From the Department of Chemistry and Biochemistry,
Arizona State University, Tempe, Arizona 85287-1604 and the
§ Department of Biochemistry and Biophysics, Oregon State
University, Corvallis, Oregon 97331
Received for publication, August 4, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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Circular permutation analysis has detected fairly
strong sites of intrinsic DNA bending on the promoter regions of the
yeast GAL1-10 and GAL80 genes. These bends lie
in functionally suggestive locations. On the promoter of the
GAL1-10 structural genes, strong bends bracket nucleosome
B, which lies between the UASG and the GAL1
TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and
start site elements in the inactive state of expression but are
completely disrupted (removed) when GAL1-10 expression is
induced. The strongest intrinsic bend (~70°) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it
to be involved in GAL1 activation in several ways,
including the recruitment of a yeast HMG protein that is required for
the normally robust level of GAL1 expression in the induced
state (Paull, T., Carey, M., and Johnson, R. (1996) Genes
Dev. 10, 2769-2781). On the regulatory gene GAL80,
the single bend lies in the non-nucleosomal hypersensitive region,
between a GAL80-specific far upstream promoter element and
the more gene-proximal promoter elements. GAL80 promoter
region nucleosomes contain no intrinsically bent DNA.
The bonds that form the backbone of DNA have significant
rotational freedom. As a result, DNA is typically a flexible
macromolecule. However, it was established some time ago that
particular DNA sequences can cause a region of DNA to assume a distinct
nonlinear curved structure (for reviews, see Refs. 2-5). This
property is referred to as intrinsic DNA bending or DNA curvature.
Intrinsically bent DNA has a preferred conformation and thus is more
rigid than a typical DNA molecule, a property that causes this DNA to
migrate more slowly than random (non-bent) DNA during polyacrylamide
gel electrophoresis (6, 7).
Intrinsic DNA bending has been found to play a significant role in the
transcriptional regulation of many prokaryotic genes (8, 9). In
prokaryotes, intrinsic bending helps implement both repression and
activation, operating by a number of distinct mechanisms. However, the
possible role of intrinsic bending in eukaryotic transcription is less
clear. Schroth et al. (10) described two intrinsically bent
regions that flank the transcription start site of rRNA in
Physarum. An intrinsic bend in the promoter region has been
detected on several human genes: For a number of years, our laboratory has been studying the yeast
GAL genes, one of the most intensely studied model systems for eukaryotic gene regulation (21-25). The structural genes,
e.g. GAL1-10, are induced to very high level
expression in galactose by the action of the activator Gal4p. Gal4p
binds to activation sequences (UASG) that lie upstream of
GAL genes (Fig. 1) and activates transcription in a process
that depends on gene-proximal TATA elements and involves numerous
coactivators and general transcription factors including TBP
(for a review see Ref. 25). The activation function of Gal4p is
modulated by Gal80p, an inhibitory regulator that binds specifically to
the activation domain of Gal4p, thus preventing gene activation in
nongalactose carbon sources (21). The gene that encodes this negative
regulator is constitutively expressed at a basal level in all carbon
sources via an Inr element located at +1 and at higher induced levels
in galactose through a UASG and TATA (26, 27). Both basal
and induced GAL80 expression depend on the
UASGAL80 (Fig. 1), a far
upstream GAL80-specific activation sequence (27).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin (11),
- and
-globin
(12), and cdc2 (13). Intrinsic bends that are binding sites
for transcription factors are found in the E2F consensus recognition
site (14) and in an enhancer of the human interferon
gene (15). Of
course, there are numerous examples of protein-induced DNA bending on
eukaryotic promoters involving both "architectural" proteins like
HMG1 (16) and specific
transcription factors (14-15, 17-20).
View larger version (10K):
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Fig. 1.
