Molecular and Cellular Biology, April 1999, p. 2977-2985, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Binding of Gal4p and Bicoid to Nucleosomal Sites
in Yeast in the Absence of Replication
Bhuvana
Balasubramanian1 and
Randall H.
Morse1,2,*
Molecular Genetics Program, Wadsworth Center,
New York State Department of Health,1 and
SUNY School of Public Health,2 Albany,
New York 12201-2002
Received 10 December 1998/Returned for modification 10 January
1999/Accepted 13 January 1999
 |
ABSTRACT |
The yeast transcriptional activator Gal4p can bind to sites in
nucleosomal DNA in vivo which it is unable to access in vitro. One
event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites
in yeast, we have used previously characterized chromatin reporters in
which Gal4p binding sites are incorporated into nucleosomes. We find
that Gal4p is able to perturb nucleosome positioning via nucleosomal
binding sites in yeast arrested either in G1, with
-factor, or in G2/M, with nocodazole. Similar results
were obtained whether Gal4p synthesis was induced from the endogenous
promoter by growth in galactose medium or by an artificial,
hormone-inducible system. We also examined binding of the
Drosophila transcriptional activator Bicoid, which belongs
to the homeodomain class of transcription factors. We show that Bicoid,
like Gal4p, can bind to nucleosomal sites in
SWI+ and swi1
yeast and in the
absence of replication. Our results indicate that some feature of the
intracellular environment other than DNA replication or the SWI-SNF
complex permits factor access to nucleosomal sites.
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INTRODUCTION |
Transcriptional activators work in
part by overcoming the repressive influence of chromatin, as suggested
by experiments both in vitro and in vivo (27, 55, 78).
However, this leaves as an open question the mechanism by which the
activators themselves gain access to sites in chromatin. To address
this question, we and others have used the yeast Saccharomyces
cerevisiae to examine the interactions of transcriptional
activators with defined chromatin structures (24, 47, 48, 61, 65,
72, 83), with particular emphasis on the yeast Gal4 protein.
Previous work has demonstrated that Gal4p can bind in vitro to
reconstituted chromatin containing five Gal4p binding sites, either as
an isolated nucleosome or in an array, and form a metastable complex,
independent of an activation domain (53, 79). However, Gal4p
binding to a single nucleosomal site is strongly inhibited when the
binding site is centered 40 or 74 bp from the nucleosome edge; when the
site is centered 21 bp from the edge, binding is effective
(75). In contrast, studies in yeast have shown that Gal4p
can gain access to sites near the center of a positioned nucleosome on
a multicopy plasmid when overexpressed (48, 83) and can bind
to a site centered 41 bp from the edge of the nucleosome even at
endogenous levels (65, 83). In all cases, binding results in
perturbation of chromatin (48, 65, 83) and is greatly
enhanced by the presence of a functional activation domain (48,
65).
The mechanism by which Gal4p gains access to nucleosomal sites in vivo
is unclear. One possibility is that a chromatin remodeling complex, such as SWI-SNF, can recognize sequence or structural elements
near the Gal4p binding site and remodel nucleosomes locally to
facilitate Gal4p binding (13, 57). However, although binding of Gal4p to a pair of nucleosomal weak binding sites in yeast has been
shown to be stronger in SWI+ than swi
cells (8), we have found that neither SWI-SNF nor GCN5 is
needed for perturbation by Gal4p of a positioned nucleosome containing
a strong Gal4p binding site in yeast (61, 67). Another
possibility is that during DNA replication, the transient removal of
the histones from DNA provides an opportunity for transcription factors
such as Gal4p to bind to nucleosomal sites in vivo (7, 70,
77). In vitro studies have shown that in some cases, repression of transcription by chromatin can be relieved by replication in the
presence of relevant transcription factors (5, 37). On the
other hand, in vivo experiments have shown that transcriptional activators can remodel chromatin in the absence of replication (58, 62, 65, 74, 78, 83, 84). However, several of these
examples involve complex promoters in which factors may contribute to
chromatin remodeling via nonnucleosomal cis-acting elements
(58, 62, 74, 78, 84), and in another case, the activator
GAL4-ER-VP16 (see Results for description) was constitutively present,
and chromatin remodeling was induced by addition of
-estradiol (65). Only recently has the binding of a single
transcriptional activator, Gal4p, to a single nucleosomal site been
examined in nonreplicating yeast cells (83). In
this case, for technical reasons, cells were first grown to stationary
phase and then arrested for 12 h with hydroxyurea, simultaneously
with induction of Gal4p synthesis, prior to examination of
chromatin perturbation by Gal4p. Thus, although Gal4p
perturbation of chromatin was observed, it was not established whether
Gal4p binding could occur to a nucleosomal site in a shorter, perhaps
more physiologically relevant interval or in cells arrested in log
phase. Furthermore, whether Gal4p can bind to a nucleosomal site in
yeast arrested in other phases of the cell cycle also remains
undetermined. This is not merely a moot point, as for example the
ability of a transcriptional activator to overcome repression of a
telomeric URA3 gene varies at different points in the cell
cycle (1).
