From the Department Biologie I, Bereich Genetik, Ludwig-Maximilians-Universität München, Maria-Ward-Strasse 1a, D-80638 Munich, Germany
Received for publication, October 25, 2002, and in revised form, December 30, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The promoter of the galactose-inducible yeast
GCY1 gene allows high rates of basal transcription and is
kept free of nucleosomes regardless of growth conditions. The
general regulatory factor, Reb1p, as well as the nucleotide sequence of
a single Gal4p-binding site, structurally cooperate to exclude
nucleosomes from about 480 bp of DNA that spans the UASGAL, the
Reb1p-binding site, the TATA-box, and the transcriptional initiation
sites. Gal4p, which induces transcription of GCY1 about
25-fold in the presence of galactose, is not required for the
alteration in chromatin configuration in the promoter upstream region
since the hypersensitive site is unchanged when Gal4p is inactive or
absent. As soon as either the Reb1p-binding site or the UASGAL
or both are mutated, nucleosomes slip into the promoter of
GCY1 paralleled by a reduction of basal transcription
activity to about 30% in either single mutant and to <10% in the
double mutant. In the mutant of the Reb1p-binding site, induction by
galactose/Gal4p restores a nucleosome-free state to an extent
resembling the GCY1 wild-type promoter, showing that, in
principle, activated Gal4p can exclude nucleosomes on its own. Northern
blots of GCY1 transcripts confirm that Reb1p modulates
basal transcription and has little influence on the
galactose-induced state.
Repression by nucleosomes represents a general principle of
transcriptional inactivation of genes in eucaryotes. In repressed promoters of regulated genes, packaging of DNA into nucleosomes usually
veils binding sites for transcription factors and/or for the basic
transcription machinery (1-5). Consequently, activation of most
regulated genes requires the removal of one or several nucleosomes
before the transcriptional preinitiation complex can be
assembled (for example, see Refs. 6-9). Revealingly, in yeast, several
promoters can be activated upon experimental nucleosome depletion even
in the absence or inactive state of the respective transactivators (10,
11). This means that, in principle, basal transcription can ensue in
the absence of specific transactivators as long as the basal
transcription machinery has access to the core promoter. On the other
hand, promoters of constitutive genes (12, 13) and a number of
inducible genes (14-16) have been described that are kept free of
nucleosomes permanently, and those nucleosomes flanking the gap, which
usually spans the cis-acting sites for regulated
transcription activators and/or the basal transcription machinery,
occupy quite distinct positions. The molecular or structural bases of
permanent nucleosome exclusion are not well understood.
Normal B-helical DNA is compatible with packaging into
nucleosomal structures. Persistent nucleosome exclusion may be
accomplished by one of at least three different strategies: (i) Either
preferred binding of two nucleosomes to two adjacent stretches of
prebent DNA positions nucleosomes in such a way that their distance is smaller than required for the accommodation of an additional nucleosome (<145 bp in yeast). Such a nucleosome arrangement, called
translational positioning of nucleosomes, has been found to be the
basis of accessibility of linker DNA to specific transactivators (8, 13, 17, 18). (ii) As an alternative, a particular nucleotide sequence,
which deviates from B conformation, may give rise to a structure that
is incompatible with packaging into nucleosomes. Several stretches of
poly(dA·dT) or poly(dG·dC) homooligomeric sequences have been found
to be incompatible with packaging into nucleosomes due to their rigid
DNA structure (19-23). (iii) As a third possibility, DNA-binding
proteins (architectural proteins) may distort the B-helical
conformation of the DNA in a way that is incompatible with wrapping
around nucleosomes and, thereby, may contribute to the positioning of
nucleosomes. The rDNA enhancer-binding protein from yeast
(Reb1p),1 which is among the
abundant multifunctional "general regulatory factors," is supposed
to play an architectural role and to exclude nucleosomes from the
flanks of its binding site on DNA. Reb1p binding to the
GAL1-GAL10 intergenic region creates a nucleosome-free gap
of about 230 bp (14, 24, 25).
