From the Department Biologie I, Bereich Genetik, Ludwig-Maximilians-Universität München, Maria-Ward-Strasse 1a, D-80638 Munich, Germany
Received for publication, February 20, 2003, and in revised form, March 10, 2003
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ABSTRACT |
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The molecular basis of constitutive gene
activation is largely unknown. The yeast profilin gene
(PFY1), encoding a housekeeping component of the actin
cytoskeleton, is constitutively transcribed at a moderate level. The
PFY1 promoter dispenses with classical transactivators and
a consensus TATA box; however, it contains a canonic site for the
abundant multifunctional nuclear factor rDNA enhancer-binding protein
(Reb1p) combined with a dA·dT element. Reb1p binds specifically
in vitro. Mutation of this site reduces PFY1
expression to about 35%. A nucleosome-free gap of about 190 bp is
centered at the genomic Reb1p binding site in vivo and
spans the presumptive core promoter and transcriptional initiation
sites. Nucleosomes at the border of the gap are positioned. Mutation of
the Reb1p motif in the genomic PFY1 promoter abolishes
nucleosome positioning, fills the gap with a non-positioned nucleosome,
and reduces transcription by a factor of 3. From permutation studies we
conclude that Reb1p induces a strong bend into the DNA. Phasing analyses indicate that it is directed toward the major groove. The data
suggest that Reb1p plays an architectural role on DNA and that
Reb1p-dependent DNA bending leads to a DNA conformation that is incompatible with packaging into nucleosomes and concomitantly facilitates constitutive transcription. In the absence of other transcription activators, Reb1p excludes nucleosomes and moderately stimulates transcription by distorting DNA.
The actin cytoskeleton is fascinating because of both the
complexity of its functions and the dynamics of its structure. In yeast, it has been shown to be involved in the establishment of cell
polarity and bud site selection, in intracellular transport, and in
signal transduction as well as in cytokinesis. According to the
plethora of functions, the organization of the microfilament system and
intracellular distribution of actin is highly dynamic and closely
linked to the progression of the cell cycle. Despite continuous cell
cycle-controlled reorganization of the cytoskeleton, the single actin
gene in yeast
(ACT1)1 is
expressed constitutively (1). The polymerization state and
organization of actin is controlled posttranscriptionally by accessory
proteins (2-4), and actin as well as most of the actin-binding
proteins are expressed constitutively.
One of the actin-binding proteins is profilin (3, 5-8), which has been
thought previously to regulate actin filament formation exclusively by
sequestering G-actin monomers and thereby to antagonize actin
polymerization (3). However, recent results point to a more complex
regulation (9). It involves the binding of phosphoinositides to
profilin and their controlled cleavage by phospholipase C, which could
provide the basis for the regulation of profilin-actin interaction (10,
11) and, as a consequence, in signaling to actin (9, 12-14). In fact,
results of recent experiments on yeast profilin and CAP, a
component of the yeast adenylyl cyclase complex, functionally link the
growth signaling pathway to the control of the cytoskeleton (15).
Previously we have isolated and characterized the single structural
gene for yeast profilin (PFY1) and shown that its deletion
leads to a temperature-conditional phenotype. PFY1 is
constitutively transcribed at a moderately high level (8, 16, 17).
Amazingly little is known about the chromatin constellation at
constitutive promoters and about transcription factors involved in
transcription of housekeeping genes. It is supposed that constitutive transcription reflects a static situation in which the promoter is in a
permanently activated state. Accordingly these promoters are presumed
to be constitutively kept free of nucleosomes (18, 19), but the
principles and mechanisms underlying nucleosome exclusion are far from
clear. Moreover it is unknown whether constitutive transcription is
basal transcription involving only the basal transcription apparatus or
whether specific transactivators are required in addition. Since
cis-acting sites for any of the classical transactivators
are absent from the 5'-flank of the PFY1 gene, we tested the
hypothesis that constitutive transcription of this gene dispenses with
classical transcription activators. We have dissected the
PFY1 promoter and demonstrate that it harbors a binding site
for the rDNA enhancer-binding
protein (Reb1p; also known as Grf2p, factor Y, and factor Q).
Reb1p is among the abundant so-called "general regulatory factors."
It is multifunctional, as it is involved in transcriptional termination
(20, 21), binds to telomeres and centromeres (22), and plays a role in
transcriptional regulation of a plethora of functionally unrelated
genes transcribed by either polymerase I or II (18, 22-26). However,
the direct transcriptional activation potential of Reb1p is marginal
compared with specific activator proteins, but combinations of Reb1p
binding sites with cognate motifs for weak transcription activators or
dA·dT elements cause considerable synergistic effects (22, 27-29).
Interactions between Reb1p and the basal transcription machinery are
discussed as well (1, 22, 26, 30, 31). Reb1p is encoded by an essential gene (32); however, the reason for its indispensability has not yet
been established. The 125-kDa protein binds as a monomer to its site on
DNA with the consensus YNNYYACCCG, and its DNA-binding domain, which
bears some similarity to the vertebrate proto-oncogene myb, is extraordinarily large (about 400 amino acids)
(32, 33). The analysis of the chromatin structure at the
GAL1-GAL10 promoter, which contains a Reb1p site overlapping
with a motif for binding of Gal4p, has revealed a nucleosome-free gap
of 230 bp. Previous studies indicated that Reb1p binding is responsible
for nucleosome exclusion from the GAL1-GAL10 promoter (34).