In the present work, we test the ability of Gal4p to perturb a
positioned nucleosome containing a Gal4p binding site in the absence of
replication. We examine two distinct nucleosomes containing Gal4p
binding sites, in one case near the nucleosome pseudodyad (i.e., near
the center) and in the other case centered 41 bp from the nucleosome's
edge; induce Gal4p synthesis by two distinct routes; and
investigate binding and chromatin perturbation at two widely separate
points in the cell cycle, G1 and G2/M. We also
increase the scope of our conclusions by performing similar experiments
with the transcriptional activator Bicoid from Drosophila melanogaster, whose DNA-binding domain is structurally
different from that of Gal4p.
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MATERIALS AND METHODS |
Strains, media, and genetic methods.
We used yeast strains
YJ0
(constructed from YJ0 [MATa trp1 ura3-52
leu2-3,112 ade2-101 gal4
gal80
MEL1], a gift from Stephen
Johnston), and FY23bar1
(MATa
bar1::LEU2 ura3-52 trp1
63 leu2
1) for
experiments involving Gal4p. For experiments with Bicoid protein, we
used strains CY296 (MATa gal4
::LEU2 lys2-801 leu2-
1 his3-
200 ura3-
99 trp1-
99
[8]), CY297b (MAT
swi1
::LEU2
gal4
::LEU2 lys2-801 leu2-
1 his3-
200 ura3-
99
trp1-
99 [61]), and YJ0bar1
(MATa trp1 ura3-52 leu2-3,112 ade2-101 gal4
gal80
MEL1 bar1::LEU2). The bar1
strains were constructed from FY23 (76) and YJ0 by one-step
gene replacement of the bar1 gene, using the
BamHI-HindIII fragment from plasmid pZV77 (a
gift from David Gross), and the gene replacement was confirmed by
Southern blotting. Yeast cells were grown in complete synthetic dropout media (Bio101) containing 2% glucose and transformed by a modification (31) of the method of Ito et al. (33).
For cell cycle arrest experiments, cells were grown in medium
containing 2% glucose or 1.5% raffinose and 25 mM phthalic acid (pH
5.5) to an optical density at 600 nm of between 0.2 and 0.5. Cells were
then arrested for 3 h, using
-factor at 0.2 to 0.5 µM or
nocodazole at 10 µg/ml. Cell cultures were supplemented with fresh
-factor or nocodazole every 3 h; for experiments involving long
nocodazole arrest, cells were spun down at 3 and 6 h and resuspended in fresh medium containing 15 µg of nocodazole per ml.
Gal4p or Bicoid synthesis was induced 3 h after initiating arrest
by addition of 100 or 200 nM
-estradiol, as indicated, or by
spinning cells down and resuspending them in 2% galactose medium
containing 25 mM phthalic acid (pH 5.5) and
-factor or nocodazole as
appropriate. Cell morphology was examined at 3 h and again at
intervals following induction of Gal4p or Bicoid. Cells exhibited <5%
budded cells and >90% shmoos from 3 to 9 h following addition of
-factor, and cell density (measured with a counting chamber) did not
increase after the initial 3-h arrest period. More than 90% of
nocodazole-arrested cells exhibited the characteristic dumbbell-shaped
(large-budded) morphology for the duration of the arrest, and cell
density did not increase. Furthermore, the copy number of the
TRP1ARS1-based plasmids did not increase relative to the genome during
the arrest, as determined by Southern blotting, consistent with
each ARS1-based plasmid replicating once per plasmid molecule and only
during S phase (15a).
Plasmids.
To construct pADH/lexA.hER.VP16, the
expression vector for LexA-ER-VP16, a 1-kb
SalI-NotI fragment containing the coding sequence for ER-VP16 (43) was ligated with pEG202, which contains a
full-length lexA gene fused to the ADH1 promoter
(26). A 7-bp linker containing a unique BstEII
site was introduced at the junction of the lexA and
ER sequences. The reporter plasmids containing LexA
operators upstream of a lacZ gene (see Fig. 1B)
(28) have been described elsewhere (22).
Induction via LexA-ER-VP16 was measured 3 h after addition of 100 nM
-estradiol.
