular mechanisms that lead
to the inducible generation of hypersensitive sites. To this end, the
PHO5 promoter in yeast became a classical model system (5). In its
repressed state this promoter region is organized into four positioned
nucleosomes with a short hypersensitive site in the middle. Upon acti-
vation by phosphate starvation this characteristic chromatin organiza-
tion becomes remodeled into an extended hypersensitive region (6).
The promoter nucleosomes in this induced state are completely disas-
sembled as assayed by the loss of histone DNA contacts (7–9). Recently,
we and others showed that this loss of nucleosomal organization corre-
sponds to a movement of histones away from the promoter in trans (10,
11), raising the mechanistic question of where the histones go.
Free histones together with DNA are notoriously aggregation prone,
and it is assumed that they occur in the cell mainly in complex with
nucleic acids or histone chaperones (12–14). Histone chaperones are a
diverse family of proteins that interact with various kinds of histones
and probably serve mainly as histone donors and acceptors during
nucleosome assembly or disassembly processes. In addition, they can
have regulatory functions as in the case of the Hir proteins or Asf1 (15,
16). Histone chaperones appear to form a redundant network as yeast
strains deleted in multiple histone chaperone genes are viable (13).
Nonetheless, histone chaperones can also be specific for certain histone
variants (17) or for certain processes, e.g. for replication-dependent or
-independent chromatin assembly (18, 19). Chromatin remodeling
complexes may work more or less specifically with, or may even contain,
histone chaperones (14, 20–23). Therefore, histone chaperones are
likely candidates for the histone acceptor in the process of histone evic-
tion in trans upon remodeling of PHO5 promoter chromatin.
In this study we investigated the effect of mutations in genes coding
for various histone chaperones on the induction of the PHO5 gene. Of
all five tested histone chaperones, only the lack of Asf1 showed an effect
on PHO5 induction. The rate of chromatin remodeling was delayed,
but, importantly, the final extent of chromatin remodeling and gene
expression was not compromised.
We also extended our study to the PHO8 gene that codes for an
alkaline phosphatase and is coregulated with PHO5 by the same trans-
activator Pho4 (24, 25). Here as well, chromatin remodeling upon
induction led to histone loss from the promoter region and Asf1 con-
tributed to the rate of histone eviction. Because PHO8 induction is
strictly dependent on Snf2 (26), we conclude that Asf1 may cooperate
with the remodeling complex SWI/SNF. However, Asf1 and Snf2 do not
exclusively work in the same pathway, as we observed a synthetic effect
of combined snf2 and asf1 mutations for the case of PHO5 induction.
MATERIALS AND METHODS
Yeast Strains, Plasmids, and Media—Yeast strain W303 asf1::HIS3
was a gift from Mary Ann Osley. Strains W303 asf1::KAN
r
(alias
*This work was supported by the Deutsche Forschungsgemeinschaft (Transregio 5), the
6th Framework programme of the European Union (Epigenome Network of Excel-
lence), and Grant 0058025 from the Ministry of Education, Science, and Technology of
the Republic of Croatia (to S. B.). The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
† This paper is dedicated to the memory of our colleague, mentor, and friend Wolfram
Ho¨ rz, who passed away during the preparation of this manuscript.
1
To whom correspondence should be addressed: Adolf-Butenandt-Institut, Universita¨t
Mu¨ nchen, Schillerstrasse 44, 80336 Mu¨nchen, Germany. Tel.: 49-89-218075435; Fax:
49-89-218075425; E-mail: pkorber@lmu.de.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 9, pp. 5539–5545, March 3, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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MAR101), W303 cac1::TRP1, W303 hir1::HIS3, W303 hir2::HIS3,
W303 hir3::HIS3, and W303 asf1::KAN
r
,hir3::HIS3 (alias YAG120)
were a gift from Alain Verreault. Strains PKY028 (wt, W303), PKY937
(asf1), and PKY1019 (asf1,cac1) were gifts from Paul Kaufman (27).
Strain BY4741 asf1::KAN
r
was obtained from EUROSCARF (web.uni-
frankfurt.de/fb15/mikro/euroscarf). Using the primers Asf1-fwd
5⬘-GGTGGCGTCTTTTGCTG-3⬘and Asf1-rev 5⬘-GGAGAG-
GTCTCCGGTTC-3⬘, we generated an asf1::KAN
r
disruption construct
by PCR with genomic DNA of BY4741 asf1::KAN
r
as template. The
ASF1 gene in the strains CY337 (aura3–52,lys2– 801,ade2–101,leu2-
⌬1,his3-⌬200 (28)), CY407 (CY337 snf2⌬::HIS3), and YS31 (29) was
disrupted by linear transformation with this PCR product, yielding the
strains CY337 asf1::KAN
r
, CY407 asf1::KAN
r
, and YS31 asf1::KAN
r
,
respectively. The disruption of the locus was confirmed by genomic
PCR with the above primers and digestion of the PCR product with
HindIII and ClaI, thus detecting an introduced ClaI site.
Plasmids pP
pho8
-lacZ, pP
pho5v33
-lacZ, and pP
gal1
-lacZ were as
described (24, 30). Yeast strains were grown in yeast extract/peptone/
dextrose/adenine (YPDA) or in yeast nitrogen base (YNB) medium sup-
plemented with the required amino acids (high phosphate conditions),
and PHO5 induction was initiated by transferring cells to phosphate-
free synthetic medium (6). Galactose induction was done as described
(30).