However, more recent results have demonstrated that the chromatin
structure in this intergenic region is not influenced by Reb1p binding
(35). Thus, the importance of Reb1p binding for the arrangement of
nucleosomes and the efficiency of transcriptional initiation is still
obscure. Whether nucleosome exclusion is a general feature of Reb1p
remains to be elucidated and is controversially discussed (22, 29, 31).
More importantly, if nucleosome exclusion is a general feature, it is
yet unknown which property of Reb1p prevents assembly of nucleosomes in
the flanks of its binding site extending over distances as long as
about 100 bp to either side.
We show that constitutive transcription at the PFY1 promoter
dispenses with classical transactivators. We demonstrate that Reb1p has
an architectural role, and its DNA binding is necessary and sufficient
to keep nucleosomes off the DNA region spanning the core promoter and
the transcription initiation sites of the PFY1 promoter.
Destruction of the Reb1p binding motif in the genomic context by
site-directed point mutation leads to occupation of the promoter by
randomly arranged nucleosomes and reduction of mRNA synthesis by a
factor of 3. Permutation studies imply that nucleosome exclusion could
be related to the strong bending of the DNA structure induced by Reb1p
binding in conjunction with a neighboring dA·dT element. We conclude
that, at the PFY1 promoter, transcription may ensue
spontaneously as long as the core promoter is prebent and accessible to
polymerase II holoenzyme.
Strains and Plasmids--
pBluescript M13 KS(+)-based vectors
(Stratagene, Heidelberg, Germany) were used as templates for promoter
truncations by PCR, for site-directed point mutation, or for tandem
ligation of the permutation construct pKS Promoter Truncations and Expression Studies--
Expression from
the PFY1 promoter was studied in the genetic background of
strain W303-1A (39) transformed with the low copy (CEN6-based)
lacZ expression vector pYLZ6 (40). Promoter truncations were
made by PCR using the 5' primers H4, H3, H2, H1, H0, and H6, each
equipped with a 5'-flanking EcoRI restriction site in
combination with the reverse primers R1 or R2, which contained a
BamHI site for ligation to the lacZ reporter of
pYLZ6. Primer R2 hybridized to sequences upstream of codon 15 (pos.
+250) and, thus, amplified the intron of PFY1 as well
(schematized in Fig. 1A). All constructs used in gene
expression had the context of the AUG translational initiation codon
from PFY1 and contained the intron of PFY1 (pos.
+14 to +222) in addition. The following forward primers were used
(nucleotides specifying restriction sites are not shown): H0,
5'-TTTGTACGAAACTCATTACCC-3'; H1, 5'-GTTTTACCATGATTTTTGGCG-3'; H2,
5'-TCGCAAAGAAATGGAGTG-3'; H3, 5'-TCATCGAGGACGACGAAGACG-3'; H4,
5'-CTCGAGAAGAAATGAATATG-3'; H6, 5'-CAGTTGACCCTTTCTCATTC-3'. Reverse
primer R1 (5'-CCTTGCCAAGACATAAT-3') contained PFY1 sequences 5' of nucleotide position +14 and was used for band shift experiments. Reverse primer R2 (5'-CCGG-TTCCTATTAAGTTATC-3') was used for
lacZ fusions.
In Vitro Mutagenesis and Constructs for Permutation and Phasing
Analyses--
Site-specific mutagenesis was performed according to
Kunkel et al. (36). The mutation of the binding site for
Reb1p, Gm (indicated in bold, TCATTACATGT), introduced an
NsiI restriction site into construct H2, and the small
promoter deletions, H2 Gel Shift Assays--
Yeast nuclear extracts were prepared from
cells (41) grown on rich medium with 3% glycerol, 2% ethanol as
carbon sources. Recombinant Reb1p was extracted from E. coli
(32). All DNA fragments used in electrophoretic mobility shift assays
were recovered from pUC19 (42) or pBluescript after cleavage with the
restriction endonucleases indicated, gel electrophoretic separation,
and electroelution. A 23-bp synthetic double-stranded oligonucleotide
that harbored the sequence of the Reb1p binding site (TCATTACCCG)
plus 2 bp of upstream and 11 bp of downstream sequences of
the PFY1 promoter was ligated to the SmaI
restriction site of pUC19. For gel retention and competition analyses,
the Reb1p binding site was excised from the polylinker as a 77-bp
EcoRI/HindIII fragment. Isolated DNA fragments
were end-labeled with [
Reb1p-induced DNA bending was visualized by Southern hybridization. Gel
shift assays were performed with unlabeled DNA fragments in 8%
polyacrylamide gels (acrylamide/bisacrylamide = 37.5:1) at
7 °C. Subsequently the DNA was denatured and transferred to nylon
membranes (Biodyne A, Pall, Dreieich, Germany) by alkaline vacuum
blotting in 0.25 N NaOH, 1.5 M NaCl for 2 h according to the manufacturer's instructions (LKB 2016 Vacugene,
Amersham Biosciences). DNA fragments used for detection of the
respective DNA-protein complexes were radioactively labeled with
[ Two-step Genomic Mutagenesis--
In the first step, the
LEO1-coding region on plasmid pUC19 was disrupted at the
XhoI restriction site by insertion of the URA3
marker gene. Ura-proficient clones were tested for correct disruption
(causing no evident phenotype) by PCR using a URA3- and a
PFY1-specific primer. One of the
leo1::URA3 clones (WLDU18) was used in
the second step in which the URA3 marker was replaced by a
PFY1 mutant promoter construct. The recipient was
co-transformed with a 790-bp EcoRI/PvuI DNA
fragment containing the same mutation of the Reb1p binding site as
above and with vector YEp351 (LEU2). Ura Analyses of Chromatin Structure--
Crude nuclei were prepared
and treated with DNase I (chromatin, 10, 15, 20, and 30 units/ml;
"naked" DNA, 0.05 and 0.1 units/ml; 5 min at 37 °C) as
described previously (45). Incubation of nuclei with increasing
concentrations of micrococcal nuclease (wild type, 15, 30, and 60 units/ml; mutant, 6, 15, and 30 units/ml; naked DNA, 0.5 units/ml) was
for 5 min at 37 °C (46). After treatment with proteinase K (20 min
at 37 °C), phenol extraction, and digestion of RNA with RNase A, DNA
was ethanol-precipitated and digested with HindIII. The
randomly primed, radiolabeled 364-bp BfrI/HindIII
fragment was used as the 3' hybridization probe. The following isolated
natural or PCR-amplified and cloned DNA fragments served as standards
for length calibration and evaluation as described previously (46) (see
Fig. 4): BfrI/HindIII (360 bp),
BglI/HindIII (590 bp),
NdeI/HindIII (950 bp), NdeI/mutant NsiI site (1270 bp), SspI (1520 bp), and
BamHI/HindIII (1830 bp).