The majority of genes that are transcribed on demand must deal with
nucleosomes that cover the respective promoter regions. Therefore, one
role of specific transcriptional activator proteins is to induce the
disruption of nucleosomal structures in core promoter regions or to
recruit accessory proteins that are able to displace nucleosomes from
the core promoter. Nucleosome displacement by regulatory proteins was
demonstrated, for instance, for the PHO5 promoter, which is
transcriptionally activated by the transactivator, Pho4p. Upon
induction by phosphate exhaustion, four out of six positioned
nucleosomes are removed during the activation process jointly by the
regulatory proteins, Pho4p and Pho2p (8, 26). Gal4p, a potent
transactivator protein, is able to disrupt nucleosomes in the core
promoter regions of galactose-inducible genes, such as the promoters of
the GAL1-GAL10 and GAL80 genes (9, 14, 15,
25, 27). Gal4p binds to its target sites in the respective UASGAL; these sites, which are permanently free of
nucleosomes, remove repressing nucleosomes from core promoter regions,
enabling the basal transcription machinery to assemble and to initiate transcription.
GCY1 (galactose-inducible
crystallin-like yeast protein) was found to
encode a glycerol dehydrogenase involved in osmoregulation and
osmotolerance of Saccharomyces cerevisiae (28, 29). We have
investigated transcriptional regulation of the galactose-inducible gene, GCY1, which is activated by the specific regulator,
Gal4p. Expression of GCY1 is induced about 25-fold by growth
on galactose as carbon source due to Gal4p binding to a single
UASGAL in the upstream control region (30). However,
GCY1 is transcribed at a relatively high basal level in the
presence of glucose or glycerol as carbon sources. Our previous studies
revealed that basal expression of GCY1 is stimulated 3-fold
by the abundant general regulatory factor, Reb1p, which binds to the
promoter of GCY1 about 100 bp upstream the canonical TATA
box (31, 32). Reb1p is supposed to exert its stimulating effect on
transcription mainly by excluding nucleosomes from the flanks of its
binding motifs and thereby to facilitate binding of other transcription factors to their target sites (14, 24). Surprisingly, the UASGAL contributes to basal transcription of GCY1 as well even in Gal4p deletion mutants (31). We have analyzed the chromatin structure of the GCY1 promoter to study the
principles of basal expression and constitutive nucleosome exclusion.
We have investigated the influence of mutations in the
GCY1 promoter, i.e. deletion of the Reb1p-binding
site or mutation of the UASGAL, or the influence of the
presence or absence of Gal4p on the nucleosomal array in the upstream
control region of GCY1. We demonstrate that in wild type,
the entire promoter region of GCY1 is constitutively free of
nucleosomes and that the nucleosome-free gap spans an unusually long
distance of about 480 bp. Mutation of either the Reb1p-binding site or
the UASGAL or both cis-acting elements results in
nucleosome packaging of the respective region, indicating that Reb1p
binding and the UASGAL are jointly responsible for the
permanent exclusion of nucleosomes from the GCY1 promoter and exert an additive effect.
Plasmids and Strains--
Vectors pBluescript KS Introduction of Genomic Mutations into the Promoter of
GCY1--
Deletion of the Reb1p-binding site, the point
mutation of the UASGAL, as well as the double mutation of both
cis-acting elements in the genomic context of the
GCY1 promoter was accomplished by a two-step gene
replacement. Construct pKS Analysis of Chromatin Structure by Digestion with DNaseI or
Micrococcal Nuclease--
Yeast cells were grown in rich medium
containing 3% glucose, 3% galactose, or 3% glycerol, 2%
ethanol as carbon sources. YM707 was cultured on 3% galactose, 3%
glycerol, 2% ethanol to simulate galactose-induced physiological
conditions. Crude nuclei were prepared and digested with increasing
concentrations of DNaseI (Roche Molecular Biochemicals) as described by
Almer and Hörz (36). Micrococcal nuclease (MBI Fermentas, St.
Leon-Rot, Germany) digests were done as described by Thoma (37).
Reactions were terminated by the addition of 0.5% SDS, 4 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 200 µg
of proteinase K (Merck Eurolab) and incubated at 37 °C for 30 min.
DNA was extracted twice with phenol/chloroform and precipitated by
ethanol. After the pellet had been resuspended in 10 mM
Tris-HCl, pH 7.5, 1 mM EDTA buffer, RNA was digested by
RNaseA (Roche Molecular Biochemicals) at 37 °C for 60 min. After
extraction once with phenol/chloroform and once with chloroform, DNA
was ethanol-precipitated. Purified DNA was digested with
EcoRV. Gel electrophoresis was at 100 V in 1.0 or 1.5%
agarose gels. After Southern transfer onto nylon membranes
(Biodyne A transfer membrane, Pall, Dreieich, Germany), DNA was
detected by a randomly primed, radiolabeled 780-bp
AsnI/EcoRV DNA probe that hybridized to the
N-terminal portion of the neighboring divergently transcribed RIO1 gene. DNA marker fragments were excised from a plasmid
that contained the entire intergenic region of the
GCY1/RIO1 gene pair and coding regions of the
genes GCY1 and RIO1. Digestions were performed
with EcoRV to generate the distal DNA fragment end and with
restriction endonucleases that cleave within the respective regions of
interest (EcoRV/AsnI, 780 bp;
EcoRV/XhoI, 1060 bp; EcoRV/HindII, 1280 bp;
EcoRV/BsmI, 1480 bp;
EcoRV/BbrPI, 1600 bp;
EcoRV/EcoRV, 2130 bp).