Functional Assays—Acid phosphatase and

-galactosidase assays
were done as described (30).
Chromatin Analysis—Nuclei preparation and chromatin analysis by
restriction enzymes or DNaseI as well as indirect end labeling, gel elec-
trophoresis, and blotting procedures were as described (6, 31, 32).
Quantification of restriction enzyme accessibility assays was done using
phosphorimaging (Fuji FLA3000, AIDA
TM
software). The probe for the
PHO5 locus corresponds to the ApaI-BamHI fragment upstream of the
PHO5 promoter (probe D in Ref. 6), and ApaI (for DNaseI mapping) or
HaeIII (for restriction enzyme accessibility) was used for secondary
cleavage. The probe for the PHO8 locus corresponds to the PvuII-XhoI
fragment downstream of the PHO8 promoter (25), and BglII (DNaseI
mapping) or BglII and EcoRV (restriction analysis) were used for sec-
ondary cleavage.
Chromatin Immunoprecipitation—Yeast cultures of a density of
1–2 ⫻10
7
cells/ml were treated with 1% formaldehyde for 20 min at
room temperature. Cross-linking was quenched by adding glycine to
125 mMfinal concentration. Cells were washed twice in ice-cold 0.9%
NaCl, resuspended in HEG150 buffer (150 mMNaCl, 50 mMHEPES, pH
7.6, 10% glycerol, 1% Triton X-100, 1 mMEDTA, 1 mMdithiothreitol, 1
mMphenylmethylsulfonyl fluoride) and treated with a French Press
three times at 1100 psi. This step lysed the cells and sheared the chro-
matin to an average size of 450 bp. Alternatively, glass beads were added
and cells were lysed by shaking for2hinanEppendorf shaker at 4 °C.
Subsequently the glass beads were removed, and chromatin was sheared
by sonication to a similar average fragment size as with the French press.
Immunoprecipitation was done as described (33). Antibodies against
the C termini of histones H3 and H4, respectively, were gifts from A.
Verreault. The anti-Pho4 antibody was commercially prepared
(Abcam) using purified Pho4. Immunoprecipitated DNA was quanti-
tated in duplicates by the ABI PRISM 7000 sequence detection system
using the following amplicons: PHO5 UASp2-A, 5⬘-GAATAG-
GCAATCTCTAAATGAATCGA-3⬘; PHO5 UASp2-B, 5⬘-GAAAA-
CAGGGACCAGAATCATAAATT-3⬘; PHO5 UASp2 probe, 5⬘-FAM-
ACCTTGGCACTCACACGTGGGACTAGC-3⬘-TAM; ACT1-A,
5⬘-TGGATTCCGGTGATGGTGTT-3⬘;ACT1-B, 5⬘-TCAAAATG-
GCGTGAGGTAGAGA-3⬘; ACT1-probe, 5⬘-FAM-CTCACGTCGT-
TCCAATTTACGCTGGTTT-3⬘-TAM; PHO5 upstream ORF-A,
5⬘-TTATTCAATTTTAGCCGCTTCTTTG-3⬘; PHO5 upstream
ORF-B, 5⬘-CAATCTTGTCGACATCGGCTAGT-3⬘; PHO5 upstream
ORF probe, 5⬘FAM-CCAATGCAGGTACCATTCCCTTAGGCA-3⬘-
TAM; PHO8 UASp2-A, 5⬘-TGCGCCTATTGTTGCTAGCA-3⬘;
PHO8 UASp2-B, 5⬘-AGTCGGCAAAAGGGTCATCTAC-3⬘; PHO8
UASp2-probe, 5⬘-FAM-ATCGCTGCACGTGGCCCGA-3⬘-TAM.
RESULTS
The Kinetics of PHO5 Induction Are Delayed in the Absence of the
Histone Chaperone Asf1—In the course of our mechanistic studies of
chromatin opening at the PHO5 promoter, we examined a possible role
of histone chaperones in the process that leads to histone eviction in
trans (10, 11). Therefore we tested whether induction of Pho5 activity,
i.e. secreted acid phosphatase activity, was impaired in strains that are
singly disrupted in various of the known histone chaperone genes in
yeast: hir1, hir2, hir3, cac1, or asf1, as well as the double mutants
asf1,hir3 and asf1,cac1 (12–14). All these strains could be induced to
wild type levels of Pho5 activity by overnight incubation in phosphate-
free medium (Fig. 1 and data not shown). As complete remodeling of the
PHO5 promoter chromatin is a prerequisite for induction of Pho5 activ-
ity (34), this result shows that neither of the tested histone chaperones is
essential for chromatin remodeling at the PHO5 promoter.
The histone chaperone Asf1 was recently reported to be required for
remodeling at the PHO5 promoter (35). Therefore we compared the
chromatin structure of asf1 and wild type (wt)
2
strains by DNaseI map-
ping and restriction enzyme digestion, both under repressive conditions
and after overnight incubation in phosphate-free medium. Under both
conditions the asf1 mutant showed the same results as the wt strain
(data not shown). We conclude that Asf1 is necessary neither for the
assembly of the repressed state nor for chromatin remodeling upon
induction.
