MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 1998, American Society for Microbiology
Feb. 1998, p. 1049–1054 Vol. 18, No. 2
Mutations in Chromatin Components Suppress a Defect of
Gcn5 Protein in Saccharomyces cerevisiae
JOSE´PE´REZ-MARTI´N,1AND ALEXANDER D. JOHNSON1,2*
Department of Microbiology and Immunology1and Department of Biochemistry and Biophysics,2
University of California, San Francisco, California 94143-0414
Received 15 September 1997/Returned for modification 22 October 1997/Accepted 18 November 1997
The yeast GCN5 gene encodes the catalytic subunit of a nuclear histone acetyltransferase and is part of a
high-molecular-weight complex involved in transcriptional regulation. In this paper we show that full activa-
tion of the HO promoter in vivo requires the Gcn5 protein and that defects in this protein can be suppressed
by deletion of the RPD3 gene, which encodes a histone deacetylase. These results suggest an interplay between
acetylation and deacetylation of histones in the regulation of the HO gene. We also show that mutations in
either the H4 or the H3 histone gene, as well as mutations in the SIN1 gene, which encodes an HMG1-like
protein, strongly suppress the defects produced by the gcn5 mutant. These results suggest a hierarchy of action
in the process of chromatin remodeling.
Nuclear processes, including transcription, require that en-
zymes gain access to the eukaryotic DNA template despite the
fact that it is complexed with histone and nonhistone proteins
to form chromatin. Genetic studies with Saccharomyces cerevi-
siae have identified two groups of genes that appear to link
transcriptional regulation to chromatin structure (40). The first
group encodes components of the SWI/SNF complex, which
has been proposed to antagonize the repressive effects of chro-
matin on transcription (24). SWI/SNF genes were identified in
genetic screens for mutants defective in the expression of var-
ious genes, including the HO and SUC2 genes (2, 18, 22, 32).
The second group of genes includes various SPT and SIN
genes, which were defined as suppressors of various types of
transcriptional defects (40). The sin2-1 mutation was found to
lie in the HHT1 gene, which encodes histone H3. Five addi-
tional different point mutations, two in histone H3 and three in
histone H4, also displayed a Sin?/Spt?phenotype (12, 25).
These mutations affect residues that are believed either to
contact DNA or to be involved in histone-histone contacts
within the histone octamer (39). The SIN1 gene was found to
encode a protein with similarities to mammalian HMG1, a
structural component of chromatin (11). Although the precise
role of yeast SIN1 is not known, the similarity of sin1 and sin2-1
mutant phenotypes has led to the inference that these two
genes have related physiological functions.
Recently, a group of genes involved in acetylation and
deacetylation of histones has been recognized. Histone acety-
lation has long been correlated with the modulation of gene
activity (37). Acetylation of lysines in histone amino-terminal
tail domains reduces the positive charge, thereby weakening
histone-DNA interactions, destabilizing higher-order struc-
ture, and rendering nucleosomal DNA more accessible to tran-
scriptional factors (4, 14). Yeast Gcn5 was originally identified
as a regulatory factor required for function of the yeast acti-
vator Gcn4 (5), and recently it has been shown that Gcn5 is a
histone acetyltransferase (3, 13, 28) that is part of at least two
high-molecular-weight complexes called ADA (8) and SAGA
(7). The recruitment of these complexes to DNA is thought to
direct the local destabilization of nucleosomes, producing
more efficient transcriptional activation on a promoter. Aside
from transcriptional regulators that function as histone acetyl-
transferases, there are also regulators that deacetylate the hi-
stones (29, 36). These deacetylases comprise part of a tran-
scriptional repression pathway conserved from yeast to
vertebrates and provide a molecular mechanism whereby tran-
scription can be continually controlled (19, 38).
In this paper we show that the expression of the HO gene is
affected by defects in histone acetylation and deacetylation.
Previous work has shown that the SWI/SNF complex and struc-
tural components of chromatin also affect HO expression (11,
12, 16, 22). We present an analysis of single and double mu-
tations in the genes encoding several of these components, and
the results suggest a hierarchy in the chromatin remodeling
MATERIALS AND METHODS
Strains and media. All strains of S. cerevisiae used in this study are described
in Table 1. Complete medium (yeast extract-peptone-dextrose [YEPD]) and
minimal medium supplemented with the required amino acids were used for
yeast growth and transformations (26). Histidine limitation was accomplished by
supplementing minimal media with 10 mM 3-amino-1,2,4-triazole (3-AT) (5).