Miscellaneous Procedures--
Molecular operations such as RNA
isolation and Northern blotting were performed according to standard
protocols (38) or as recommended by the manufacturer. Northern blots
were quantified using the ImageQuant analysis software (Amersham
Biosciences).
Effect of Genomic Promoter Mutations on Transcription of
GCY1--
Previous molecular analyses of the galactose-inducible
GCY1 promoter (30-32) have revealed the presence of three
cis-acting elements: an upstream binding motif for Gal4p
(positions In Vivo Footprinting of the GCY1 Wild-type Promoter--
To study
chromatin structure and the effect of transcriptional induction by
galactose on the nucleosomal array at the GCY1 promoter,
nuclear footprints were performed in vivo using DNaseI or
micrococcal nuclease digestion of native chromatin. Since expression of
GCY1 is induced by galactose and weakly repressed by
glucose, and to analyze the influence of the carbon source on the
arrangement of nucleosomes, analyses of the GCY1 promoter
were performed after growth of yeast cells on galactose, glucose, or
glycerol/ethanol (Fig. 3A and
B). DNaseI- or micrococcal nuclease-treated chromatin from
yeast cells after growth on all three carbon sources (glucose, galactose, or glycerol/ethanol) displayed no obvious differences in the
nucleosomal organization of the GCY1 promoter and revealed that under any condition, the upstream control region of
GCY1 was free of nucleosomes. The nucleosome-free gap
extends over a region of about 480 bp that contains the transcriptional
initiation sites, the TATA element, the Reb1p-binding site, and the
UASGAL. The protected regions flanking the gap on the
3'-GCY1-coding side as well as over the two distal
poly(dA·dT) blocks at the 5'-side are narrow (about 140 bp),
indicating that these nucleosomes are positioned.
Evidently, in the GCY1 wild-type promoter, the Gal4 protein
does not contribute to the nucleosome-free state of the GCY1
upstream promoter region since the nucleosomal array is identical after growth on glucose (Fig. 3A), where the expression level of
Gal4p is extremely low, or on glycerol/ethanol, where Gal4p is
essentially inactive and complexed with Gal80p, or on galactose (Fig.
3B). To confirm this observation and test directly whether
Gal4p has any influence on the array of nucleosomes, we analyzed the
chromatin structure of the GCY1 promoter in a
gal4 background (Fig. 3B). Indeed, the large
nucleosome-free gap was detected likewise in wild type and a strain
that lacked functional Gal4p, corroborating that nucleosome exclusion
from the regulatory GCY1 upstream region is not caused by
binding of Gal4p to DNA. As an alternative possibility, we examined
whether destruction of the binding sites for Gal4p or Reb1p influence
the array of nucleosomes.
Mutations of Reb1p-binding Site or UASGAL Result in
Packaging of the GCY1 Promoter into Nucleosomes--
Our previous
studies on expression of Gcy1/
In vivo, DNaseI or micrococcal nuclease digests were
performed with each of the two single GCY1 promoter
mutants (YMA2
In the double mutant, the patterns of nucleosomes are similar to each
of the single mutants with the exception that the three protected
regions are short (about 150 bp) and slightly more distinct as compared
with the single mutants, and nucleosomes are separated by narrow
linkers. This implies that that three positioned nucleosomes occupy the
GCY1 upstream region in the double mutant.
The molecular mechanisms allowing basal transcription are poorly
understood. Our previous analyses demonstrated that GCY1 is
expressed at a relatively high basal level in the absence of galactose
as a carbon source. Basal expression in the absence of active Gal4p was
found to be in part dependent on the general regulatory factor, Reb1p,
which binds to its target site about 100 bp 5' of the TATA box and
about 140 bp 3' of the UASGAL (31). The UASGAL
contributes to basal transcription of GCY1 as well, an
effect that is independent of Gal4p binding. In the present work, we
analyzed the chromatin structure of the Gal4p-inducible gene,
GCY1. Surprisingly, the complete promoter region of
GCY1, 480 bp in length, is permanently free of nucleosomes independent of growth conditions. DNaseI as well as micrococcal nuclease digestions of native chromatin revealed no obvious differences in the chromatin organization after growth of yeast cells on glucose, galactose, or glycerol/ethanol. The nucleosome-free gap of about 480 bp
comprises the UASGAL, the Reb1p recognition site, the TATA box,
and the transcriptional initiation sites. Although GCY1 is
transcriptionally regulated by Gal4p in the presence of galactose as
carbon source, no alterations of the chromatin structure can be
detected upon Gal4p activation. These observations have been
corroborated by analyses of the chromatin structure of the GCY1 promoter region in a Gal4p-deficient yeast background.