We and others observed in the past that the absence of chromatin-
related factors like Gcn5 or Snf2, although not essential for chromatin
opening at the PHO5 promoter, could lead to a delay in the induction
process
3
(7, 30, 36, 37). We also tested this possibility for the above
histone chaperone mutant strains by monitoring induction kinetics.
The strain deleted in the ASF1 gene showed a marked delay in PHO5
2
The abbreviations used are: wt, wild type; ChIP, chromatin immunoprecipitation; ORF,
open reading frame.
3
T. Luckenbach, S. Barbaric, P. Korber, D. Blaschke, and W. Ho¨ rz, manuscript in
preparation.
FIGURE 1. The kinetics of PHO5 induction are delayed in an asf1 strain. Acid phos-
phatase activity was monitored during the induction of a wt (CY337; closed circles) and
the isogenic asf1 strain (open circles) in phosphate-free medium. Error bars indicate the
S.D. of two to four independent experiments per time point.
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induction (Fig. 1). An equivalent delay was observed in two other strain
backgrounds if deleted for ASF1 (W303 and BY4741, data not shown).
The cac1 mutant also showed a significant delay in PHO5 induction.
However, by monitoring restriction enzyme accessibility during induc-
tion kinetics (see below), we found that the delay in induction was not
because of retardation on the level of chromatin opening (data not
shown) as was analogously observed by Adkins et al. (35) in histone-loss
kinetics during induction of a cac2 mutant. Accordingly, the induction
of phosphatase activity in the asf1,cac1 double mutant was more
delayed than in either single mutant but did not show a significant
further delay on the level of chromatin opening as compared with the
asf1 single mutant (data not shown). All other tested mutants did not
significantly affect PHO5 induction (data not shown), and we focused
our further studies on the role of Asf1.
The Absence of Asf1 Delays the Kinetics of PHO5 Induction on the
Level of Chromatin Opening—We tested whether the kinetic effect of
the asf1 mutation as measured on the level of PHO5 activity was because
of a kinetic delay at the chromatin transition step. The course of chro-
matin remodeling at the PHO5 promoter during induction kinetics in
asf1 and wt strains was monitored by measuring the accessibility of the
intranucleosomal ClaI site in the PHO5 promoter region (6, 32). A clear
delay in nucleosome remodeling was observed (Fig. 2A). In addition, we
followed the loss of histones from the PHO5 promoter region by ChIP
assay either with antibodies directed against the C terminus of histone
H3 in the CY337 background (Fig. 2B) or with antibodies directed
against the C terminus of histone H4 in the W303 background (data not
shown). The ChIP assay was internally controlled for region specificity
as amplicons corresponding to an open reading frame upstream
(“upstream ORF”) of PHO5 (Fig. 2B) did not detect significant changes
in histone levels (7). This chromatin assay also showed a clear delay for
the asf1 strain in both cases, but after prolonged incubation in phos-
phate-free medium no difference between the wt and asf1 strains was
observed (Fig. 2B). Therefore, both quantitative chromatin assays con-
firmed that the rate of chromatin opening, rather than only other steps
downstream in the gene expression process, is affected in the absence of
Asf1.
It was shown previously that stable binding of Pho4 to its intranucleo-
somal binding site UASp2 requires chromatin opening (38). We there-
fore hypothesized that the observed delay in chromatin remodeling
should also cause a delay in Pho4 binding to the promoter. Indeed, by
ChIP assay using anti-Pho4 antibodies, we found in asf1 strains that
Pho4 recruitment to the promoter was equally delayed as chromatin
remodeling (Fig. 2C). Equivalent results were obtained in a W303 back-
ground (data not shown).
The Kinetic Delay in asf1 Strains Is Not Due to Effects Upstream of the
Chromatin Remodeling Process—Previously, we took advantage of the
PHO5 promoter variant 33 (P
pho5v33
), in which both Pho4 binding sites
are replaced by Gal4 binding sites, to control for effects that a mutation
might have upstream of the chromatin remodeling step at the PHO5
promoter (30, 39). In such a variant, the same chromatin transition and
resulting promoter activation take place upon galactose induction as
with the wt promoter upon phosphate starvation (40). Therefore, any
mutation that is thought to directly affect the chromatin remodeling
step should also affect the chromatin transition in this variant.
The induction of the promoter P
pho5v33
through the GAL pathway
was similarly delayed in an asf1 strain (Fig. 3A) as the induction of the wt
promoter through the PHO pathway (Fig. 1). This delay was not because
of possible effects on the GAL signaling pathway or on Gal4 activity as
the induction kinetics of the GAL1 promoter was unaffected by the asf1
mutation (Fig. 3B). The asf1 strain even showed a much higher extent of
final induction for the P
gal1
-lacZ construct than the wild type strain
(data not shown). We note that asf1 strains tend to yield higher final
values than the wt strains for basal and fully induced levels of PHO5
induction (Fig. 1) or for final Pho4 binding (Fig. 2C). However, such
higher expression levels are not associated with changes in the chroma-
tin structure (data not shown) and are probably due to downstream
effects on gene expression.