Strain constructions. Single mutants were obtained either by gene disruptions
performed by using the one-step replacement method (27) or by gene conver-
sions carried out by a two-step gene replacement procedure (31). Double and
triple mutants were obtained by crossing single mutants of opposite mating types
and selecting segregants carrying the desired mutations.
A strain carrying a swi5::LEU2 null allele was generated as described in
reference 34. The gcn5::hisG strain was generated as described in reference 15.
The HO-lacZ fusion allele is described in reference 30. The histone mutations
were introduced in the chromosome by a two-step replacement procedure (31)
using integrating plasmids marked with the URA3 gene (obtained from R. K.
Tabtiang and I. Herskowitz); as these mutations are partially dominant, it is
possible to observe their effects, even in the presence of another histone gene
copy (12). The rpd3?::LEU2 strain was generated by transforming yeast with
pDM176 digested with BamHI. This plasmid carries the RPD3 locus with a
replacement of the entire RPD3 open reading frame (ORF) with the LEU2 gene
(15a). Correct integration was tested by PCR analysis using oligonucleotides
flanking the RPD3 locus. The sin1?::TRP1 deletion strains were generated by
transforming yeast with pUC-SIN1::TRP linearized with EcoRI-SphI. This plas-
mid carries a replacement of the SIN1 ORF with the TRP1 gene (11). Correct
integration was tested by PCR analysis using oligonucleotides flanking the SIN1
RNA analysis. Strains were grown to mid-log phase in YEPD medium. Total-
yeast RNA was isolated and fractionated on formaldehyde gels, transferred to
nylon membranes (Genescreen; DuPont), and hybridized with random-primed
32P-labeled fragments. The DNA probes used were obtained as PCR fragments
by amplification of the desired ORF with specific primers (MapPairs; Research
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, University of California, 513 Parnassus Ave.,
San Francisco, CA 94143-0859. Phone: (415) 476-8783. Fax: (415)
476-0939. E-mail: firstname.lastname@example.org.
Genetics Inc.), with the exception of the HO probe, which was obtained as a
2.6-kb HindIII fragment from the plasmid pGAL-HO (9).
Other methods. Yeast cells were transformed by the LiOAc method (6).
?-Galactosidase assays were performed as described elsewhere (26).
The Gcn5 protein is required for HO expression. HO gene
expression is dependent on SWI5. This gene encodes a zinc
finger DNA-binding protein which binds specifically, along
with the PHO2 protein, to the upstream region of the HO
promoter (1, 34). Genetic studies have described a series of
extragenic suppressor mutations that permit expression of HO
in the absence of the SWI5 gene product (17, 33). Two of the
genes identified in this screen, RPD3 and SIN3, encode, re-
spectively, a histone deacetylase and a protein tightly associ-
ated with it (10, 29, 35). The fact that mutations in the gene
pair SIN3/RPD3 are able to suppress the absence of the Swi5
protein suggests that one of the roles of the Swi5-Pho2 het-
erodimer is the recruitment, either directly or indirectly, of a
histone acetyltransferase activity. A likely candidate is the
GCN5 gene, which encodes a protein with histone acetyltrans-
ferase activity (13). To test this idea, we examined the levels of
HO mRNA produced in wild-type and isogenic gcn5 mutant
strains (obtained by disruption of the GCN5 gene; see Mate-
rials and Methods). We found that a gcn5 mutant strain pro-
duced significantly less HO mRNA (Fig. 1A). By contrast, the
absence of the Gcn5 protein did not impair the normal levels
of PHO2 and SWI5 mRNA.