In the absence of the specific transactivator Gal4p, the nucleosomal array is identical to that of the GAL4 wild-type yeast
strain, suggesting that the Gal4 protein on its own has no bearing on the chromatin organization of the GCY1 upstream region.
Investigations on the GAL1-GAL10 intergenic region and the
GAL80 promoter demonstrated that the UASGAL sites
are constitutively free of nucleosomes as well (15, 25, 27). However,
Gal4p levels are very low, and binding to its target site is not
detectable when yeast cells are grown on glucose (15), whereas the
factor binds to its recognition site on glycerol/ethanol, but with its
activation domain blocked by Gal80p, and is active only when bound
under conditions of galactose induction (reviewed by Johnston (40)).
Therefore, nucleosome exclusion cannot be attributed to Gal4p action
but must be due either to structural properties of the DNA at the
UASGAL per se or to another DNA-binding protein that
binds to the same or an overlapping motif and inhibits the assembly of
nucleosomes. We have excluded the possibility that the UASGAL
region is strongly bent or kinked, and as a consequence, incompatible with packaging into nucleosomes by using permutation analyses (data not
shown). However, we cannot decide whether this cis-acting element displays a quite rigid DNA structure that cannot be assembled into chromatin or whether an additional protein binds to an overlapping sequence within the UASGAL apart from Gal4p. No indication in
favor of the latter possibility could be detected by electrophoretic mobility shift assays with nuclear extracts from glucose-grown cells.
In contrast to the GCY1 promoter, the hypersensitive regions
within the UASGAL of the GAL1-GAL10 and
GAL80 promoters span only about 150-230 bp and do not
include the TATA elements or the transcriptional start sites (15, 25).
Gal4p action induces alterations of the chromatin structure in these
promoters, i.e. repressing nucleosomes are disrupted in the
core promoters (9, 15, 25). Since at the GCY1 promoter, the
TATA box and the initiation sites are not concealed in chromatin, there
is no need for Gal4p to remove nucleosomes from the basal promoter of
GCY1. Therefore, the permanent exclusion of nucleosomes from the core promoter of GCY1 provides a plausible explanation
for the relatively high basal transcription rate.
In addition to Gal4p, the general regulatory factor, Reb1p, has been
shown to bind to the upstream control region of GCY1. This
abundant DNA-binding protein stimulates basal transcription of
GCY1 about 3-fold (31). Reb1p is supposed to act mainly by nucleosome exclusion from the flanks of its binding site over a region
of about 230 bp (13). One way of its action may be linked to the recent
observation with Reb1p action on the promoter on the
profilin gene, where it was found that the binding of Reb1p strongly
bends DNA. Therefore, it has been concluded that the DNA
deviates from the normal B conformation and assumes a structure that is
incompatible with packaging into
nucleosomes.2 Whether in
addition Reb1p assists recruitment of chromatin remodeling machines or
histone deacetylases to the promoter site, however, remains to be
elucidated. Nevertheless, the effect of Reb1p on expression of
GCY1 is small and restricted to basal transcription of the
gene (31).
On the other hand, the nucleosome-free gap in the GCY1
promoter comprises about 480 bp and thus cannot be attributed to Reb1p binding alone. Our investigations imply that Reb1p binding to the
upstream region of the GCY1 gene and the UASGAL
together contribute to the permanent exclusion of nucleosomes.
To analyze the importance of the Reb1p recognition site and the
UASGAL for the organization of the chromatin in the GCY1 upstream control region, we introduced mutations of the
respective cis-acting elements into the original genomic
context. Mutation of either the Reb1p target sequence or the
UASGAL resulted in a loss of the nucleosome-free gap of 480 bp.