Miscellaneous Procedures--
Promoter Truncations and Reporter-based Expression
Studies--
The PFY1 gene is encoded on chromosome XV in
tandem with LEO1, a non-essential gene coding for an acidic,
highly polar protein (50), the function of which has not yet been
established. The intergenic distance comprises 287 bp, and the
non-transcribed region between the presumed transcriptional termination
motif of LEO1 and the most upstream initiation site of
PFY1 at pos.
PFY1 is expressed constitutively at a moderate level (8).
Computer analysis of the PFY1 upstream region revealed only
two candidate sequences for cis-acting motifs: a presumptive
canonic binding site for the general regulatory factor Reb1p (sequence TCATTACCCG, pos.
As an approach to identify cis-acting elements relevant for
constitutive transcription of PFY1, the complete 5'-upstream
region together with the N-terminal 14 triplets of the 5'-coding region (reverse primer R2, see "Materials and Methods") was fused to the
bacterial lacZ gene as a reporter. The promoter region was truncated stepwise from the 5'-end, and
The longest PFY1 promoter-lacZ fusion
construct, H4R2, started in the coding region of the 5'-adjacent gene
LEO1 and, thus, contained the termination region of
LEO1 as well as the complete non-transcribed upstream region
of PFY1. With progressing promoter truncation, expression
remained at a relatively constant level until it dropped to about 12%
of the original value in the interval between positions
Two-base pair exchanges in the most conserved part of the recognition
sequence of Reb1p in mutant H2GmR2 (see "Materials and Methods"),
which abolished Reb1p binding (Fig.
2C, below), reduced PFY1 expression to about one-third of the wild type. The
comparison of the activities obtained with H2GmR2 and H6R2 reveals that
some additional element 3' of the Reb1p site is important for
expression of the reporter. We tested the oligo(dT) element (pos. Reb1p Binds Specifically to the PFY1 Promoter--
To verify
binding of Reb1p to the presumptive site in the PFY1
promoter, a 77-bp double-stranded oligonucleotide containing this motif
(see "Materials and Methods") was incubated with yeast nuclear
extracts (Fig. 2A) or recombinant Reb1p from E. coli (Fig. 2B) and used in electrophoretic mobility
shift assays. Specificity of Reb1p binding was demonstrated by
homologous competition with a 25-, 50-, and 100-fold molar excess of
the respective unlabeled DNA fragment (Fig. 2, A and
B) or by heterologous competition with a 100-, 200-, and
400-fold molar excess of an unlabeled DNA fragment containing the
functional Reb1p binding site of the GCY1 promoter (25)
(Fig. 2B, right panel). Both recombinant Reb1p from E. coli or from yeast nuclear extracts specifically
binds to the oligonucleotide, and the Reb1p site of the PFY1
promoter is a more efficient competitor than that of the
GCY1 promoter.
Since the intrinsic potential of Reb1p to activate transcription
directly is negligible as with any of the so-called general regulatory
factors, we tested whether we could detect any additional non-histone protein binding to this region as a candidate for a
transcriptional activator (Fig. 2C). We used a set of
promoter fragments with variable extensions into the 5'-flank of the
PFY1 gene and harboring a functional Reb1p site (H0R1, H1R1,
H2R1, and H3R1 using reverse primer R1) or lacking it (H2GmR1
containing a mutated Reb1p site) (see Fig. 1 for the constructs). DNA
fragments were incubated with either recombinant Reb1p isolated from
E. coli or with nuclear extracts from yeast, and DNA-protein
complexes were separated by native polyacrylamide gel electrophoresis.
With all wild type DNA fragments very similar retention signals were found using either recombinant Reb1p or yeast nuclear extracts (similar
to the oligonucleotide in Fig. 2, A and B). Since
constitutive transcription reflects a static situation of permanent
promoter activation, one would expect to detect by this assay the
binding of any additional transactivator protein even if its cellular concentration is low. Together with the deletion data, this result strengthens the conclusion that no transcription factor binds to the
5'-upstream region of PFY1 in addition to Reb1p.