Maps of the GAL1-10 and GAL80 promoter
and 5' coding regions. The intergenic region of
GAL1-10 (panel A) and the upstream region of
GAL80 (panel B) are shown. The numbers
below the thick line are base pairs measured from a
reference EcoRI (GAL1-10) or MboII
site (GAL80). Upstream activation sequences
(UASG) are delineated by brackets, TATA
boxes by a boxed T, a GAL80-specific activation
sequence (UASGAL80) by an inverted
triangle, an ~20-bp purine-pyrimidine tract by a short
dashed line, and the regions used in the cyclic permutation
experiments of Figs. 4 and 5 by rectangles. The
transcription start sites are located at the origins of the
horizontal, labeled arrows.
During the course of working with GAL promoter region DNA,
we have noticed gel migration anomalies. The results presented below
confirm the source of these anomalies, intrinsic DNA bends on the
GAL1-10 and GAL80 promoter DNAs. The extensive
knowledge base for GAL regulation allows us to correlate
quite accurately the locations of the intrinsic bends with the
locations of known DNA regulatory sites on these promoters. In
addition, the chromatin structure of both the GAL1-10 (28)
and GAL80 promoter regions (29) are known, as well as the
role that promoter nucleosome structure plays in regulation of the
GAL genes (cf. Refs. 25 and 30). Thus, perhaps
more importantly, we are also able to correlate bend locations with
promoter nucleosome locations, a comparison that has not been possible
with the intrinsic bends detected on other eukaryotic promoters.
Indeed, this comparison provides some very interesting results
regarding the relationship of bends and nucleosomes.
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EXPERIMENTAL PROCEDURES |
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Plasmids containing the promoter regions of GAL1-10 or GAL80 (29, 31) were isolated according to Birnboim (32) and the insert DNA excised from the plasmids by restriction digestion as follows: EcoRI to release a 907-bp fragment containing the GAL1-10 intergenic region; EcoRI-NcoI to release a 1005-bp fragment containing the upstream and 5' coding region of GAL80. The insert DNA was separated from the plasmid vectors by preparative gel electrophoresis, the bands of interest were identified by UV shadowing, and the DNA was eluted from the gel by a gel crush method (33).
Gel Mobility--
Restriction digests of promoter region DNA
were electrophoresed on analytical gels (7.5%, 29.4:1
acrylamide:bisacrylamide-TBE), along with DNA size standards
(pBR322-MspI, x174-HaeIII) at 4 °C and 1.5 V/cm for 96 h in the absence or presence of 1 µg/ml ethidium
bromide in the gels (34). The relative migrations
(Rf) of DNA fragments were calculated by dividing
band migration distances by the migration distance of a DNA standard,
which would correspond to the same number of base pairs, determined
from a plot of migration distance versus size for standard
reference digests, e.g. pBR-MspI. This method
avoids the possibility of an intrinsically bent standard DNA fragment
affecting promoter Rf values.
Circular Permutation--
The desired DNA fragment was cut out
from the promoter region DNA by restriction digestion and isolated
using polyacrylamide gel electrophoresis, UV shadowing, and gel crush
as described above. To make concatamers of the fragment, a T4 ligase
blunt end reaction was carried out at 16 °C for 2-6 h, and
BamHI linkers were then ligated to the ends of these
fragments (T4 ligase, 16 °C for 16 h). The resulting products
were digested with BamHI and analyzed on a 1.5% agarose gel
to estimate the DNA concentration. This concatamer mixture was ligated
into a BamHI cut (and dephosphorylated) pBSK+
vector under sticky end ligation conditions (1:1-4:1 insert to plasmid
molar ratio), transformed into a UV-XL1Blue Escherichia coli
host and grown in L broth to stationary phase with ampicillin selection
(35). The resulting library was screened for the desired head-to-tail
dimer insert using standard -complementation and miniprep
restriction digest screening techniques (35). The desired dimeric
fragments were isolated using polyacrylamide gel electrophoresis/UV shadowing/gel crush.