Plasmid pGAL1-10/4 lexA sites/GAL4 was constructed by
ligating together (i) a BamHI-HindIII
fragment containing a modified GAL1-10 promoter deleted for
the four Gal4 binding sites and URS B and C regions and containing four
lexA sites (28), (ii) a 40-bp
HindIII-SphI oligonucleotide, containing a
unique NdeI site at the beginning of the GAL4
open reading frame, (iii) a 2.6-kb SphI-KpnI
fragment containing the remainder of the GAL4 coding and
termination sequences, and (iv) the shuttle vector pRS426 (11).
The yeast plasmids TALS and TA17
80 were excised from bacterial
vectors and religated before being transformed into yeast, as described
previously (47, 48). To create TABic4
80, the sequence
5'-TCCCTATCTAATCCCTATCTAATCCCTATCTAATCCC-3' was inserted into pRS104 (47) between residues corresponding to 859 and 860 map units of TRP1ARS1 by PCR to create pRS104Bic4; an
80-bp deletion was then made by PCR (63) and verified by DNA
sequencing, to create pRS104Bic4
80. Yeast sequences were then
excised from this plasmid with SacI and
HindIII (yielding a fragment carrying the 3' end of the
TRP1 gene) and ligated with the complementary
SacI-HindIII fragment from pRS110 (containing
the 5' end of the TRP1 gene [47]) and
transformed into yeast to create TABic4
80, which was verified by
Southern analysis.
The multicopy plasmid pRS426GAL4 contains the GAL4 gene
under control of its own promoter (61). The Bicoid
expression vector was created by ligating a 3-kb
KpnI-XbaI fragment from pDB1 (9) (a
gift from D. S. Burz) containing the Bicoid gene under
control of the GAL1 promoter into the polylinker of pRS416
(11). Bicoid expression was induced by GAL4-ER-VP16, using
100 nM
-estradiol (Fig. 6), or by GAL4-ER-VP16F442P (66),
using 200 nM
-estradiol (Fig. 7).
Northern analysis.
Total cellular RNA was extracted,
electrophoresed on formaldehyde-containing gels, blotted, and
hybridized as described previously (12, 85). A 2.9-kb
SphI-HindIII fragment from the
GAL4 gene was used to probe for the GAL4
transcript. The GAL1 and GAL10 transcripts were
visualized by probing with EcoRI-BamHI and
SalI-EcoRI restriction fragments, respectively,
from pBM48 (a gift from Mark Johnston), which contains the
SalI-BamHI fragment (SC4918) from the
GAL1-10 locus (69).
Enzyme assays and chromatin characterization.
Assays for
-galactosidase (the MEL1 gene product) and
-galactosidase were performed as described previously (59,
61). For topoisomer analysis, DNA was prepared and
electrophoresed on topoisomer-resolving gels, and Gaussian
centers of the resulting topoisomer distributions were
determined as described previously (46, 49).
For indirect end-label analysis (50, 80), chromatin was
prepared by combining two previously described methods (16, 38). Cells (100 to 200 ml) were grown in medium containing 25 mM
phthalic acid (pH 5.5) to an optical density at 600 nm of 0.5 to 1.5 and harvested by centrifugation at 4,000 × g for 5 min. The pellet was resuspended in 50 mM Tris (pH 7.4)-0.1%
-mercaptoethanol at a concentration of 5 × 107
cells/ml and incubated with gentle shaking in a water bath for 15 min
at 30°C. Cells were centrifuged as before; the pellet was resuspended
in medium containing 1 M sorbitol at a concentration of 109
cells/ml and spheroplasted by addition of 1/10 volume of Zymolyase 100T
(10 mg/ml; Seikagaku America, Inc., Ijamsville, Md.) and gentle shaking
at 30°C for 15 min. The spheroplasted cells were then diluted with 15 volumes of chilled medium containing 1 M sorbitol and collected by
centrifugation (2,000 × g for 5 min). Cells were
washed with medium containing 1 M sorbitol and spun down in an HB-4
rotor at 3,500 rpm (2,000 × g) for 5 min. The pellet
was then taken up at 2 × 109 cells/ml in 1 M
sorbitol-50 mM NaCl-10 mM Tris-HCl (pH 7.4)-5 mM
MgCl2-1 mM CaCl2-1 mM
-mercaptoethanol (or
10 mM dithiothreitol)-0.5 mM spermidine and transferred in 100- or
150-µl aliquots into 1.5-ml microcentrifuge tubes on ice. Micrococcal
nuclease (MNase) or restriction enzyme was added to each tube to an
appropriate concentration, and digestion was begun by addition of an
equal volume of the same buffer containing 0.15% Nonidet P-40, mixing gently, and immediately placing the tube in a 37°C bath. Digestions were halted (after 5 min for MNase and after 15 and 30 min for PstI) by addition of 55 µl of proteinase K (5 mg/ml)
sodium dodecyl sulfate (SDS; 5%). Naked DNA samples were
processed as above but immediately digested with proteinase
K-SDS, cleaned and precipitated, and digested with MNase in 300 µl of 10 mM HEPES (pH 7.5)-2 mM CaCl2-5 mM
MgCl2 for 5 min. Samples were cleaned and precipitated and
then analyzed as described below.