Effect of the Mutation of the Reb1p Site on PFY1 Expression in
Vivo--
The above data show that a 2-base pair point mutation in the
Reb1p motif diminishes
The concentration of PFY1 mRNA was standardized relative
to that of the ACT1 messenger (both probes of comparable
length were labeled to about the same specific radioactivity and then
mixed for hybridization and detection). In wild type and two different mutant clones the ACT1 mRNA was constant, whereas the
concentration of the PFY1 mRNA differed between wild
type and mutants (Fig. 3A). The densitometric evaluation
revealed that in the wild type PFY1-specific mRNA
amounted to ~35-40% of ACT1 mRNA. In the mutant clones this value dropped to about one-third of the wild type (about
14% of ACT1 mRNA). This value is in good agreement with the data obtained from the Western blot (Fig. 3B) and from
reporter expression studies with PFY1
promoter-lacZ fusion constructs (compare with Fig.
1). Taken together, the destruction of the presumptive motif for Reb1p
binding both on a plasmid and in the genomic context reduces
transcription of PFY1 by a factor of about 3.
Chromatin Array at the PFY1 Promoter--
We studied the
nucleosome arrangement at the PFY1 promoter in the genomic
context to test whether Reb1p excludes nucleosomes from this region.
The chromatin of wild type cells was digested either with DNase I (Fig.
4A, left panel) or
with micrococcal nuclease (Fig. 4A, right panel)
and visualized using a probe hybridizing 756-1120 bp downstream of the
5'-end of the PFY1-coding region. The results obtained with
the two nucleases were very similar and, due to the slightly different
preferences of DNA cleavage, mutually complementary. In wild type
chromatin, the array of nucleosomes displayed a pronounced gap of
hypersensitive DNA spanning about 190 bp. Standardization and sequence
alignment revealed that, with the exception of about 90 bp in the
center of the gap, the PFY1 promoter region was unprotected
in the chromatin context. In chromatin, the center of the gap was
resistant to both nucleases to a similar extent, but it displayed
normal sensitivity to either nuclease with protein-free naked DNA.
Thus, the protection to degradation in wild type chromatin was not
attributable to the cleavage specificity of one of the respective
nucleases but rather was a protein-dependent
property of the chromatin at the PFY1 promoter. Most likely
it was related to the binding of a protein or protein complex. The
protected region within the gap was centered at the Reb1p binding
site.
The nuclease digestion pattern of chromatin on the 5' side of the gap
revealed a positioned nucleosome as the protected region was short
(about 145 bp) and the signals of the linker sequences were distinct
and narrow. The farther upstream adjacent nucleosomes were more mobile
since the linkers were wider and the boundaries of the nucleosomes were
more fuzzy. The 3'-end of the LEO1 gene lies in a moderately
sensitive region, presumably comprising the transcriptional terminator
region of this gene.
The first nucleosome on the 3' side of the gap has a fixed position as
well according to the same criteria. Farther PFY1-proximal, i.e. 3'-adjacent to the first nucleosome in the
PFY1-coding region, a wide nuclease-sensitive linker
coincided with the 3'-end of the intron. The downstream adjacent
non-positioned two nucleosomes were bounded by two consecutive
hypersensitive sites that lay in the bidirectional overlapping
termination region of the two convergently transcribed genes
PFY1 and GCY1 (51, 52), the 3'-mRNA ends of
which overlap in this region by 37 nucleotides (40).
To analyze the contribution of Reb1p to the creation of the observed
nucleosome-free gap in the PFY1 promoter and, in addition, to examine whether the protected region in the center of the
nucleosome-free promoter region was attributable to Reb1p binding, the
genomic PFY1 promoter mutant, which had been used in the
expression studies described in Fig. 2, was analyzed. It carried a
2-base pair change of the Reb1p binding motif in the promoter region of
the genomic PFY1 gene so that Reb1p binding was abolished
(see "Materials and Methods"). The 2-base pair change did not
influence the cleavage pattern of naked DNA by either DNase I (not
shown) or micrococcus nuclease (Fig. 4B).
In the mutant, the pattern of digested chromatin resembled that of
naked DNA in the region of the PFY1 core promoter, and the
190 bp gap is absent. This reveals that, in the absence of a binding
site for Reb1p, the nucleosomes are randomly arranged and not
positioned (compare with Ref. 53). This interpretation is in agreement
with the expression data that showed that destruction of the Reb1p
binding site did not abolish PFY1 expression completely but
reduced it to about 35%. In addition, the 90-bp stretch of uncleaved
DNA found in the wild type in the center of this region is not detected
in mutant chromatin. This shows that Reb1p binding is responsible
simultaneously for exclusion of nucleosomes from 190 bp of promoter DNA
and for protection (or distortion to a non-cleavable conformation) of a
stretch of DNA comprising its own binding site in the wild type.
Analyses of Reb1p-induced DNA Bending--
To elucidate the
mechanism by which Reb1p effects nucleosome exclusion, we
investigated by permutation analyses (54) whether Reb1p induces a DNA
bend upon binding to its recognition site that might lead to a DNA
conformation that is incompatible with wrapping around nucleosomes. The
PFY1 promoter fragment containing the high affinity Reb1p
target site was ligated in tandem to the identical fragment (Fig.
5A and see "Materials and
Methods"). Separate digestions with the restriction enzymes indicated
gave rise to a set of DNA fragments of identical lengths that were permutated with respect to the location of the Reb1p binding site relative to the ends of the fragments (Fig. 5A). The
individually isolated restriction fragments were incubated with
recombinant Reb1p from E. coli and separated in
non-denaturing polyacrylamide gels. As a control, the mobilities of the
free DNA fragments were determined. DNA fragments were detected by
Southern hybridization. The relative mobilities of the permutated
DNA-protein complexes differed (Fig. 5B). When the Reb1p
recognition sequence was located in the center of the DNA fragment, the
DNA-protein complexes were maximally retarded. The closer the Reb1p
target sequence was to one of the ends of the DNA fragment the less the
mobilities of the respective DNA-protein complexes were affected.