The Combined Disruption of the SNF2 and ASF1 Genes Has a Syn-
thetic Effect on the Induction Kinetics of PHO5—So far, no chromatin
remodeler has been identified that is essential for opening PHO5 pro-
moter chromatin. However, the absence of Snf2 leads to a marked
kinetic delay in PHO5 induction
3
(7, 36, 37), indicating a significant role
of the SWI/SNF complex in chromatin remodeling at the PHO5 pro-
moter. We wanted to test whether the SWI/SNF complex cooperates
with Asf1 in this remodeling process and, if so, whether they function
FIGURE 2. The absence of Asf1 delays the kinetics of PHO5 induction on the level of
chromatin opening. The wt (CY337; closed symbols) and isogenic asf1 strains (open
symbols) were monitored during the course of induction in phosphate-free medium with
the following assays: accessibility of the ClaI site in the PHO5 promoter region (A), ChIP
using antibodies directed against the C terminus of histone H3 (
␣
H3) and amplicons for
either the PHO5 promoter region (UASp2; circles) or an upstream control region
(upstream ORF; triangles)(B), and chromatin immunoprecipitation using anti-Pho4 anti-
bodies (
␣
Pho4), otherwise as in panel B (C). The data of panels B and Care normalized to
input DNA and an amplicon for the ACT1 coding region. Error bars indicate the S.D. of
three independent experiments. Equivalent results as in panel B were obtained using the
W303 background and anti-histone H4 antibodies (not shown).
Asf1 Function at the Yeast PHO5 and PHO8 Promoters
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exclusively in the same pathway or whether Asf1 also has a role in the
alternative pathway through which PHO5 promoter chromatin is
remodeled in the absence of Snf2.
We constructed an asf1,snf2 double disruption strain and compared
PHO5 induction kinetics in this strain with the kinetics in the single
mutants (Fig. 4A). While the kinetics of PHO5 induction were strongly
delayed in the snf2 strain, the additional absence of Asf1 caused a sig-
nificant further delay, showing that Asf1 contributes to the rate of the
alternative, Snf2-independent remodeling process. After overnight
induction, however, the final activity level of the double mutant was
similar to that of the single mutants (data not shown), arguing for full
opening of PHO5 promoter chromatin even in the absence of both Snf2
and Asf1.
Deletion of the negative regulator Pho80 has previously been shown
to lead to full opening of the chromatin structure at the PHO5 promoter
even under otherwise repressive high phosphate conditions (41, 42).
Under such conditions the absence of Asf1 had no effect, or even slightly
increased Pho5 activity, and the absence of Snf2 led to a modest
decrease in activity. However, the level of induction of the asf1,snf2
double mutant was strongly reduced in comparison to that of the snf2
mutant (Fig. 4B). This demonstrated again a synthetic effect of this
double deletion, suggesting that under such conditions the contribution
of Asf1 to chromatin remodeling becomes important only in the
absence of Snf2, i.e. as part of an alternative pathway. Nonetheless, the
double mutant was still able to significantly induce PHO5 in a pho80
background under high phosphate conditions as compared with the
corresponding repressed PHO80 strain (compare asf1,snf2,pho80 to
asf1,snf2 in Fig. 4B).
The Kinetics of PHO8 Induction Are Also Delayed at the Level of
Chromatin Remodeling in the Absence of Asf1—Induction of the PHO8
promoter is triggered by the transactivator Pho4 as well and also leads to
a pronounced chromatin transition (25). Intriguingly, chromatin
remodeling and the induction of this promoter depend critically on the
activities of Snf2 and Gcn5 (26), much in contrast to the chromatin
transition at the PHO5 promoter.
Adkins et al. (35) reported that Asf1 was essential for induction of
PHO8. We measured PHO8 promoter activity by using a PHO8 promot-
er-driven lacZ reporter gene because alkaline phosphatase activity in
yeast is due not only to the PHO8 but also to the PHO13 gene product
(43). The PHO8 induction kinetics in phosphate-free medium were
delayed in an asf1 strain, whereas the final level of activity was close to
wild type levels (Fig. 5A).
We have shown here that induction of the PHO8 gene also led to
histone depletion in the promoter region in agreement with recently
published data (35). Furthermore, the kinetics of histone loss in asf1
strains were significantly delayed in comparison to wt strains (Fig. 5B),
corresponding with the delay in promoter induction. The same immu-
noprecipitated DNA as in Fig. 2Bwas used, making additional control
regions not necessary. Normalization of the data in Figs. 2Band 5Bto
the pre-induction values allows one to compare more directly the
kinetic delay of histone loss at the PHO5 and PHO8 promoter regions
(Fig. 5C). For unknown reasons, the histone occupancy at the PHO8
promoter prior to induction as measured by ChIP started off with lower
FIGURE 3. The kinetic delay in asf1 strains is not due to effects upstream of the
chromatin remodeling process. A, the wt (CY337; closed circles) and isogenic asf1
strains (open circles), both carrying the plasmid pP
pho5v33
-lacZ, were monitored for

-ga-
lactosidase activity during the time course of galactose induction. B,asinpanel A, but
cells contained the plasmid pP
gal1
-lacZ. Error bars indicate the S.D. of two to six inde-
pendent experiments per time point. o/n, overnight induction times (15–18 h).