After >2 h of incubation with proteinase K-SDS, samples were cleaned
with phenol and chloroform and precipitated. Pellets were taken up in
100 µl 10 mM Tris-Cl (pH 8.0)-1 mM EDTA and treated with RNase A. One-third to one-half of each sample was digested with
EcoRV, precipitated, and electrophoresed on a 1.2% agarose gel for 5.5 h at 135 V (4.5 V/cm). Blotting and hybridization were
as described previously (12, 47). Samples were probed with
an EcoRV-HindIII probe from TRP1ARS1
sequences, prepared by PCR. At least two independent analyses were done
for each indirect end-label experiment.
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RESULTS |
Hormone-dependent induction of Gal4p synthesis.
To investigate
the ability of Gal4p to bind to nucleosomal sites in the absence of
replication, we used two chromatin reporter plasmids, TALS and
TA17
80, depicted in Fig. 1A. In yeast
cells not expressing Gal4p (e.g., cells grown in glucose
medium), each of these reporters contains a nucleosomal Gal4p
binding site (48, 65). Our strategy was to prevent cells
harboring TALS or TA17
80 from replicating by arresting them in late
G1 (with
-factor) (15) or G2/M
(with nocodazole) (1, 25, 34) and then to express Gal4p
while maintaining cell cycle arrest and analyze plasmid chromatin
structure. Perturbation of chromatin structure accompanying Gal4p
expression as is seen in unsynchronized cells (48, 65) would
indicate that Gal4p was able to recognize and bind to its site in a
positioned nucleosome in the absence of replication.

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FIG. 1.
Experimental strategy. (A) Schematic depiction of two
chromatin reporter plasmids, TALS and TA17 80. UASGAL3 is
a single Gal4p binding site from the GAL3 promoter
(3), and UAS17 is a single near-consensus
Gal4p binding site (21) introduced in the TRP1ARS1
derivative TA17 80 (48). Only nucleosomes I and II have
been shown to be well positioned in TA17 80, and so the remaining
nucleosomes are not numbered. (B) Scheme for placing Gal4p
synthesis under hormonal control. aa, amino acids. See text for
details. (C) The chimeric activator LexA-ER-VP16 was tested for
activity at a CYC1-lacZ reporter gene having two,
four, or eight LexA binding sites upstream. Yeast cells (YJ0 )
grown in raffinose medium to mid-log phase were incubated for
3 h after addition of 100 nM -estradiol before measurements of
-galactosidase activity for plus-hormone samples. The results shown
are an average from three independent colonies from each of two
independent transformations.
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We first sought to achieve rapid, inducible expression of Gal4p by
placing the Gal4p coding sequence under hormone control. This strategy
was based on previous work with the chimeric activator GAL4-ER-VP16,
which contains the Gal4p DNA-binding domain, the human estrogen
receptor hormone-binding domain (ER), and the strong VP16 activation
domain (43). Expression of GAL4-ER-VP16 in yeast allows
rapid, hormone-dependent expression from promoters containing Gal4p
binding sites (43, 65). To allow Gal4p induction by a
similar strategy, we replaced the coding sequence for the Gal4p DNA-binding domain of GAL4-ER-VP16 with that for the bacterial LexA
protein to create pADH/lexA.hER.VP16 (Fig. 1B). This
expression vector was introduced into yeast and was first tested for
activity in assays using lacZ reporters containing two,
four, or eight lexA sites (Fig. 1C). LexA-ER-VP16 showed low
activity in the absence of estradiol, which increased 12- to 33-fold
after 3 h induction with
-estradiol.
The GAL4 gene was then placed under control of the same
promoter containing two, four, or eight lexA sites in place
of the lacZ gene (Fig. 1B). This plasmid and the
LexA-ER-VP16 expression vector were introduced into a gal4
gal80
MEL1+ yeast strain, and the induction of
Gal4p upon the addition of hormone was demonstrated by induction of
-galactosidase (the product of the Gal4p-regulated MEL1
gene) (data not shown). The Gal4p expression vector having four LexA
binding sites was found to have the best induction characteristics and
was used in all subsequent experiments. Proper induction was confirmed
in each experiment by monitoring
-galactosidase activity.
Induction of the GAL1 and GAL10 genes in
nonreplicating yeast.