However, the mobilities of the corresponding free DNA fragments varied
slightly as well. These results indicate that the PFY1
promoter region contains a slight intrinsic bend on its own that likely
is attributable to the oligo(dA·dT) stretch, which is slightly phased
(e.g. Refs. 55-57). A bending angle of ~25° for the
respective protein-free PFY1 promoter DNA was calculated
(58). After the relative mobilities of the DNA-protein complexes had
been corrected for the differences in the mobilities of the respective
free DNA fragments, the bending angle caused by Reb1p binding to its
target site was calculated (58) to be ~120°. By correlating the
relative mobilities of the distinct DNA-protein complexes with respect
to the position of the Reb1p target site on the DNA fragment the
bending center was determined to be in the immediate 5'-flank of the
canonic Reb1p binding sequence (Fig. 5C).
To corroborate these findings we repeated the permutation analysis
using a truncated version of recombinant Reb1p that exclusively harbored the 364 amino acids comprising the DNA-binding domain (33). The bending angle caused by the DNA-binding domain of Reb1p was
calculated to be in the range of 100° and thus comparable to that of
the full-length protein (Fig. 6). As with
full-length Reb1p, the bending center caused by the DNA-binding domain
of Reb1p was determined to be in the 5' vicinity of the canonic
consensus sequence (Fig. 6B).
To determine the relative direction of the apparent bend induced by
Reb1p binding, a DNA fragment containing the Reb1p binding site was
fused to a set of fragments of phased oligo(dA) tracts of known
magnitude and direction of bending in which the spacing between the two
sites was varied in increments of 2 bp over a full helical turn
(59-62). The phasing analyses with both wild type and Reb1p
truncation-derived protein-DNA complexes demonstrated that Reb1p
binding bends the DNA toward the major groove (data not shown).
Reb1p Influences Transcription and Nucleosome Arrangement
at the PFY1 Promoter--
Yeast general regulatory factors constitute
an interesting class of DNA-binding proteins. On the one hand, they
abound and have many target sequences throughout the genome, explicitly
in a great number of promoters. On the other hand, their potential to
activate transcription directly is negligible, and deletion of their
binding elements from a particular promotor in most instances leads to
only marginal reduction of transcription capacity of the respective
gene (e.g. Refs. 25 and 31). Nevertheless, apart from
CBF1, the genes encoding general regulatory factors are
essential, but the molecular basis of the mode of action of Cbf1p,
Rap1p, or Abf1p is still far from clear, and, in the case of Reb1p, it
is even an enigma.
As an approach to understand the role of Reb1p, we have analyzed the
constitutive PFY1 promoter. We show that the relatively short activation region of the PFY1 gene dispenses with
binding sites for classical transactivators. Since the constitutive
PFY1 promoter reflects a static chromatin array, the
presence of additional activators would have been detected. Instead the
PFY1 promoter contains only a single canonic motif for
DNA binding of Reb1p to which Reb1p binds specifically so that the
impact of this factor on chromatin structure and transcription
activation can be studied free of the influence of other factors.
PFY1 is moderately strongly transcribed, and we have shown
that Reb1p modestly enhances PFY1 mRNA transcription.
We also show that the core promoter of PFY1 is
nucleosome-free and that binding of Reb1p to its cognate site is
responsible for the exclusion of nucleosomes. Mutation of the
recognition motif of Reb1p leads to encroachment of the promoter by
randomly arranged nucleosomes. So Reb1p has two effects on the
PFY1 promoter: (i) exclusion of nucleosomes and (ii) subtle
stimulation of transcription.
Contradictory Results at Other Promoters Suggest
Context-dependent Action of Reb1p--
Reb1p binding sites
have been found both in regulated and constitutive promoters. Much work
has been devoted to the analysis of Reb1p in regulated promoters in
which its importance is mainly restricted to a moderate stimulation of
basal, but not of induced, transcription (31, 52, 63).
In the case of the GAL1-GAL10 intergenic region, Reb1p
binding was supposed to be responsible for nucleosome exclusion (34). However, more recent analyses demonstrate that Reb1p binding has no
bearing on the chromatin structure or on the transcription of
GAL1 or GAL10 (35). Similarly it has been found
that Reb1p binding does not influence chromatin organization at the
ILV1 promoter but positively influences Gcn4p-independent
transcription in combination with two AT-rich elements (29). At the
HSC82 promoter, Reb1p has only a mild effect on nucleosome
positioning but rather facilitates and stabilizes binding of the heat
shock transcription factor and the TATA-binding protein TBP (31). However, at the Gal4p-regulated GCY1 promoter, containing a
Reb1p site remote from a Gal4p binding site, it has recently been
demonstrated that Reb1p and the nucleotide sequence, i.e.
presumably a particular DNA structure comprising the dG·dC-rich Gal4p
binding site, contribute simultaneously to activation of basal
transcription and nucleosome exclusion at the GCY1 promoter
in an additive fashion (25, 52).
In constitutive promoters, the role of Reb1p is not well established.
The ACT1 gene (1) encoding a cytoskeletal protein like
profilin (PFY1) also is expressed constitutively. Strikingly both promoters lack binding sites for classical transcription activators and instead contain sites for the binding of the general regulatory factor Reb1p in combination with poly(dA·dT) elements (1).