Three unpositioned nucleosomes occupy this region in the respective
promoter single mutants. The nucleosomal arrays are essentially
identical in both single mutants. For the promoter double mutant, in
which the Reb1p-binding site and the UASGAL have been destroyed
simultaneously, the pattern is quite similar to the one in the single
mutants. However, some hypersensitivity is still detectable in the
single mutants, whereas in the double mutant, the protected regions are more pronounced, and the linkers between the nucleosomes are more distinct, indicating that the additional nucleosomes that cover the
double mutant promoter are positioned. We propose that mutations of
either the Reb1p recognition motif or the UASGAL result in
packaging into chromatin of the upstream control region but that the
nucleosomes are not tightly fixed to a definite position but rather
allow to some extent the basal transcription machinery to assemble at
the TATA box. In contrast, in the promoter double mutant, the
transcriptional initiation complex is largely excluded from its target
since the nucleosomes seem to be positioned. Both the Reb1p target site
as well as the UASGAL are essential for nucleosome exclusion
over a stretch of 480 bp in the GCY1 promoter. Neither of
the two elements is sufficient to perform this task on its own. As soon
as one of the cis-acting elements is eliminated, basal
transcription drops to about 1/3 of the wild-type level. Gal4p
does not play a role in nucleosome exclusion from the GCY1
promoter since the chromatin structures are identical in the wild type
and in a Gal4p-deficient background. This indicates that Gal4p acts
mainly by stimulating recruitment of the basal transcription machinery
to the GCY1 promoter. However, the Reb1p-site mutation of
the GCY1 promoter is loosely covered with nucleosomes in the
absence of galactose but displays the same nucleosome-free gap as the
wild-type promoter after growth of yeast cells on galactose. This
demonstrates that, once the chromatin is closed as in the mutant at the
Reb1p site, active Gal4p is required to open the chromatin and to make
the promoter accessible, whereas in the wild type, this task is
constitutively fulfilled by the architectural activity of Reb1p. This
simultaneously explains why Reb1p stimulates basal transcription of
GCY1 but does not affect induction of GCY1 by
Gal4p action since Gal4p, in its active state, does not depend on the
nucleosome excluding function of Reb1p because it is able to disrupt
nucleosomes on its own.
In summary, and when the transcription data are compared with the
chromatin analyses, the following conclusions can be drawn: (i) In the
wild type, the chromatin is constitutively open (independent of growth
conditions and independent of the presence of Gal4p). The level of
GCY1 mRNA synthesis exclusively reflects the cellular concentration and activation status of Gal4p. (ii) Analysis of the
mutant promoters reveals a particular DNA conformation that is due to
the binding of Reb1p and a sequence element comprising the
UASGAL. Both cis-acting elements cooperate to
exclude nucleosomes from the core promoter and promote assembly of the basal transcription machinery and, thus, allow high rates of basal transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Stratagene, Heidelberg, Germany) or pUC19 were used in ligation
reactions. SURE was used as the Escherichia coli host strain
(Stratagene). Yeast strain W303-1A (33) served to introduce
genomic mutations into the promoter region of GCY1, generating the yeast strains YMA1, YMA2, YMA3, or YMA4 (see Fig. 1).
YM707 gal4-542 (obtained from M. Johnston,
Washington University, Medical Center, Department of Genetics, St.
Louis, MO) served for analyzing the chromatin structure of the promoter
region of GCY1 in a gal4-deficient genetic background.
-GCY
R, in which the natural
Reb1p-binding site of the GCY1 promoter had been replaced by
a HindIII restriction site (30), served to insert a 1170-bp
HindIII restriction DNA fragment encoding URA3 to
yield the Ura-prototrophic strain YMA1. After a PvuII restriction site had been introduced into the promoter proximal region
of GCY1 to generate homologous DNA fragment ends, the
respective URA3-containing GCY1 promoter fragment
was excised by PvuII and XhoI and inserted into
the genome by homologous recombination using the yeast transformation
protocol described by Gietz et al. (34). Recombinants were
verified by PCR and restriction digestion. Constructs
pKS
-GCY
REB1, pKS
-GCY-UASmut,
and pKS
-GCY
REB1/UASmut served to replace
the URA3 gene from the promoter region of GCY1
and to introduce the respective mutations of cis-acting elements into the promoter of GCY1 in the genomic context.
DNA fragments carrying the mutation were excised by PvuII
and XhoI (see above) and co-transformed with YEp351
(LEU2). Transformants were initially selected by growth on
selective media lacking leucine. URA3-auxotrophic
recombinants were identified by replica plating on
5-fluoroorotate-containing media (35) and analyzed by PCR using whole
yeast cells. Genomic promoter mutations were verified by restriction
digests of the amplificate with HindIII (YMA2 = GCY
REB1) or XbaI (YMA3 = GCY-UASmut) or with HindIII and XbaI (YMA4 = GCY
REB1/UASmut), respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
369 to
353 with the adenine of the translational start
triplet as +1), a canonic Reb1p site (CATTCACCCG, positions
224 to
215), and a consensus TATAAA core promoter sequence
(positions
111 to
116) (Fig.