Figure 2A
shows directly the induction of GAL4 transcripts via
LexA-ER-VP16 (lanes 1 and 2) 3 h after addition of 100 nM
-estradiol. Hormone induction of Gal4p synthesis in raffinose medium
in the gal4
gal80
strain YJ0
resulted in strong
induction of the GAL1 and GAL10 genes, indicating
that the artificially induced Gal4p protein functions normally (Fig.
2A, lane 2). Some expression of GAL1 and GAL10
transcripts was observed even in uninduced cells (Fig. 2A, lanes 1 and
3), corresponding to low levels of Gal4p (weakly detectable at the RNA
level and also when assayed for
-galactosidase activity) induced by
LexA-ER-VP16 in the absence of
-estradiol. Importantly, this level
of Gal4p was not sufficient to cause significant changes in chromatin
structure in our reporters (see below).

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FIG. 2.
Induction of mRNAs by Gal4p in arrested and
unsynchronized yeast cells. (A) Induction of GAL4 mRNA under
control of LexA-ER-VP16, and consequent induction of GAL1
and GAL10 mRNAs by Gal4p, from unsynchronized and
nocodazole-arrested YJ0 yeast cells grown in raffinose medium in the
presence (for 3 h) and absence of -estradiol. The
PYK1 message was examined as a control. (B) Induction of
GAL4, GAL1, and GAL10 mRNAs under
endogenous control in unsynchronized and arrested yeast cells.
Cells (FY24 yeast cells containing TALS and pRS426GAL4) were grown in
glucose medium and mRNA was harvested (glu lanes), or the
cells were spun down and transferred to galactose medium, and mRNA was
harvested at indicated times (gal lanes). The
PRC1 message was examined as a control.
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Figure 2A also shows that the GAL1 and
GAL10 genes are induced upon hormone addition in
cells arrested in G2/M phase with nocodazole. Similarly,
galactose induction of the wild-type GAL4 gene caused
approximately equivalent induction of the GAL1 and GAL10 genes in unsynchronized cells and in cells arrested
with nocodazole or
-factor (Fig. 2B). These results indicate that GAL4 functions normally in nonreplicating yeast cells,
consistent with previous work showing that a lacZ reporter
under gal control could be induced in
-factor-arrested
cells (32). Our results have additional significance,
however, as induction of both the GAL1 and GAL10
genes is normally accompanied by remodeling (albeit subtle
[17]) of chromatin structure (2, 42). Thus,
our results suggest that Gal4p is capable of remodeling the chromatin structure of the GAL1-10 promoter in the absence of replication.
Remodeling of TALS chromatin by Gal4p.
The results of
Fig. 2 demonstrate that Gal4p can induce transcription and
therefore, by inference, remodel chromatin at the GAL1-10 locus in nonreplicating yeast. However, since
the Gal4p binding sites in the GAL1-10 promoter are
nonnucleosomal (41), this experiment yields no information
on binding of Gal4p to a nucleosomal site. We therefore next examined
Gal4p binding to the TALS chromatin reporter. TALS is a TRP1ARS1-based
episome in which nucleosomes are strongly positioned by the
2-MCM1
complex in yeast
cells (60). A single Gal4p binding
site, derived from the GAL3 gene (3), is centered
41 bp from the left edge in nucleosome IV, which is immediately
adjacent to the
2-MCM1 binding site (Fig. 1A). This region is
inaccessible to Escherichia coli Dam methyltransferase
expressed in yeast, in the absence of Gal4 protein (39). In
the presence of Gal4p, nucleosome IV is perturbed and TALS chromatin is
remodeled, as shown by changes in MNase cleavage, SacI
accessibility, and plasmid topology (65).
We examined TALS remodeling by monitoring plasmid topology. This assay
is based on the fact that packaging of DNA in chromatin causes a change
in linking number of
1 per nucleosome, which can be readily
visualized and quantified in a closed circular plasmid (20, 49,
64). Perturbation of TALS chromatin by Gal4p expressed in
galactose medium results in a loss of nearly one negative supercoil per
plasmid (65) (Fig. 3B, lanes 1 and 2). An approximately equivalent change in topology is seen upon hormone induction of Gal4p synthesis via LexA-ER-VP16 (Fig. 3A, lanes 1 and 2; Table 1). Thus, although the LexA-ER-VP16-mediated induction of
Gal4p is less stringent than that of the native GAL4 promoter, it closely mimics native induction with respect to TALS remodeling.

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FIG. 3.