For the circular permutation assay, a head-to-tail dimer fragment was cut with various single cut (per monomer unit) restriction enzymes and analyzed on polyacrylamide gels (8%, 1-mm thick) run at 4 °C and 1.5 V/cm for 120-200 h. Rf values for the monomer fragments were calculated by dividing the migration distance for the fragment by the migration distance of a standard DNA fragment of the same length (obtained from a plot of log bp versus migration for a standard restriction digest). The bend center was determined as the intersection of two lines on the plot of restriction cut site versus Rf from the circular permutation analysis. Each line was a best fit of a weighted least squares formula for data approaching the maximum Rf from the left or right, respectively. Where there was a scarcity of data approaching from one side, the data were wrapped from the other side of the plot. A minimum of four independent electrophoretic determinations were carried out for each bend site.
DNA Simulations--
The program LORDS (Long Range DNA
Simulator, P. S. Ho laboratory), which incorporates the helical
parameters of rise, twist, roll, and tilt from any number of different
bending models, was used to construct low resolution, three-dimensional
structures for the GAL1-10 and GAL80 promoter
DNA (cf. Fig. 2). In this
study, we used the parameters derived by Bolshoy et al.
(36), the parameters derived by Goodsell and Dickerson (37), or the
parameters from Brukner et al. (40) to construct DNA models
with LORDS.
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RESULTS |
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We initially applied several computational bending algorithms
(36-38) to the promoter DNA sequences of the GAL1-10 and
GAL80 genes. Although all models predicted intrinsic
bending, the models differed in the predicted locations and strengths
of the bends within the promoter regions (cf. Fig.
2A). Thus, a preliminary experimental screening method was
utilized to identify intrinsically bent regions of DNA on which to
carry out circular permutation analysis. The screen involved
carrying out several restriction digests of GAL1-10 and
GAL80 promoter DNA and testing the resulting sets of small
(100-400-bp) fragments for anomalous migration during polyacrylamide
gel electrophoresis (see "Experimental Procedures"). Two such sets
of results are shown in Fig. 3.
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The data identify some GAL1-10 promoter DNA fragments that consistently demonstrate anomalously slow migration under these electrophoresis conditions. That the anomalous migration is due to intrinsic bending is confirmed by the observation that the anomalies completely disappear when electrophoresis is carried out in the presence of ethidium bromide, a treatment that has been shown to remove migration anomalies that are caused by bending (34). DNA fragments containing the region immediately upstream of the GAL1 TATA consistently show the most anomalous migration behavior (b or 1, Fig. 3A). However, in a fragment with an upstream terminus closer to GAL1, at 659 bp, the anomalous migration is largely abolished (not shown). This observation would suggest that an intrinsic DNA bend lies between 600 and 659 bp.
Based on those types of observations, we carried out a circular
permutation analysis (6) on a tandem head-to-tail dimer of a fragment
containing DNA from 550 to 896 bp on the GAL1-10 promoter
(Fig. 1), as described under "Experimental Procedures." In this
type of analysis, the dimer is cleaved with restriction enzymes that
cut once within each monomer unit, producing a set of identically sized
(monomer) DNA molecules but with varying internal locations of any
intrinsic bends. The fragments with the highest relative mobility arise
when the restriction site that is cut lies at or near the center of the
bend; monomer fragments produced by this enzyme will not contain the
entire internal bend (6). The results are shown in Fig.
4. To locate the bend center, we carried
out a best-fit analysis on each of the two distinct arms of the plot.
By this analysis, the bend center maps to 645 bp. Again, the presence
of ethidium bromide removes all anomalous migration and all
Rf differences among the fragments (Fig. 4,
open circles), indicating that intrinsic curvature is responsible for the migration anomalies. Based on the slopes of the
lines in these plots, one can estimate the average bending angle (39).
For the 645-bp bend, this average angle is ~70° (Table
I).
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The bending models and the restriction digests predict two other strong
bends, at sites around 500 bp on the GAL1-10 promoter and
around 500 bp on the GAL80 promoter. Circular permutation analysis confirms both of these intrinsic bending sites (Fig. 5), at 536 bp on GAL1-10 and
at 551 bp on GAL80. Again, these migration anomalies are not
observed in the presence of ethidium bromide (not shown). The estimated
bending angles for these bends are ~50° for each (Table I).