In principle, SWI5 and GCN5 gene products could act in the
same pathway or through different pathways to activate HO
expression. If two genes act in the same pathway, then the
phenotype of the double mutant should be the same as that of
one of the single mutants. On the other hand, if two genes act
through different pathways, then the phenotype of the double
mutant should be more severe than that of either single mu-
tant. To distinguish between these two possibilities, we mea-
sured the ?-galactosidase activity produced by a chromosomal
HO-lacZ gene fusion in a swi5 gcn5 double mutant and com-
pared it to those in the single mutants (Fig. 1B). HO-lacZ
expression in the gcn5 and swi5 mutants was reduced 50- and
200-fold, respectively. In the double mutant, HO-lacZ expres-
sion was reduced 200-fold. The ?-galactosidase values of the
swi5 mutant are so low (0.5 Miller units) that we cannot make
a conclusive argument about the relationship of SWI5 and
GCN5. However, since both defects are suppressed by the
same mutations (i.e., by rpd3, sin1, and sin2 mutations; see
below) and since the levels of mRNA for SWI5 and PHO2
genes are not affected by gcn5 mutations (Fig. 1A), these facts
support the idea that SWI5 and GCN5 act in the same pathway
to stimulate HO expression.
Deletion of the RPD3 gene suppresses the gcn5 mutation.
The results described above are compatible with the idea that
histone acetylation is required for maximal HO transcriptional
activation. According to this hypothesis, a mutation in a gene
encoding a deacetylase should be able to suppress a gcn5 mu-
tation. A likely candidate is the RPD3 gene, since mutations in
this gene suppress the Swi5 requirement in the HO gene (35).
We therefore measured HO-lacZ activity in single and double
mutants carrying null alleles of the GCN5 and RPD3 genes.
FIG. 1. Gcn5 is required for HO expression. (A) Effects of gcn5 disruption on the mRNA levels of the HO, PHO2, and SWI5 genes. Total RNA was extracted from
FY120 (GCN5) and JJY54 (gcn5::hisG) grown in YEPD medium to mid-log phase. ACT1 mRNA was used as a control. (B) Genetic relationships between SWI5 and
GCN5. ?-Galactosidase activity was measured in strains carrying an HO-lacZ reporter gene integrated in the chromosome at the HO locus. The strains used were JJY12
(wild type [wt]), JJY28 (gcn5::hisG), JJY13 (swi5::hisG), and JJY60 (gcn5::hisG swi5::hisG). Values are averages of three independent measurements with less than 10%
TABLE 1. Yeast strains used in this study
FY120...........................MATa ura3-52 leu2?1 his4-912? Lys2-128?
RT238...........................MAT? ura3-52 leu2?1 his3 trp1 HO-lacZ
JJY12............................MAT? ura3-52 leu2 ?1 trp1 lys2-128? HO-lacZ
JJY13............................Same as JJY12, plus swi5::hisG
JJY28............................Same as JJY12, plus gcn5::hisG
JJY36............................Same as JJY12, plus sin1?::TRP1
JJY42............................Same as JJY28, plus hhf2-8
JJY43............................Same as JJY28, plus hhf2-13
JJY44............................Same as JJY28, plus sin2-1
JJY45............................Same as JJY28, plus sin1?::TRP1
JJY54............................Same as FY120, plus gcn5::hisG
JJY60............................Same as JJY28, plus swi5::hisG
JJY64............................Same as JJY12, plus rpd3?::LEU2
JJY65............................Same as JJY28, plus rpd3?::LEU2
JJY72............................Same as JJY41, plus rpd3?::LEU2
JJY73............................Same as JJY42, plus rpd3?::LEU2
JJY74............................Same as JJY43, plus rpd3?::LEU2
JJY75............................Same as JJY44, plus rpd3?::LEU2
1050 PE´REZ-MARTI´N AND JOHNSONMOL. CELL. BIOL.
The results (Fig. 2) show that a deletion of the deacetylase
gene RPD3 alleviates the requirement for the histone acetyl-
transferase gene GCN5 in HO gene expression. The level of
suppression of a gcn5 mutation by the deletion of RPD3 is
similar to that observed in the case of swi5 mutations (35) and
is also similar to the suppression observed in a triple swi5 gcn5
rpd3 mutant, again supporting the view that SWI5 and GCN5
function in the same pathway.
One of the defects originally observed in gcn5 mutant strains
was their inability to grow in media imposing amino acid lim-
itation (20). Thus, a strain carrying a deletion of the GCN5
gene is defective in growth in media containing 3-AT, a con-
dition that mimics histidine starvation (5). To address whether
a deletion in the RPD3 gene suppresses other defects in gcn5
strains, we also tested the ability of the RPD3 deletion to allow
growth of a gcn5 strain in the presence of 3-AT. As shown in
Fig. 2, the gcn5 strain exhibited a growth defect under such
conditions compared with an isogenic wild-type strain. Dele-
tion of the RPD3 gene indeed alleviates this defect, allowing
growth of the gcn5 strain under these conditions.