FIGURE 4. Combined disruption of the SNF2 and ASF1 genes has a synthetic effect on
the induction kinetics of PHO5.A, wt (CY337; closed circles) and asf1 (open circles), snf2
(closed triangles)orasf1,snf2 (open triangles) mutants were monitored for acid phospha-
tase activity during induction kinetics in phosphate-free medium. B, acid phosphatase
activity of the indicated mutant strains isogenic to CY337 (wt) was measured in high
phosphate medium. Error bars indicate the S.D. of at least three independent
experiments.
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absolute values in asf1 strains either by using antibodies directed against
the H3 (Fig. 5B) or the H4 C terminus (data not shown).
Further support for a delay of PHO8 induction on the level of chro-
matin opening came from restriction enzyme accessibility measure-
ments. Using the same nuclei preparations as in Fig. 2Awe also tested
the accessibility of restriction enzymes in the PHO8 promoter region
that are known to change due to the chromatin transition upon induc-
tion (25). After 2 h of induction all three restriction sites tested were less
accessible in asf1 than in wt cells (Fig. 5D). Nonetheless, we confirmed
in several strain backgrounds by HpaI restriction site accessibility that
remodeling in asf1 cells, even though delayed after2hofinduction in
phosphate-free medium, reached wild type levels after overnight induc-
tion (Fig. 5E). In addition, the constitutive induction of an asf1,pho80
double mutant in high phosphate medium led to the same open chro-
matin structure. Furthermore, the DNaseI pattern of the asf1 mutant
after full induction was indistinguishable from the wild type pattern
(Fig. 5F).
As was the case for the PHO5 promoter, at the PHO8 promoter no
significant effect on the level of chromatin opening was observed with
the hir3 or cac1 histone chaperone mutant strains (data not shown).
Altogether, these data demonstrate that the rate of chromatin remod-
eling at the PHO8 promoter was compromised in the absence of Asf1,
although not as strongly as at the PHO5 promoter (compare Figs. 2A
and 5Eand Figs. 2Band 5B), and that full remodeling was achieved after
prolonged incubation.
Asf1 Is Critically Required for PHO5 Induction Only under Submaxi-
mal Inducing Conditions—As already mentioned, Adkins et al. (35)
reported that Asf1 is essential for PHO5 and PHO8 induction, whereas
we have shown here that full induction of both promoters was achieved
in asf1 strains. Adkins et al. use phosphate-depleted medium for induc-
tion, and it is known that the phosphate depletion protocol (44) gener-
ates low phosphate medium that contains residual amounts of phos-
phate, in contrast to the no phosphate synthetic medium used in our
study. Therefore, the apparent discrepancy between the results from
our and the Tyler group could be due to the different induction media
used.
Dhasarathy and Kladde (36) showed recently that the cofactors Gcn5
and Snf2, which are not necessary for complete PHO5 induction and
chromatin opening under fully inducing conditions
3
(7, 30, 36, 37),
become essential under submaximal inducing conditions. The sub-
maximal induction conditions were generated by allowing various
amounts of residual phosphate in the induction medium. We used the
same approach to examine the Asf1 requirement for PHO5 induction at
various low phosphate concentrations. By increasing the phosphate
concentration the induction of PHO5 was more severely affected in the
asf1 mutant than in the wt cells (Fig. 6). At certain amounts of phos-
phate the induction of the asf1 mutant was entirely repressed, whereas
induction of the wt remained mostly unaffected.
DISCUSSION
In this study we have shown that the histone chaperone Asf1
increases the rate of histone eviction during induction of the yeast
PHO5 and PHO8 promoters. At both promoters the kinetics of chro-
matin remodeling and consequent induction were delayed in asf1
strains. Such a phenotype is reminiscent of earlier findings with PHO5
induction in strains deleted in the genes for the histone acetyltransferase
Gcn5 or the ATPase subunit Snf2. Both deletions also lead to a kinetic
delay in PHO5 induction on the chromatin level
3
(7, 30, 36, 37). Inter-
estingly, in the case of the coregulated PHO8 promoter that is critically
dependent on Snf2 and Gcn5 (26) no mutations causing a kinetic delay
are known so far. The asf1 mutation described here is the first of this
kind.
Importantly, prolonged induction of the asf1 mutant under fully
inducing conditions, i.e. in the absence of phosphate, led to the full
extent of remodeling, and the resulting final activity levels were as high
FIGURE 5. The kinetics of PHO8 induction are also delayed at the level of chromatin
remodeling in the absence of Asf1. A,

-galactosidase activity was measured for the wt
(CY337; closed circles) and isogenic asf1 strain (open circles), both carrying the plasmid
pP
pho8
-lacZ, during induction kinetics in phosphate-free medium. Error bars indicate the
variation of two independent experiments. B, histone H3 occupancy at the PHO8 pro-
moter during induction kinetics for wt (closed circles) and asf1 strain (open circles). The
same immunoprecipitated DNA as in Fig. 2Bwas used for ChIP analysis but with primers
for an amplicon in the PHO8 promoter region. Equivalent results were obtained using the
W303 background and anti-histone H4 antibodies (not shown). C, the data for the PHO5
promoter in Fig. 2Band for the PHO8 promoter in panel B were normalized to the respec-
tive values at 0-h induction. D, using a nuclei preparation after2hofinduction as in Fig.