Remodeling of TALS chromatin assessed by changes in
topology in unsynchronized and arrested yeast cells. (A) Cells (YJ0 )
harboring TALS and having GAL4 under control of LexA-ER-VP16
were grown in raffinose medium in the presence or absence of hormone,
either unsynchronized or arrested as indicated, and DNA was isolated
for analysis of TALS topoisomer distributions. Hormone
induction was for 3 h at 100 nM -estradiol. The band near the
top is nicked circular DNA, and the lower bands represent
topoisomers differing in linking number from adjacent bands by
one; under the conditions used, faster-migrating species are more
positively supercoiled. Values shown for Lk indicate the differences
between the calculated centers of the Gaussian distributions in the
lanes indicated. (B) Topoisomer distributions of TALS from cells (FY24)
harboring TALS and a multicopy plasmid bearing the GAL4 gene
grown in glucose medium in the presence of nocodazole (10 µg/ml) for
3 h or in its absence, as indicated. Cells were spun down and
taken up in galactose medium with or without nocodazole and incubated
for the additional intervals indicated. The uppermost band corresponds
to nicked circular TALS, and faster-migrating topoisomers are
more positively supercoiled. The linking number changes between samples
grown in glucose and galactose are indicated at the bottom. o/n,
overnight.
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When yeast cells harboring TALS were arrested with nocodazole,
induction of Gal4p via LexA-ER-VP16 again resulted in loss of negative
supercoiling (Fig. 3A, lanes 3 and 4; Table
1). Similar results were observed upon
induction of the native GAL4 gene in nocodazole-arrested
cells (Fig. 3B, lanes 3 and 4; Table 1). MNase cleavage sites in TALS
chromatin induced by Gal4p (65) were also observed when
Gal4p was induced by LexA-ER-VP16, in both unsynchronized and
nocodazole-arrested cells (4). Thus, a nucleosomal Gal4p
binding site could be accessed by Gal4p, with consequent chromatin
remodeling, in the absence of replication.
Remodeling of TA17
80 chromatin by Gal4p.
Because nucleosome
positioning in TALS requires the
2 protein, we could not determine
whether Gal4p could bind to a positioned nucleosome in TALS in
-factor-arrested yeast cells. To circumvent this problem and to
investigate a different nucleosomal Gal4p binding site, we used a
different chromatin reporter, TA17
80 (48). This
TRP1ARS1-based episome contains a single 17-bp near-consensus Gal4p
binding site near the center of a positioned nucleosome (Fig. 1A). In
the presence of Gal4p, nucleosome positioning in TA17
80 near the
Gal4p binding site is perturbed, indicating that Gal4p is able to
outcompete histones for occupancy of this site in cycling yeast cells
(48). As with TALS, the Gal4p binding site is in a region of
the nucleosome which is inaccessible to Gal4p in vitro (75),
and so we wished to test whether its accessibility to Gal4p in vivo
would occur during replication.
We first attempted to use LexA-ER-VP16 mediated induction of Gal4p as
described above. However, for reasons that we do not understand,
introduction of TA17
80 into yeast harboring the expression vectors
for LexA-ER-VP16 and Gal4p resulted in loss of inducible Gal4p
activity. We therefore instead turned to galactose induction of Gal4p
from the native GAL4 gene on a multicopy plasmid in cells first grown in glucose. This protocol differs from one commonly used
for Gal4p-dependent induction from cells grown in raffinose or
glycerol-lactate medium. In raffinose or glycerol-lactate medium, the GAL4 gene is active, but Gal4p is repressed by Gal80p
(36, 44); addition of galactose to the cells releases
Gal4p from Gal80p repression, and genes responsive to Gal4p are rapidly
induced (40). In contrast, the GAL4 gene is repressed in
glucose medium, and a considerable lag occurs before
Gal4p-responsive genes are induced following a shift to galactose
medium (23, 35, 51).
Before undertaking experiments with arrested cells, we first determined
the time of galactose induction needed to perturb nucleosome
positioning in TA17
80 in cycling yeast cells. TA17
80 shows little
if any change in topology upon galactose induction, in contrast to TALS
(4). This probably means that nucleosomes are rearranged
without net loss upon Gal4p binding to TA17
80, whereas Gal4p binding
to TALS causes both rearrangement and net loss of nucleosomes. We
therefore monitored chromatin remodeling of TA17
80 by MNase
digestion followed by indirect end labeling (50, 80). Only
slight perturbation of nucleosome positioning was seen at 3 and
4.5 h following galactose induction; however, after 6 h,
changes in the MNase cleavage pattern reflecting perturbation of
nucleosome positioning could clearly be seen (4) (see
below). Similarly, between 5 and 6 h of growth in galactose medium
sufficed for the maximal change in topology of TALS induced by Gal4p
(Fig. 3B). Based on these results, we examined remodeling of TA17
80 chromatin by Gal4p in noncycling yeast cells 6 h following
galactose induction. FY23bar1
cells were grown to mid-log
phase and incubated with
-factor or nocodazole for 3 h to
ensure arrest. (The bar1 deletion removes a protease which
degrades
-factor, allowing
-factor arrest to be maintained for
long periods.) Cells were then spun down and resuspended in glucose or
galactose medium for an additional 6 h of incubation in the
continued presence of
-factor or nocodazole prior to isolation and
digestion of chromatin. Cell density was unchanged during the 6-h
induction period, and the morphology remained consistent with the
arrest (see Materials and Methods).