Disruption of SIN1, a gene encoding an HMG1-like protein,
also suppresses gcn5 defects. In addition to sin3 and rpd3
mutations, defects in other genes are well-known suppressors
of transcriptional deficiencies in HO. One of these genes is
SIN1. This gene encodes a protein with similarities to the
mammalian HMG1 protein, and it is believed to be a compo-
nent of chromatin (11). We have monitored both HO-lacZ
expression and the ability to grow in the presence of 3-AT of a
double mutant defective in both GCN5 and SIN1. The results
shown in Fig. 3 indicate that the absence of Sin1 protein re-
lieves the requirement of Gcn5 both for HO expression and for
growth on 3-AT.
Histone mutations also suppress gcn5 defects. An explana-
tion for the results obtained with the sin1 mutant is that the
suppression we observed is caused by a defect in chromatin
structure, such that this defective chromatin bypasses the re-
quirement for histone acetylation. If this is the case, then other
mutations which produce defective chromatin might also be
expected to suppress the gcn5 defects. Certain amino acid
changes (sin mutations) in either histone H3 or histone H4
alleviate the same set of transcriptional defects as does the sin1
mutation (12, 23). These sin mutations lie in the histone fold
domain of histones H3 and H4, and they are in close proximity
to one another on the surface of the histone octamer. It has
been proposed that residues altered by these mutations may
define a functional domain (the SIN domain) that behaves
formally as a negative regulator of transcription (12).
To address if defective histones also suppress gcn5 muta-
tions, the following histone mutant alleles were tested for their
ability to suppress a deletion of the GCN5 gene: sin2-1 (R116H
in HHT1), hhf2-7 (R45C in HHF4), hhf2-8 (V43I in HHF4),
and hhf2-13 (R45H in HHF4). In spite of the fact that the
targets for GCN5 protein are the histone tails, mutations in the
histone fold are able to efficiently suppress the defects caused
by the absence of the GCN5 gene product (Fig. 4A).
We also determined the effects of combining a deletion of
the RPD3 gene with the histone sin mutations. Levels of HO-
lacZ activity were determined in single and double mutants,
and we found in the double mutants a strong synergistic effect;
that is, the activity displayed by the double mutant is higher
than the sum of the activities displayed by the single mutants
(Fig. 4B). The same synergistic effect is also seen in combina-
tions of rpd3 and sin1 mutations (data not shown).
FIG. 2. A deletion of the RPD3 gene partially suppresses the defects caused by a disruption of the GCN5 gene. Cultures of JJY1 (wild type [wt]), JJY64
(rpd3?::LEU2), JJY28 (gcn5::hisG), and JJY65 (rpd3?::LEU2 gcn5::hisG) cells (approximately 5 ? 106/ml) were spotted in 10-fold serial dilutions on medium lacking
histidine (SD-HIS) and on medium lacking histidine and containing 10 mM 3-AT. Plates were incubated at 30°C for 3 days. The same cultures were used to measure
?-galactosidase activity (in Miller units). Values are averages of three independent measurements with less than 10% deviation.
FIG. 3. Deletion of the SIN1 gene alleviates the defects associated with disruption of the GCN5 gene. Cultures of JJY12 (wild type [wt]), JJY36 (sin1?::TRP1),
JJY28 (gcn5::hisG), and JJY45 (sin1?::TRP1 gcn5::hisG) cells (approximately 5 ? 106/ml) were spotted in 10-fold serial dilutions on medium lacking histidine (SD-HIS)
and on medium lacking histidine and containing 10 mM 3-AT. Plates were incubated at 30°C for 3 days. The same cultures were used to measure ?-galactosidase activity
(in Miller units). Values are averages of three independent measurements with less than 10% deviation.
VOL. 18, 1998Gcn5 AND CHROMATIN1051
Regulation of the yeast HO gene is complex, and many genes
that regulate HO have been identified (16). These include
genes encoding the SWI/SNF complex (22, 32); SIN1, which
encodes an HMG1-like protein (11); SIN2, which encodes
histone H3; HHF4, which encodes histone H4 (12); and SIN3,
which, along with RPD3, is involved in the deacetylation of
histones (35). In this paper, we show a requirement for the
GCN5 gene, which encodes a histone acetyltransferase (3), for
optimal transcription of the HO gene.