2A, the accessibility of the indicated restriction sites in the PHO8 promoter region was
assayed. The left and right lanes for each restriction enzyme show the results obtained
with 0.3 or 1.2 units/
l, respectively. Numbers underneath the lanes give the percentage
of cleavage as quantitated by phosphorimaging analysis. E, nuclei of wt (black bars) and
asf1 mutant (white bars) after 2 h and after overnight (o/n) induction in phosphate-free
medium were analyzed for HpaI restriction enzyme accessibility. Error bars indicate the
S.D. of three independent experiments per time point and include results with CY337,
W303, and BY4741 as strain backgrounds. Results with YS31 (pho80) under high phos-
phate conditions (constitutive induction) were also included with the o/n induction data
set. F, the chromatin structure of the repressed (⫹P
i
) and fully induced (⫺P
i
) state at the
PHO8 promoter region is not affected in an asf1 strain. Nuclei prepared from wt (CY337)
and asf1 mutant grown in full medium (⫹P
i
) or incubated overnight in phosphate-free
medium (⫺P
i
) were analyzed by limited DNaseI digestion and indirect end labeling.
Wedges on top of the lanes denote increasing DNaseI concentrations. Ovals on the left of
the gel mark the positions of nucleosomes ⫺1to⫺4 in the promoter region in the
repressed state. The coding region is represented by a black vertical line (PHO8 ORF). The
arrows point to short hypersensitive sites (sHS) that correspond to accessible upstream
activating sequence elements in the repressed state. Upon induction remodeling results
in an extended hypersensitive site (eHS,dashed line on the right). The marker lane (M)
shows positions of fragments generated with restriction enzymes that cut in the region
of the PHO8 promoter (XhoI,HindIII, and EcoRV).
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MARCH 3, 2006•VOLUME 281•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5543
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or even higher than in wt strains. Therefore there is an alternative,
Asf1-independent pathway for chromatin remodeling at the PHO5 and
PHO8 promoters. This is not unexpected as histone chaperones consti-
tute a notoriously redundant system in yeast (13). However, the normal
mechanism of chromatin remodeling at both promoters in the wt situ-
ation appears to have some kind of specificity for Asf1 as the absence of
four other histone chaperones; i.e. Hir1, Hir2, Hir3, or Cac1, did not
show any effect.
A role for a histone chaperone in remodeling of PHO5 promoter
chromatin agrees with the prediction that a remodeling mechanism
leading to histone eviction in trans (10, 11) should, in some way or other,
involve a histone acceptor in trans as well. An involvement of histone
chaperones would be less likely if histones were to leave the promoter
region by a mechanism in cis. In turn, we use this argument to suggest
that the observed contribution of the histone chaperone Asf1 to PHO8
promoter chromatin remodeling speaks for a mechanism of histone
eviction in trans at this promoter as well.
Interestingly, induction of the GAL10 promoter was also shown to
lead to the depletion of histones (45, 46), but we have shown here that
Asf1 does not influence the rate of induction of the coregulated GAL1
promoter. This may suggest that histone eviction at the GAL promoters
does not occur via a trans pathway or that there is a specificity for a
different histone chaperone. It clearly shows that Asf1 does not neces-
sarily have a role in transcriptional activation in general.
Recently, another study also addressed the role of Asf1 in the induc-
tion of PHO5 and PHO8 (35). The authors came to the conclusion that
neither the PHO5 nor the coregulated PHO8 gene could be induced in
asf1 strains at all and therefore considered Asf1 as essential for tran-
scriptional activation of these genes. As outlined above, our results are
in agreement with the general conclusion that Asf1 plays a role in chro-
matin remodeling and the resulting activation of both genes, but we
have shown here that chromatin remodeling at both genes can be
achieved to wild type levels in the absence of Asf1. The apparent dis-
crepancy between the two data sets can be explained by differences in
the induction conditions used. It is reported that the extent of induction
of the PHO regulon can be controlled by the phosphate concentration in
the medium and that submaximally inducing conditions (low instead of
no phosphate) lead to more pronounced dependences of the PHO5 gene
on cofactors like, for example, Snf2 or Gcn5 (36). We obtained analo-
gous results comparing the induction of PHO5 in an asf1 and wt strain
at various levels of residual phosphate in the induction medium. There
were intermediary induction conditions in which a wt strain is substan-
tially induced while an asf1 mutant is uninduced. Adkins et al. (35) made
use of induction medium that is prepared by a chemical depletion pro-
tocol (44). In contrast to phosphate-free synthetic medium, such phos-
phate-depleted medium contains residual amounts of phosphate and
may even allow slow but continued cell proliferation. Therefore, such
medium probably corresponds to intermediary inducing conditions as
described above and by two other groups (36, 47) and result in the more
stringent dependence on Asf1. In our experiments, we examined the
requirement for Asf1 during induction in phosphate-free medium that
let cells go into growth arrest after overnight incubation (6), or we used
the pho80 mutation that allows constitutive PHO induction even for
continuously dividing cells in high phosphate media (41, 42). Both pro-
tocols led to complete opening of the PHO5 and PHO8 promoters even
in the absence of Asf1.