1A). A poly (dA·dT) block,
frequently found adjacent to Reb1p-binding sites, is absent. To study
the bearing of Reb1p or the UASGAL on the transcriptional
activity of the GCY1 promoter, the respective
cis-acting elements were mutated in the genomic context. The
mutations of the Reb1p site,
REB1, or the Gal4p site,
UASmut, or the double mutant,
REB1/UASmut, were brought into the correct genomic context by a two-step gene replacement yielding the respective mutant strains, YMA2
REB1, YMA3
UASmut, or YMA4
REB1/UASmut (Fig. 1,
B and C). Transcription was measured by Northern
blotting, and the signals were quantified. Total RNA was isolated from
wild-type cells grown on glucose, glycerol/ethanol, or galactose or
from the three promoter mutants, YMA2
REB1, YMA3
UASmut, or YMA4
REB1/UASmut (Fig.
2A). The signals were
quantified relative to the maximum activity displayed by the
galactose-induced wild type (Fig. 2B). In addition, all
values were normalized to the respective signal of the mRNA of
major adenylate kinase, which is expressed constitutively (39) and used
as a loading control. It must be considered that a 25-30-fold induction (which has been measured with lacZ reporter
constructs (31)) is outside the linear range of signal evaluation in
Northern blots so that basal expression levels are overestimated
relative to induced mRNA synthesis. Each of the single mutants,
REB1 or UASmut, reduce basal GCY1
transcription to about 1/3 of the wild type, whereas the
galactose-induced level of mRNA synthesis in strain
REB1 is
hardly affected at all, suggesting that Reb1p exclusively endorses
basal transcription and has a marginal effect on induced transcription.
The
REB1/UASmut double mutation reduces GCY1
transcription to about 10%, showing that the contributions of both
elements to transcriptional activation of the GCY1 promoter are about additive (Fig. 2B).
View larger version (17K):
[in a new window]
Fig. 1.
GCY1 promoter
and genomic mutations. A, GCY1
wild-type promoter (black bar). Positions of UASGAL
(striped box), Reb1p-binding site (stippled box),
TATA box (black box), transcriptional (asterisk),
and translational initiation sites are indicated by numbers below
the bar. B, YMA1, disruption construct for two-step
gene displacement. C, genomic promoter mutants: YMA2 REB1, YMA3
UASmut, YMA4
REB1/UASmut.
View larger version (35K):
[in a new window]
Fig. 2.
Transcriptional analysis of wild-type and
mutant GCY1 promoter. A, upper
panel, Northern analysis of GCY1 mRNA from wild
type grown on glucose (Glc), glycerol/ethanol
(Gro/EtOH), or galactose (Gal) or
mutants YMA2 REB1 or YMA3 UASmut (Um)
or YMA4
REB/UASmut
(
R/Um), respectively. Lower
panel, AKY2 mRNA, loading control. B,
quantification of the Northern blot data from panel A
normalized to the loading control.
View larger version (73K):
[in a new window]
Fig. 3.
Chromatin structure at the GCY1
Promoter. A, chromatin structure at the
GCY1 promotor in cells grown on glucose. Chromatin and
"naked" DNA were digested with increasing concentrations of DNase
I (symbolized by wedges) as described under "Experimental
Procedures." Hypersensitive regions of DNA and arrangement of
nucleosomes are schematized on the right. Reb1p-binding
site, UASGAL, distal oligo(dA:dT) blocks, core promoter
(T), and transcriptional initiation sites (i) are
indicated. B, wild-type cells were grown on the carbon
sources indicated (Glc, glucose; Gal, galactose;
Gro/EtOH, glycerol/ethanol), the gal4
strain was grown on galactose/glycerol/ethanol, and chromatin or
naked DNA was digested by increasing concentrations of DNase I
(left) or micrococcus nuclease (right). Positions
of marker fragments are indicated on the margins. The
nucleosomal arrangement is schematized in the center as
described in the legend for Fig. 2.