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These experimental results show fairly good agreement with the Goodsell and Dickerson bending model (37), which is based on nucleosome positioning data. For example, the experimentally determined bend centers and magnitudes of the GAL1-10 536- and 645-bp bends and the 551-bp bend of GAL80 show excellent agreement with model predictions (Table I). Moreover, this model predicts a broad curvature above 900 bp on GAL80 (well within the coding region), and we find evidence for a bend centered at 960 bp (not shown). Also, in our initial screen (gel migration anomalies), we did not detect evidence for any intrinsic promoter region bends besides those predicted by the Goodsell and Dickerson model. Thus, 300-500 bp on the GAL1-10 promoter and 300-500 and 600-800 bp on the GAL80 promoter are bend-free.
The Goodsell and Dickerson model (Fig. 2B) also predicts two
weaker bends just upstream of GAL10 (at ~220 and ~280
bp) and a bend in the GAL10 5' coding region (at ~130 bp).
Another bending model, Brukner et al. (40), also predicts
bends at ~130, ~190, and ~ 280 bp (not shown). Migration
anomaly and circular permutation studies yield results that are
consistent with the presence of bends at ~130 and ~200 bp
(cf. fragments 32 c in Fig. 3A
and data not shown), but there is a much greater variability in the
mobility studies, and the circular permutation signals are much weaker for these bends than for the 536- and 645-bp bends. Thus, it is likely
that there are intrinsic bends at ~130 and ~200 bp, but these bends
are probably weaker than the GAL1 proximal bends, as
predicted (Table I). The close proximity of the three
GAL10-proximal bends might also affect the strength of the
bending signal from this region and definitely complicates circular
permutation studies. We feel that these GAL10-proximal bends
are not likely to be as important as the stronger bends upstream of
GAL1 and GAL80 and therefore have not pursued
their study. The bend centered at ~ 280 bp is predicted
to be the weakest of the GAL10-proximal bends (Fig.
2B) and lies in a region with few restriction sites.
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DISCUSSION |
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Using gel mobility and circular permutation analyses, we have
detected fairly strong (50°) sites of intrinsic DNA bending in the
promoters of the yeast GAL1-10 structural genes and the GAL80 regulatory gene. In vivo, these
GAL promoters contain two distinct types of chromatin
regions: constitutively non-nucleosomal hypersensitive regions in which
the major promoter elements, the UASG, reside; and
nucleosomal regions, where the TATA, Inr, and transcription start sites
are found (30). The single intrinsic DNA bend on the
GAL80 promoter lies in the non-nucleosomal region, between
the UASGAL80 and the more gene-proximal promoter elements (Fig. 6). The GAL80-specific
UASGAL80 activation sequence is the most upstream element
on GAL80 and is required for both basal and induced
expression. The intrinsic DNA bend might help to bring this
constitutively functioning promoter element closer to the gene or to
the other promoter elements. Intrinsic bends on prokaryotic promoters
often function in such an architectural role (9). There are no
intrinsic bends in the nucleosomal regions of the GAL80
promoter (
or D, Fig. 6).
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In contrast, the intrinsic bends in the GAL1-10 promoter are all found within the promoter nucleosomal regions. These nucleosomes (A-C, Fig. 6) cover the TATA and transcription start sites of GAL1 and GAL10 under inactive (noninducing) conditions, and their presence helps to implement gene repression in those carbon sources (cf. Refs. 25 and 30). The strongest bends lie at 536 and 645 bp within a nucleosome referred to as B (Fig. 6). In vivo, nucleosome B clearly has a strong locational preference, although it is not clear whether nucleosome B is absolutely positioned (25). The dyad center of this nucleosome should lie around 595 bp (28), roughly midway between the two bending sites. Based on the nucleosome positions estimated by recent nuclease digestion studies (41, 42), the bend centers would lie within the terminal 20 bp or so on each end of a 147-bp core nucleosome B.