Our findings are in agreement with the recent results of a genome-
wide screen for yeast mutants defective for PHO5 regulation (48). This
screen identified nine new genes that were not known before to be
involved in PHO5 regulation. However, also in this study phosphate-
free synthetic medium was used and the ASF1 gene was not found to be
essential for PHO5 induction, either in the screen or when tested indi-
vidually and in various strain backgrounds.
It is reported and discussed that chromatin remodeling complexes,
for example the SWR or Ino80 complexes that catalyze the exchange of
histones, work in concert with histone chaperones (14, 21, 22). The
same is likely to be true for chromatin remodelers that catalyze the
eviction of histones in trans. This eviction mechanism has not yet been
properly reconstituted in vitro; however, there is ample evidence for its
relevance in vivo (45, 49), and chromatin remodeling at the PHO5 and
PHO8 promoters were among the first examples (7, 9–11).
The induction of both PHO5 and PHO8 is mediated by Snf2 with
PHO8 induction being totally abolished (26) and PHO5 induction
severely compromised in the absence of Snf2
3
(7, 36, 37). Here we have
provided evidence that the SWI/SNF complex can work together with
the histone chaperone Asf1, suggesting that the SWI/SNF complex is an
example of a chromatin remodeler that can catalyze histone eviction in
trans in vivo. However, our results indicate that the role of Asf1 in
nucleosome eviction is not exclusively connected with the remodeling
function of the SWI/SNF complex, i.e. that the SWI/SNF complex and
the histone chaperone Asf1 work in distinct parallel pathways that func-
tionally overlap. The SWI/SNF complex and Asf1 can cooperate with
alternative histone chaperones or remodelers, respectively. The argu-
ment is as follows. Chromatin remodeling at the PHO8 promoter has to
proceed via a SWI/SNF-dependent pathway. This pathway is delayed in
asf1 strains, implying that Asf1 usually contributes to the outcome of
this pathway but can be replaced by some other factor. Conversely,
chromatin remodeling at the PHO5 promoter usually involves a SWI/
SNF-dependent pathway as well but can also be achieved in the absence
of Snf2 through another pathway, albeit at a slower rate. This alternative
pathway in turn can also cooperate with Asf1, as a deletion of Asf1 in a
snf2 background leads to a synthetic kinetic effect with even slower
induction rates and even lower steady state induction levels during
induction via the pho80 mutation in high phosphate media. Strictly
speaking, we cannot be sure whether the alternative pathways make use
of alternative remodelers or histone chaperones at all. However, the
alternative pathways in each case led to the same final outcome of
remodeled chromatin structure, suggesting that the main features of the
overall mechanism, e.g. histone eviction in trans through the combined
FIGURE 6. Asf1 becomes critically required for PHO5 induction only under submaxi-
mal inducing conditions. YPDA, or phosphate-free medium supplied with phosphate
(KH
2
PO
4
) to the indicated final concentrations, was inoculated with logarithmically
growing cultures of wt (CY337) or asf1 strains to a final A
600
of 0.01 and further incubated
at 30 °C overnight. Acid phosphatase activity was determined for each culture, and the
error bars indicate the variation between two independent cultures.
Asf1 Function at the Yeast PHO5 and PHO8 Promoters
5544 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 9•MARCH 3, 2006
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action of a chromatin remodeler and a histone chaperone, are
maintained.
Acknowledgments—We thank Mary Ann Osley, Alain Verreault, and Paul
Kaufman for sharing strains and for helpful discussions. We thank Alexander
Brehm for critical reading of the manuscript.
REFERENCES
1. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997)