-galactosidase reporter proteins
demonstrated that basal expression of GCY1 is mainly
influenced by binding of the general regulatory factor Reb1p to the
upstream control region on the one hand and on the other hand by the
single UASGAL. Basal expression of the Gcy1/
-galactosidase
reporter (absence of galactose) is diminished to one-third after
deletion of the Reb1p-binding site and reduced to about the same
degree by point mutations in the UASGAL in line with the
Northern blots shown in Fig. 2. In the double mutant, which was
simultaneously deleted for the Reb1p-binding site and for the 5'-flank
of the UASGAL, basal transcription of GCY1 was
reduced to 1/10 of wild type (30), suggesting that the two sites
operate independently of one another and that their effects are about
additive. We also showed that the stimulating effect of the
UASGAL was not due to Gal4p activity in the absence of
galactose as carbon source as it was also observed in a gal4
genetic background. To investigate whether Reb1p binding or/and the
UASGAL activate transcription of GCY1 via an
alteration of the chromatin configuration to yield a nucleosome-free gap, the respective promoter mutations were introduced into the original genomic context by a two-step gene replacement (see above and
also see Fig. 1, B and C), and the arrangement of
nucleosomes was analyzed. The introduction of these mutations into the
genomic situation by homologous recombination was necessary to exclude artifacts that could result from plasmid constructs or ectopic insertion of the respective promoter into the genome by means of
integrative plasmids.
REB1 and YMA3 UASmut) as well as with the
double mutant (YMA4
REB1/UASmut). In each of the single
mutants as well as in the double mutant, the chromatin structure was
strikingly different from the wild-type situation (Fig.
4). When only one of the
cis-acting elements was eliminated, we observed that the
hypersensitivity decreased, and two bands of slightly sensitive linker
DNA signals emerged instead. With micrococcal nuclease, we observed
similar patterns as with DNaseI with both single mutants. In the
presence of galactose, the chromatin structure of the
REB1 single
mutant was identical to that of the wild-type promoter. This
demonstrates that, once nucleosomes cover the promoter as in the
REB1 mutant, Gal4p is able to bind to nucleosomal DNA and to disrupt
repressing nucleosomes at the core promoter in vivo on its
own, i.e. independently of Reb1p. This property is not
required in the wild-type situation of the Reb1p site due to the
architectural activity of Reb1p.
View larger version (44K):
[in a new window]
Fig. 4.
Chromatin structure of promoter mutants of
GCY1. Chromatin or naked DNA of the mutants REB1,
UASmut, or
REB1/UASmut were digested with
either DNase I (left) or micrococcus nuclease
(right) as described under the legend for Fig. 2. Three
nucleosomes cover the GCY1 upstream region, the most distal
of which is mobile (double arrow). The protection by these
nucleosomes is incomplete in the single mutants (symbolized by
shaded nucleosomes).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We thank M. Johnston, St. Louis, MO, for yeast strain YM707 gal4-542.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 190, TP B6 (to W. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-89- 2180-6176; Fax: 49-89-2180-6160; E-mail:
M.Angermayr@lrz.uni-muenchen.de.
Published, JBC Papers in Press, January 20, 2003, DOI 10.1074/jbc.M210932200
2 M. Angermayr and W. Bandlow, unpublished.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Reb1p, rDNA enhancer-binding protein; GCY1, galactose-inducible yeast gene encoding an aldo/keto reductase; RIO1, yeast gene encoding the protein kinase, Rio1p.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kornberg, R. D., and Lorch, Y. (1999) Cell 98, 285-294[Medline] [Order article via Infotrieve] |
2. | Struhl, K. (1999) Cell 98, 1-4[Medline] [Order article via Infotrieve] |
3. | Workman, J. L., and Kingston, R. E. (1998) Annu. Rev. Biochem. 67, 545-579[CrossRef][Medline] [Order article via Infotrieve] |
4. | Gregory, P. D., and Hörz, W. (1998) Curr. Opin. Cell Biol. 10, 339-345[CrossRef][Medline] [Order article via Infotrieve] |
5. | Becker, P., and Hörz, W. (2002) Annu. Rev. Biochem. 71, 247-273[CrossRef][Medline] [Order article via Infotrieve] |
6. | Piña, B., Barettino, D., Truss, M., and Beato, M. (1990) J. Mol. Biol. 216, 975-990[Medline] [Order article via Infotrieve] |
7. | Archer, T. K., Cordingley, M. G., Wolford, R. G., and Hager, G. L. (1991) Mol. Cell. Biol. 11, 688-698[Medline] [Order article via Infotrieve] |
8. | Fascher, K. D., Schmitz, J., and Hörz, W. (1990) EMBO J. 9, 2523-2528[Abstract] |
9. | Axelrod, J. D., Reagan, M. S., and Majors, J. (1993) Genes Dev. 7, 857-869[Abstract] |
10. | Han, M. H., and Grunstein, M. (1988) Cell 55, 1137-1145[Medline] [Order article via Infotrieve] |
11. | Durrin, L. K., Mann, R. K., and Grunstein, M. (1992) Mol. Cell. Biol. 12, 1621-1629[Abstract] |
12. | McLean, M., Hubberstey, A. V., Bouman, D. J., Pece, N., Mastrangelo, P., and Wildeman, A. G. (1995) Mol. Microbiol. 18, 605-614[Medline] [Order article via Infotrieve] |
13. |
Angermayr, M.,
Oechsner, U.,
Gregor, K.,
Schroth, G. P.,
and Bandlow, W.