However, it has not been investigated yet whether Reb1p influences the
organization of the chromatin structure of the ACT1
promoter. Another example for a constitutively transcribed strong
promoter is that of the triose-phosphate isomerase (TPI1) gene (18). Reb1p moderately stimulates basal transcription of TPI1 and is supposed to exclude nucleosomes, thereby
allowing binding of additional activators such as Gcr1p or Rap1p.
However, an immediate involvement of Reb1p in nucleosome exclusion has not been demonstrated.
Taken together, a general feature of Reb1p action is stimulation of
basal or constitutive transcription (18, 25, 31, 52, 63). However, the
mechanisms by which Reb1p exerts these positive effects seem to differ
at the respective promoters. At some promoters, e.g.
PFY1 and GCY1, Reb1p has the capacity to prevent
nucleosome formation in the vicinity of its binding site and to
position nucleosomes at a distance (52), whereas in other Reb1p-containing promoters, this function is evidently not required since other mechanisms, i.e. specific transactivators or the
intrinsic DNA sequence (see below), fulfill this task (31, 64, 65).
Reb1p Bends DNA upon Binding--
Based on permutation studies, we
have shown that Reb1p induces a bend into its target sequence with the
bending center in the immediate vicinity 5' of the canonic binding
sequence. Because the extent of curvature is important to exclude
nucleosomes from DNA, the Reb1p site cannot be replaced by the soft
curvature of phased oligo(dA·dT)
sequences.2 The induced bend
angle, although its magnitude may have been overestimated by the
procedure used, is relatively large but comparable to other general
regulatory factors from yeast such as Abf1p, Cpf1p, and Rap1p (66-69).
The general regulatory factors from yeast may differ from many other
DNA-binding proteins in that they have an extraordinarily large DNA
interaction domain and, in contrast to certain mammalian basic
helix-loop-helix transcription factors like the AP1-binding Fos-Jun
heterodimer or yeast Gcn4p that were found to cause only a small DNA
deformation (62, 70), induce large bend angles.
Nucleosome exclusion from core promoters facilitates transcriptional
initiation. In line with the above conclusions, it has been observed
that at least some promoters can be maximally activated even in the
absence of transactivators by experimental deprivation of nucleosomes
(71, 72). Moreover constitutive promoters have been found that dispense
with any classical gene activators or other DNA-binding proteins and
yet display nucleosome-free core promoters. These apparently rely on
structural peculiarities of the DNA that render it incompatible with
packaging into nucleosomes, e.g. the promoter of the major
adenylate kinase gene AKY2 (19) and the RIO1
promoter.2 These results together demonstrate that
loosening (73, 74), removal (75, 76), or absence (19, 52) of
nucleosomes from the core promoters is necessary and, at least at some
promoters, sufficient to allow transcription even in the absence of
transcriptional activators. In addition, this points out that, apart
from translational positioning of nucleosomes as found in many
promoters (55, 73-77), several alternative mechanisms exist to keep
nucleosomes off a certain cis-site. Exclusion of nucleosomes
in the vicinity of a Reb1p binding site due to
protein-dependent distortion of the DNA may add to this
list. Thus, Reb1p plays its role mainly by distorting normal DNA structure.
The distortion of the DNA structure may facilitate and/or stabilize
binding of additional DNA-binding proteins, presumably including TBP.
Prebending of the core promoter structure has been shown to facilitate
binding of transcription factor IID and to allow spontaneous
assembly of the basal transcription machinery in the absence of
transcription activators (78-81). Since TBP-TATA box complexes
induce a strong bend in the core promoter that also is directed toward
the major groove (78, 81), it appears plausible to assume that
prebending effected by Reb1p binding causes a synergistic effect.
Moreover it could stabilize binding and facilitate interactions between
DNA-bound proteins that are separated by long distances as the
intervening DNA sequences would loop out. Reb1p, binding to the vertex
of the loop, could bring several weak activator proteins into close
proximity. Thereby it could promote direct protein-protein contacts
between them or facilitate interactions with bridging coactivators or
with components of the basal transcription machinery. In line with this
conclusion, it has been found that Reb1p binding stabilizes
interactions of TBP with core promoter DNA and/or binding of other
factors such as heat shock transcription factor or Gcr1p (18, 31).
Thus, the architectural influence of Reb1p binding on DNA structure,
possibly endorsed by the combination with a dA·dT element (82),
simultaneously may lead to nucleosome exclusion and facilitated binding
of the basal transcription machinery to the core promoter in the
vicinity of a Reb1p binding site. In line with this interpretation, the
core promoter of the PFY1 gene, presumably a dC- and dT-rich
stretch of DNA, has been found <40 bp downstream of the Reb1p site and
about 40 bp upstream of the first (major) transcriptional
initiation site (8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-REB-tandem.
Escherichia coli strains CJ236 (36), XL1 Blue, or SURE (both
from Stratagene) served as hosts. Yeast were grown on standard
medium (37). Nuclear extracts were prepared from the
protease-deficient yeast strain ABYS1 (38). Recombinant full-length
Reb1p or truncated Reb1p was recovered from E. coli strains
that had been transformed with plasmids pET11a-REB1 or pET11a-PCR7
(33).
1, H2
2, and H2
3, also were obtained by
site-directed mutagenesis. To obtain the construct for permutation
studies, sequences of the PFY1 promoter (pos.
150 to pos.
+14) harboring the Reb1p binding site were amplified by PCR
simultaneously introducing restriction sites for EcoRI and
BamHI for ligation into vector pBluescript KS
.