Nature 389, 251–260
2. Roeder, R. G. (2005) FEBS Lett. 579, 909 –915
3. Fyodorov, D. V., and Kadonaga, J. T. (2001) Cell 106, 523–525
4. Narlikar, G. J., Fan, H. Y., and Kingston, R. E. (2002) Cell 108, 475– 487
5. Svaren, J., and Ho¨rz, W. (1997) Trends Biochem. Sci. 22, 93–97
6. Almer, A., Rudolph, H., Hinnen, A., and Ho¨rz, W. (1986) EMBO J. 5, 2689 –2696
7. Reinke, H., and Ho¨rz, W. (2003) Mol. Cell 11, 1599 –1607
8. Reinke, H., and Ho¨rz, W. (2004) Biochim. Biophys. Acta 1677, 24 –29
9. Boeger, H., Griesenbeck, J., Strattan, J. S., and Kornberg, R. D. (2003) Mol. Cell 11,
1587–1598
10. Korber, P., Luckenbach, T., Blaschke, D., and Ho¨rz, W. (2004) Mol. Cell. Biol. 24,
10965–10974
11. Boeger, H., Griesenbeck, J., Strattan, J. S., and Kornberg, R. D. (2004) Mol. Cell 14,
667–673
12. Verreault, A. (2000) Genes Dev. 14, 1430 –1438
13. Tyler, J. K. (2002) Eur. J. Biochem. 269, 2268 –2274
14. Loyola, A., and Almouzni, G. (2004) Biochim. Biophys. Acta 1677, 3–11
15. Osley, M. A., and Lycan, D. (1987) Mol. Cell. Biol. 7, 4204 –4210
16. Sutton, A., Bucaria, J., Osley, M. A., and Sternglanz, R. (2001) Genetics 158, 587–596
17. Tagami, H., Ray-Gallet, D., Almouzni, G., and Nakatani, Y. (2004) Cell 116, 51– 61
18. Ray-Gallet, D., and Almouzni, G. (2004) Methods Enzymol. 375, 117–131
19. Ray-Gallet, D., Quivy, J. P., Scamps, C., Martini, E. M., Lipinski, M., and Almouzni, G.
(2002) Mol. Cell 9, 1091–1100
20. Ito, T., Bulger, M., Kobayashi, R., and Kadonaga, J. T. (1996) Mol. Cell. Biol. 16,
3112–3124
21. Shen, X., Ranallo, R., Choi, E., and Wu, C. (2003) Mol. Cell 12, 147–155
22. Mizuguchi, G., Shen, X., Landry, J., Wu, W. H., Sen, S., and Wu, C. (2004) Science 303,
343–348
23. Loyola, A., LeRoy, G., Wang, Y. H., and Reinberg, D. (2001) Genes Dev. 15, 2837–2851
24. Mu¨nsterko¨tter, M., Barbaric, S., and Ho¨rz, W. (2000) J. Biol. Chem. 275,
22678–22685
25. Barbaric, S., Fascher, K. D., and Ho¨ rz, W. (1992) Nucleic Acids Res. 20, 1031–1038
26. Gregory, P. D., Schmid, A., Zavari, M., Mu¨ nsterko¨ tter, M., and Ho¨ rz, W. (1999)
EMBO J. 18, 6407–6414
27. Sharp, J. A., Rizki, G., and Kaufman, P. D. (2005) Genetics 171, 885– 899
28. Richmond, E., and Peterson, C. L. (1996) Nucleic Acids Res. 24, 3685–3692
29. Svaren, J., Schmitz, J., and Ho¨ rz, W. (1994) EMBO J. 13, 4856– 4862
30. Barbaric, S., Walker, J., Schmid, A., Svejstrup, J. Q., and Ho¨ rz, W. (2001) EMBO J. 20,
4944–4951
31. Svaren, J., Venter, U., and Ho¨ rz, W. (1995) Methods Med. Genet. 6, 153–167
32. Gregory, P. D., Barbaric, S., and Ho¨ rz, W. (1999) Methods Mol. Biol. 119, 417–425
33. Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. (1997) Genes Dev. 11,
83–93
34. Fascher, K. D., Schmitz, J., and Ho¨ rz, W. (1993) J. Mol. Biol. 231, 658– 667
35. Adkins, M. W., Howar, S. R., and Tyler, J. K. (2004) Mol. Cell 14, 657– 666
36. Dhasarathy, A., and Kladde, M. P. (2005) Mol. Cell. Biol. 25, 2698 –2707
37. Neef, D. W., and Kladde, M. P. (2003) Mol. Cell. Biol. 23, 3788 –3797
38. Venter, U., Svaren, J., Schmitz, J., Schmid, A., and Ho¨ rz, W. (1994) EMBO J. 13,
4848–4855
39. Gregory, P. D., Schmid, A., Zavari, M., Lui, L., Berger, S. L., and Ho¨ rz, W. (1998) Mol.
Cell 1, 495–505
40. Ertinger, G. (1998) The Role of Transcription Factors for Opening of the Chromatin
Structure at the PH05 Promoter in Saccharomyces cerevisiae. Ph.D thesis, Universita¨t
Mu¨ nchen, Germany
41. Schmid, A., Fascher, K. D., and Ho¨ rz, W. (1992) Cell 71, 853–864
42. O’Neill, E. M., Kaffman, A., Jolly, E. R., and O’Shea, E. K. (1996) Science 271, 209 –212
43. Kaneko, Y., Toh-e, A., Banno, I., and Oshima, Y. (1989) Mol. Gen. Genet. 220,
133–139
44. Han, M., Kim, U. J., Kayne, P., and Grunstein, M. (1988) EMBO J. 7, 2221–2228
45. Lee, C. K., Shibata, Y., Rao, B., Strahl, B. D., and Lieb, J. D. (2004) Nat. Genet. 36,
900–905
46. Schwabish, M. A., and Struhl, K. (2004) Mol. Cell. Biol. 24, 10111–10117
47. Springer, M., Wykoff, D. D., Miller, N., and O’Shea, E. K. (2003) PLoS Biol. 1, E28
48. Huang, S., and O’Shea, E. K. (2005) Genetics 169, 1859 –1871
49. Bernstein, B. E., Liu, C. L., Humphrey, E. L., Perlstein, E. O., and Schreiber, S. L. (2004)
Genome Biol. 5, R62
Asf1 Function at the Yeast PHO5 and PHO8 Promoters
MARCH 3, 2006•VOLUME 281•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5545
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Hörz
Schermer, Dorothea Blaschke and Wolfram
Luckenbach, Andrea Schmid, Ulrike J.
Philipp Korber, Slobodan Barbaric, Tim
PromotersPHO8 and PHO5
Rate of Histone Eviction at the Yeast
The Histone Chaperone Asf1 Increases the
Genes: Structure and Regulation:
doi: 10.1074/jbc.M513340200 originally published online January 4, 2006
2006, 281:5539-5545.J. Biol. Chem.
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