(2002)
Nucleic Acids Res.
30,
4199-4207 |
14. | Fedor, M. J., Lue, N. F., and Kornberg, R. D. (1988) J. Mol. Biol. 204, 109-127[Medline] [Order article via Infotrieve] |
15. | Lohr, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10628-10632[Abstract] |
16. |
Moreira, J. M. A.,
Hörz, W.,
and Holmberg, S.
(2002)
J. Biol. Chem.
277,
3202-3209 |
17. | Travers, A. A. (1990) Cell 60, 177-180[Medline] [Order article via Infotrieve] |
18. | Straka, C., and Hörz, W. (1991) EMBO J. 10, 361-368[Abstract] |
19. | Struhl, K. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8419-8423[Abstract] |
20. | Brandl, C. J., and Struhl, K. (1990) Mol. Cell. Biol. 10, 4256-4265[Medline] [Order article via Infotrieve] |
21. | Tanaka, S., Zatchej, M., and Thoma, F. (1992) EMBO J. 11, 1187-1193[Abstract] |
22. | Tanaka, S., Livingstone-Zatchej, M., and Thoma, F. (1996) J. Mol. Biol. 257, 919-934[CrossRef][Medline] [Order article via Infotrieve] |
23. | Iyer, V., and Struhl, K. (1995) EMBO J. 14, 2570-2579[Abstract] |
24. | Chasman, D. I., Lue, N. F., Buchman, A. R., LaPointe, J. W., Lorch, Y., and Kornberg, R. D. (1990) Genes Dev. 4, 503-514[Abstract] |
25. | Lohr, D., and Hopper, J. E. (1985) Nucleic Acids Res. 13, 8409-8423[Abstract] |
26. | Schmid, A., Fascher, K. D., and Hörz, W. (1992) Cell 71, 853-864[Medline] [Order article via Infotrieve] |
27. | Cavalli, G., and Thoma, F. (1993) EMBO J. 12, 4603-4613[Abstract] |
28. | Costenoble, R., Valadi, H., Gustafsson, L., Niklasson, C., and Franzen, C. J. (2000) Yeast 16, 1483-1495[CrossRef][Medline] [Order article via Infotrieve] |
29. | Norbeck, J., and Blomberg, A. (2000) Yeast 16, 121-137[CrossRef][Medline] [Order article via Infotrieve] |
30. | Magdolen, V., Oechsner, U., Trommler, P., and Bandlow, W. (1990) Gene (Amst.) 90, 105-114[CrossRef][Medline] [Order article via Infotrieve] |
31. | Angermayr, M., and Bandlow, W. (1997) Mol. Gen. Genet. 256, 682-689[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Angermayr, M.,
and Bandlow, W.
(1997)
J. Biol. Chem.
272,
31630-31635 |
33. |
Crivellone, M. D.,
Wu, M.,
and Tzagoloff, A.
(1988)
J. Biol. Chem.
263,
14323-14333 |
34. | Gietz, R. D., Schiestl, R. H., Willems, A. R., and Woods, R. A. (1995) Yeast 11, 355-360[Medline] [Order article via Infotrieve] |
35. | Boeke, J. D., LaCroute, F., and Fink, G. R. (1984) Mol. Gen. Genet. 197, 345-346[Medline] [Order article via Infotrieve] |
36. | Almer, A., and Hörz, W. (1986) EMBO J. 5, 2681-2687[Abstract] |
37. | Thoma, F. (1996) Methods Enzymol. 274, 197-214[Medline] [Order article via Infotrieve] |
38. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
39. | Oechsner, U., Magdolen, V., Zoglowek, C., Häcker, U., and Bandlow, W. (1988) FEBS Lett. 242, 187-193[CrossRef][Medline] [Order article via Infotrieve] |
40. | Johnston, M. (1987) Microbiol. Rev. 51, 458-476 |