Oligonucleotide 5'-TATCTTTTCTCGAGGATCAC-3' was used to introduce an
XhoI restriction site into this PFY1 promoter
fragment, H0-R1, yielding the plasmid
pKS
-PFY-H0-R1-XhoI.
pKS
-PFY-H0-R1-XhoI was restricted by
EcoRI. Protruding 5'-ends were removed by treatment with
mung bean nuclease; subsequently the plasmid was digested by
BamHI. In a second reaction
pKS
-PFY-H0-R1-XhoI was restricted by
BamHI, and after 5'-protruding ends had been filled in by
Klenow polymerase the plasmid was digested by EcoRI. The DNA
fragments from both reactions were mixed and ligated in tandem
generating an additional BamHI recognition site at the
fusion point. The resulting tandem dimer was inserted as an
EcoRI/BamHI fragment into the polylinker of
pBluescript KS
(pKS
-REB-tandem). From this
construct a set of fragments permutated with respect to the position of
the Reb1p binding site was obtained by using naturally occurring
restriction sites (SspI, HindII, DraI,
and PvuI) and the introduced sites for XhoI and
BamHI. Correctness of all constructs was controlled by
nucleotide sequencing.
-32P]dATP using Klenow
polymerase and incubated on ice for 20 min with 20 µg of yeast
nuclear extract or 200 ng of recombinant Reb1p from E. coli,
respectively, in 10 mM HEPES-KOH, pH 8.0, 5 mM
MgCl2, 50 mM NaCl, 1 mM
dithiothreitol, 1 mM EDTA, 5% glycerol, 0.005% bromphenol
blue. Poly(dI-dC) (Sigma) or fragmented E. coli DNA served as unspecific competitor DNA. Heterologous competition experiments were performed using a 93-bp fragment containing the functional Reb1p binding site of the GCY1 promoter (24).
DNA-protein complexes were separated on prerun non-denaturing
polyacrylamide gels (4.5% acrylamide; acrylamide/bisacrylamide = 45:1) at 7-8 V/cm for 3-4 h at 7 °C.
-32P]dATP using the High Prime DNA labeling kit from
Roche Applied Science, and hybridization reactions were carried out
according to standard procedures (43).
Leu+ cells were replica-plated on
5-fluoroorotate-containing medium (44) to increase selective pressure
in favor of Ura-deficient clones. Proper replacement was tested by
Southern blotting and restriction analysis with NsiI and by
DNA nucleotide sequencing. Two independent transformant clones were
used, WPGm1 and WPGm2.
-Galactosidase activities of
promoter-lacZ fusion proteins were determined as described
previously (26, 47). Values give the mean expression activity of at
least four independent clones. Generally the values varied in the order
of 5-15%. Protein concentrations were determined according to
the method described by Bradford (48). Yeast were transformed using the
procedure described by Gietz et al. (49). Other molecular
operations were performed according to standard procedures (43) or as
recommended by the manufacturer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
41 (with respect to the adenine residue of
the translational start codon of PFY1 as +1) is only 172 bp
(Fig. 1A).
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Fig. 1.
Schematic drawing of the LEO1-PFY1
intergenic region. A, positions of 5'-ends of
primers used in truncation constructs are indicated by
arrows and numbers (adenine residue of the
translational initiation codon as +1). Stippled ellipse,
Reb1p binding site; small striped box, dT element; *,
transcriptional initiation sites; wide open bars, coding
regions. B, promoter truncations used for
lacZ fusions (reverse primer R2, pos. +223) or gel shift
assays (reverse primer R1, pos. +13). C, promoter deletions
and mutation of the recognition motif of Reb1p (ellipse) or
dT element (striped box). The right
part displays -galactosidase (
-Gal)
activities of transformant strains harboring the respective
promoter-lacZ fusion constructs on low copy plasmid pYLZ6
(nmol/mg·min).
136 to
127) and a dA·dT element (16 T
residues of 20 bases, pos.
124 to
105). A TATA box obeying
the consensus is absent, and no motif for binding of any of the
classical transcription activators is apparent. No signal in addition
to the one caused by Reb1p binding to PFY1 promoter DNA
could be detected by gel shift assays.
-galactosidase activities were measured in homogenates of the respective yeast transformants (Fig. 1B).
150 and
108
(Fig. 1B, compare activities with constructs H0R2 and H6R2).
This segment contains the presumptive Reb1p binding site and the
dA·dT element. This indicated that sequences 5' of position
150
altogether are dispensable for PFY1 transcription and that
the element(s) deleted in the interval between
150 and
108 is
important for the expression of PFY1. To examine more
directly the role of the presumptive Reb1p element in transcription of
PFY1, the sequence was mutated by site-directed in
vitro mutagenesis.
124
to
105) that is immediately 3'-adjacent to the Reb1p binding motif. Indeed deletion of this element (construct H2
1) reduced reporter activity to about 30% of the wild type promoter (Fig. 1C),
i.e. this element has about the same importance as the Reb1p
binding site. Simultaneous deletion of both sites and farther extension in the 3' direction in construct H2
3 (pos.
136 to
75) reduced expression to near background, although the transcriptional initiation sites were left untouched. Since in yeast the core promoter usually is
40-120 bp upstream of the first transcriptional initiation site
(in PFY1 at pos.
41 relative to the start triplet), these data imply that the core promoter has been deleted in this construct in
addition to the Reb1p site and the oligo(dT) tract (interval between
pos.
104 and
75). The presence of the core promoter in this segment
is in line with the lowered activity obtained with construct H2
2 in
which the Reb1p site and the oligo(dT) element have been left
intact.
View larger version (39K):
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Fig. 2.
Electrophoretic mobility shifts confirming
specific Reb1p binding to the upstream promoter region of
PFY1. A, homologous competition
experiments after incubation with 20 µg of yeast nuclear extract.