Figure 4 shows indirect end-label
analysis of MNase-digested TA17
80 chromatin from cycling cells,
and cells arrested with nocodazole or
-factor, grown in glucose and
galactose. In cycling cells, enhanced MNase cleavage is seen in the
region of nucleosome II following 6 h of growth in galactose
medium compared to cells grown in glucose (Fig. 4A, lanes 1 to 6; note
the two bands marked by asterisks). The upper of these two new cleavage
sites does not correspond to any of the cleavage sites seen in naked
DNA (lane 7) and is the most prominent. This cleavage most likely reflects a rearrangement of nucleosome positioning that results from
Gal4p binding to its site near the center of nucleosome I, as it
depends on growth in galactose and is not seen at endogenous levels of
Gal4p (4). Furthermore, a PstI site near the
Gal4p binding site which is blocked in cells grown in glucose becomes accessible after 6 h of growth in galactose (Fig. 5; see below), consistent with perturbation of nucleosome positioning by
Gal4p. A somewhat different MNase cleavage pattern is seen after longer (overnight) growth in galactose medium, in which enhanced cleavage is
seen in both nucleosomes I and II, while the upper cleavage site seen
in nucleosome II after 6 h becomes less prominent (4, 48). We do not at present understand the reason for this apparent change in perturbation of TA17
80 chromatin occurring between 6 and
24 h. However, since the perturbation seen in the region of
nucleosome II was completely reproducible, we used this MNase cleavage
pattern to compare with that seen in arrested cells 6 h following
galactose induction.

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FIG. 4.
Remodeling of TA17 80 chromatin by Gal4p in
unsynchronized and arrested cells assayed by indirect end-label
analysis of MNase cleavage sites. (A) Yeast cells
(FY23bar1 ) harboring TA17 80 and pRS426GAL4 were grown
in glucose, either unsynchronized or arrested with nocodazole as
indicated, or shifted from glucose to galactose medium (still
containing nocodazole for arrested cells) and incubated an additional
6 h prior to harvesting of chromatin for MNase digestion. MNase
cleavage sites were mapped counterclockwise from the EcoRV
site (Fig. 1A). Lanes: C, chromatin; D, naked DNA; M, X DNA digested
with HaeIII. The locations of nucleosomes I and II are
indicated at the left, with the rectangle in nucleosome I representing
the Gal4p binding site. Cleavage sites induced in galactose medium
after 6 h are indicated by asterisks (lanes 4 and 13); the upper
site is much more prominent and is not cleaved in naked DNA. MNase was
used at 0 (lanes 8, 9, and 17), 2 (lanes 1, 6, 10, and 15), 5 (lanes 2, 5, 11, and 14), and 20 (lanes 3, 4, 12, and 13) U/ml for chromatin and
at 4 (lane 7) and 10 (lane 16) U/ml for naked DNA. (B) Cells were grown
in glucose and arrested with -factor, and chromatin was isolated
before and after 6 h of additional incubation in galactose medium.
n.a., not applicable. MNase concentrations used are given in
units per milliliter.
|
|
When cells were arrested with nocodazole or with
-factor and then
grown in galactose medium for an additional 6 h, a perturbation in
the MNase cleavage pattern similar to that in cycling cells was seen
(Fig. 4A, lanes 9 to 15; Fig. 4B). These results suggest that Gal4p can
access a nucleosomal binding site even cells arrested in late
G1 by
-factor or in G2/M by nocodazole. We
also examined accessibility to the restriction enzyme PstI
in TA17
80 chromatin from cells grown in glucose and galactose. The
PstI recognition site is in nucleosome I, 30 bp from the
edge of the Gal4p binding site (Fig. 1A). This site is strongly
protected against digestion in chromatin from cells grown in glucose
and is strongly cleaved after 6 h of growth in galactose (Fig.
5, lanes 7 to 12), corroborating the
MNase cleavage data that indicate chromatin remodeling by Gal4p in
galactose. Enhanced PstI cleavage is also seen in cells arrested with nocodazole in galactose but not in glucose (Fig. 5, lanes
1 to 6), again in agreement with MNase cleavage results (Fig. 4).

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FIG. 5.