These two intrinsic DNA bends might help determine the location of nucleosome B in its preferred position. For example, it has been suggested that nucleosomes have a preference to locate more flexible DNA in their central region and that this can be a determinant of nucleosome positioning (43, 44). The preferred position of nucleosome B is consistent with this suggestion; it places the more rigid (intrinsically bent) DNA near the nucleosome termini, in regions where DNA is less constrained by nucleosome structure (45, 46) and locates the more flexible interbend DNA within the nucleosome. However, nucleosome B does not show positioning in vitro (47). Thus, these intrinsic bends are apparently not capable by themselves of imposing locational boundaries on nucleosome B. Other features that could also contribute to the in vivo positioning of nucleosome B have been discussed elsewhere (25). Despite the presence of these intrinsic bends, nucleosome B is stable, perhaps because the 110 bp of DNA between the two bending sites is close to the length of DNA (~120 bp) that is strongly associated with the core histones in the nucleosome (45, 46). Nevertheless, these intrinsic DNA bends might interfere enough with the normal wrapping of DNA on the nucleosome to cause B to be less stable than a nucleosome without such bends.
These two intrinsic bends may play their most important roles in the
processes that are associated with the transcriptional activation of
GAL1. 1) When GAL1-10 expression is activated in galactose, the promoter nucleosomes (AC, Fig. 6)
appear to be completely removed (28, 48). The fact that the strongest
bends on the GAL1-10 intergenic region lie in nucleosome B
may reflect the central role of B in the nucleosome disruption process
(for a review, see Ref. 25). For example, the bends could
preferentially destabilize nucleosome B, either intrinsically (see
above) or by attracting factors (see below), thus facilitating its
complete disruption. The 645-bp bend also lies very near the nucleosome terminus that shows accessibility changes when histone H4 N termini are
deleted or the acetylation sites are removed (41). These H4 mutations
decrease GAL1 induction levels by severalfold, indicating that the H4 tails normally play a role in GAL1 activation.
Perhaps that role involves the intrinsic bend.
2) It has been shown that placing an intrinsically bent DNA sequence between an upstream activation element and a TATA element will enhance transcription activation in vivo (20), and a bent sequence in phase with the TATA strongly enhances TBP-TATA binding (49). The center of the 645-bp bend lies 31 bp from the first TA step of the GAL1 TATA, roughly three helical repeats of 10.5 bp, the solution repeat of DNA (50). Thus, the 645-bp bend could function to enhance TBP-TATA binding and GAL1 expression, as observed in the model systems. The intrinsic bend and the bend induced by TBP would, if combined in phase, produce an ~150° bend.
3) The 645-bp bend may also recruit the yeast HMG analogues NHP 6A/6B,
which are absolutely required for high-level GAL1 induced expression in vivo (1). NHP6A displays an enhanced affinity for distorted DNA structures and bends DNA significantly upon binding
to it (51, 52). Complete removal of nucleosome B (during gene
induction) will expose both the 536- and 645-bp intrinsic bends to
NHP6A, which could then bind to one or both of these bent sequences.
Competition of NHP6A for these binding sites could even aid the
nucleosome disruption process, at least thermodynamically. Bending
induced by NHP6A binding should significantly shorten the distance
between the UASG sequences and the TATA-bound TBP. Perhaps,
more importantly, it has been shown in vitro that NHP6A stimulates polymerase II transcription by promoting the
formation of an especially favorable TBP initiation complex (1). The bend at 645 bp would appear to be a suitable site from which NHP6A could promote the TBP complex assembly on GAL1. Both the
TATA and UASG functions on GAL1 depend on
NHP6A/6B (1). Thus, the strong bends upstream of GAL1 may
function in nucleosome location in the inactive state and in gene
activation during induction.
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ACKNOWLEDGEMENT |
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We thank Stefan Dieckmann for stimulating discussions.
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FOOTNOTES |
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* 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.
¶ Supported by Grant MCB-9728240 from the National Science Foundation.
Supported by Grant ES00210 from the National Institute of
Environmental Health Sciences.
** To whom correspondence should be addressed. Tel.: 480-965-5020; Fax: 480-965-2747; E-mail: dlohr@asu.edu.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M007070200
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ABBREVIATIONS |
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The abbreviations used are: HMG, high mobility group; bp, base pairs; TBP, TATA-binding protein; UAS, upstream activation sequence.
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