B, gel shift assay after incubation with 200 ng of
recombinant Reb1p from E. coli. Specific binding of Reb1p
was confirmed by homologous competition with a 25-, 50-, and 100-fold
molar excess of unlabeled DNA fragment (REB1-Oligo) or by
heterologous competition with a 100-, 200-, and 400-fold molar excess
of unlabeled DNA fragment containing the functional Reb1p binding site
of the GCY1 promoter (REB1-GCY). C,
DNA binding of recombinant (E) or yeast (Y) Reb1p
to a set of DNA fragments differing in length on their 5' side. In
fragment H2GmR1, the binding site of Reb1p has been mutated. Retention
signals due to binding of Reb1p are indicated.
-galactosidase expression from plasmid-borne PFY1 promoter-reporter fusion constructs (Fig.
1C). To corroborate that this element is also important
in vivo, we have replaced the Reb1p motif in the genuine
genomic context of the PFY1 promoter with the same 2-base
pair point mutation as above. Although deletion of the PFY1
gene has been reported to be conditionally lethal (16), this promoter
mutation is correlated with no temperature-sensitive phenotype,
although cells are slightly enlarged resembling
pfy1 cells in this respect. This suggests that the mutant promoter is partly
functional despite the fact that Reb1p does not bind, which is in line
with the in vitro data. We have analyzed the cellular
concentration of PFY1 mRNA in wild type and mutant by Northern blot analysis (Fig.
3A) and of Pfy1p in a Western
blot using anti-profilin antibodies (Fig. 3B).
View larger version (40K):
[in a new window]
Fig. 3.
Expression of PFY1 in
(genomic) mutants with altered Reb1p binding site in the
PFY1 promoter. A, Northern blot of
total RNA from wild type (wt) and two mutants hybridized
with radioactive probes labeled to about the same specific
radioactivity. B, Western blot of the same three strains
decorated with anti-profilin antiserum and detected with
peroxidase-coupled anti-rabbit secondary antiserum. * denotes a
cross-reacting yeast protein used as internal loading control.
View larger version (64K):
[in a new window]
Fig. 4.
Chromatin structure analysis at the promoter
and coding region of PFY1 in the genomic context.
Native chromatin of the wild type (A) and a strain mutated
at the Reb1p binding site of the genomic PFY1 copy
(B) was digested with increasing concentrations of
micrococcal nuclease or DNase I (wild type only), and DNA was then
treated, electrophoresed, and detected as described under "Materials
and Methods." DNA fragment length standards (M)
are given at the margins. The results are illustrated by the
corresponding schematic drawings in the center
(A, wild type; B, mutant). Ellipses
symbolize nucleosome-protected DNA, thick black bars
indicate coding, thin bars indicate non-coding regions, and
wide black boxes reflect hypersensitive sites. Double
arrows, mobile nucleosomes; stippled ellipse, Reb1p
binding site; Gm, mutation of the Reb1p motif;
TAA, termination codon of LEO1; i,
AUG; t, transcriptional and translational initiation sites
and bidirectional transcriptional termination region of
PFY1, respectively.
View larger version (21K):
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Fig. 5.
Permutation analysis of the Reb1p binding
site. A, digestion of the permutation construct
pKS -REB-tandem by several restriction enzymes generated
DNA fragments that contained the Reb1p binding site (striped
box) at different positions relative to the DNA fragment ends.
B, gel shift assay to investigate Reb1p-induced DNA bending.
The respective DNA restriction fragments were incubated without
(left part of the panel) or with (right part of
the panel) recombinant Reb1p from E. coli, separated on 8%
polyacrylamide gels, and detected by Southern hybridization.
S, SspI; D, DraI;
H, HindII; X, XhoI;
P, PvuI; B, BamHI.
C, determination of the Reb1p-induced bending center. The
mobilities of the Reb1p-DNA complexes (µ complex)
were corrected for variations in mobility of the free DNA (µ free) and normalized to the fastest migrating complex. The
relative mobilities (µ complex/µ free) were plotted as a function
of the position of the Reb1p binding site relative to the DNA fragment
ends. Standard deviations were calculated from three independent
experiments.
View larger version (24K):
[in a new window]
Fig. 6.
Permutation analysis with a truncated version
of Reb1p. A, retention assay to test DNA bending
induced by the DNA-binding domain of Reb1p. DNA fragments (see Fig.
5A) were incubated with truncated Reb1p from E. coli, separated on 8% polyacrylamide gels, and detected by
Southern hybridization. B, determination of the bending
center induced by the Reb1p DNA-binding domain. Relative mobilities
were plotted as a function of position of the Reb1p target site within
the DNA fragment. S, SspI; D,
DraI; H, HindII; X,
XhoI; P, PvuI; B,
BamHI.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
The skillful technical assistance of G. Strobel is gratefully acknowledged. E. coli transformant strains either expressing recombinant Reb1p or harboring the cloning vector pET11a were donated to us by J. Warner (Bronx, NY). Anti-profilin and anti-actin antisera were kindly provided by S. Brown (Ann Arbor, MI) and A. Adams (Tucson, AZ), respectively.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich Grant 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.
§ Present address: Lichtenstein Pharmaceutica GmbH und Co., Industriestrasse 10, D-82256 Fürstenfeldbruck, Germany.
Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.M301806200
2 M. Angermayr, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ACT1, yeast gene encoding actin; PFY1, yeast gene encoding profilin; pos., position(s); Ura, uracil; TBP, TATA-binding protein.
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REFERENCES |
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