The identification of histone acetyltransferases and histone
deacetylases as transcriptional regulators provides molecular
FIG. 4. Histone sin mutations suppress gcn5 defects. (A) Cultures of JJY12 (wild type [wt]), JJY28 (gcn5::hisG), JJY41 (hhf2-7 gcn5::hisG), JJY42 (hhf2-8
gcn5::hisG), JJY43 (hhf2-13 gcn5::hisG), and JJY44 (sin2-1 gcn5::hisG) cells (approximately 5 ? 106/ml) were spotted in 10-fold serial dilutions on medium lacking
histidine (SD-HIS) and on medium lacking histidine and containing 10 mM 3-AT. Plates were incubated at 30°C for 3 days. The same cultures were used to measure
?-galactosidase activity (in Miller units). Values are averages of three independent measurements with less than 10% deviation. (B) Effects of rpd3 deletion on the
suppression of gcn5 defects by histone sin mutations and sin1 mutations. The strains used were JJY12, JJY28, and JJY41 through JJY44 (all as described for panel A),
as well as JJY65 (rpd3?::LEU2 gcn5::hisG), JJY72 (hhf2-7 rpd3?::LEU2 gcn5::hisG), JJY73 (hhf2-8 rpd3?::LEU2 gcn5::hisG), JJY74 (hhf2-13 rpd3?::LEU2 gcn5::hisG),
and JJY75 (sin2-1 rpd3?::LEU2 gcn5::hisG). Values are averages of three independent measurements with less than 10% deviation.
1052PE´REZ-MARTI´N AND JOHNSONMOL. CELL. BIOL.
mechanisms whereby transcription might be turned up or down
(38), but so far no such interplay between acetylase and
deacetylase activities at a single gene has been reported. The
suppression of the gcn5 defects by deletion of one of the genes
encoding a deacetylase activity provides clear support for such
interplay at the HO promoter. The suppression we observed is
only partial, suggesting a functional redundancy in the deacety-
lase activity. Another protein with deacetylase activity is en-
coded by the gene HDA1, and three additional ORFs with high
levels of homology with RPD3 and HDA1 have also been de-
scribed (29). However, we observed that deletion of HDA1 or
of one of these additional ORFs (HOS1) does not suppress the
GCN5 requirement in HO expression (data not shown). An-
other explanation for the fact that suppression is only partial is
that the rpd3 deletion may destabilize additional proteins with
which it is complexed (7), and these additional proteins may
contribute to the activation of HO.
The pattern of genetic interactions described in this work
suggests a hierarchy of gene function that pertains to chroma-
tin components, histone acetylation, and the SWI/SNF com-
plex. Loss of Swi5 (the major activator protein for the HO gene
) can be partially suppressed by sin1, sin2 (histone H3),
sin3, and rpd3 mutations (33, 35). Loss of GCN5 (a histone
acetyltransferase, also required for HO transcription) can be
suppressed by these same mutations (Fig. 2, 3, and 4A). How-
ever, while defects in the SWI/SNF complex can be suppressed
by sin1 (which is thought to be a target of the SWI/SNF com-
plex  and sin2 mutations (11, 12), they cannot be sup-
pressed efficiently by sin3 or rpd3 mutations (33, 35). These
results indicate that histone acetylation at the HO promoter
functions upstream of the SWI/SNF complex. Consistent with
this view is the strong synergy seen between rpd3 mutations
(which affect the acetylation of histone tails) and sin1 and sin2
mutations (which circumvent the need for the SWI/SNF com-
plex) (Fig. 4B). One hypothesis consistent with this genetic
hierarchy is that, at the HO promoter, histone acetylation pre-
cedes and enables the action of the SWI/SNF complex. A
similar view has recently been developed independently by
Pollard and Peterson (24a).
We thank D. Moazed, R. K. Tabtiang, and R. Candau for providing
indispensable strains and plasmids throughout the course of this work.
D. Moazed is also acknowledged for critical reading of the manuscript.
This work was supported by an NIH grant to A.D.J. and by an
EMBO long-term postdoctoral fellowship to J.P.-M.
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