Remodeling of TA17 80 chromatin by Gal4p in
unsynchronized and arrested cells assayed by restriction enzyme
accessibility. Chromatin from cells treated as for Fig. 6 (an aliquot
from the same preparation) was incubated in the absence (for 30 min) or
presence of PstI at 200 U/ml for 15 min or 30 min, as
indicated. Purified DNA was secondarily digested with EcoRV
and analyzed by indirect end labeling, probing counterclockwise from
the EcoRV site (Fig. 1A). The band at the top is the
EcoRV-cut intact plasmid, and the band indicated by the
arrow corresponds to cleavage at the PstI site in nucleosome
I. The cleavage site at about 1,400 bp corresponds to a second
PstI site in TA17 80. Lane M contains X DNA digested
with HaeIII.
|
|
We conclude from these experiments that Gal4p is able to remodel a
preexisting positioned nucleosome in TA17
80 in nonreplicating yeast
cells, in agreement with results obtained with TALS as the chromatin reporter.
The Drosophila transcriptional activator Bicoid can
bind to nucleosomal sites in SWI+ and in
swi yeast, and in the absence of replication.
Transcriptional activators bind to DNA via domains that exhibit
considerable structural diversity (30). The DNA-binding domain of Gal4p is characterized by six cysteine residues that coordinate two Zn2+ ions in a bimetal-thiolate cluster; the
two Gal4p molecules comprising the biologically active dimer contact
CCG triplets at either end of the 17-bp recognition site through
contacts with the major groove (45, 54). Gal4p can bind to
nucleosomal sites in yeast, in swi as well as
SWI+ cells (61) and in the absence of
replication (this work). To determine whether these properties are
shared with transcriptional activators having other kinds of
DNA-binding domains, we chose to examine interaction of the
Drosophila transcriptional activator Bicoid with nucleosomal
sites in yeast.
Bicoid is a member of the homeodomain class of transcriptional
activators and binds to the consensus site TCTAATCCC
(14). However, whereas a binding site for a single
Gal4p dimer allows maximal transcriptional activation in yeast
(82), maximal activation by Bicoid in yeast requires more
than two binding sites (9). To simulate a strong binding
site, we therefore replaced the Gal4p binding site of TA17
80
with four copies of the Bicoid consensus binding site spaced 11 bp apart (29) to generate TABic4
80, which was
introduced into yeast. Nucleosomes I and II retained the strong
positioning seen with TA17
80 in this episome, as seen by MNase
digestion followed by indirect end-label analysis (Fig. 6A, lanes 1 to 4).

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FIG. 6.
Remodeling of TABic4 80 chromatin by Bicoid in
unsynchronized SWI+ and swi1 yeast cells. (A)
MNase cleavage sites were mapped counterclockwise from the
EcoRV site in chromatin from yeast cells (CY296) harboring
TABic4 80 and the Bicoid (Bic) expression system grown in the absence
of hormone (lanes 3 and 4) or 4.5 h after addition of 100 nM
-estradiol (lanes 5 and 6). Also shown is chromatin treated with
MNase from cells harboring TA17 80, grown in glucose (lane 7) or
galactose medium (lane 8). DNA samples are TABic4 80 (lanes 1 and 2).
The locations of nucleosomes I and II are indicated at the top and
the right, with the small rectangle in nucleosome I
corresponding to binding sites for Bicoid or Gal4p in the various
episomes. MNase was used at 5 (lanes 3 and 6) and 20 (lanes 4, 5, 7, and 8) U/ml for chromatin and at 4 (lane 1) and 10 (lane 2) U/ml for
naked DNA. (B) MNase cleavage sites were mapped counterclockwise from
the EcoRV site in chromatin from swi1 yeast
cells (CY297b) harboring TABic4 80 and the Bicoid expression system
grown in the absence of hormone (lanes 3 to 5) or 4.5 h after
addition of 100 nM -estradiol (lanes 6 to 8). MNase
was used at 0 (lanes 3 and 8), 2 (lanes 4 and 7), and 5 (lanes 5 and 6)
U/ml for chromatin and at 4 (lane 1) and (lane 2) 10 U/ml for naked
DNA.
|
|
Bicoid expression was placed under hormone control by
introducing into yeast cells a plasmid having the Bicoid coding
sequence fused to the GAL1 promoter along with an expression
vector for GAL4-ER-VP16 (9, 43). Administration of 100 nM
-estradiol induced activation of a reporter gene containing four
Bicoid sites to near maximal levels in 3 to 4 h
(4). Induction of Bicoid for 4.5 h in yeast cells
harboring TABic4
80 resulted in perturbation of nucleosome I, which
contains the four Bicoid binding sites, as well as the neighboring
nucleosome II (Fig. 6A, lanes 5 and 6), similar to the perturbation of
TA17
80 caused by Gal4p expression (Fig. 6A, lanes 7 and 8).
Although the SWI-SNF complex can assist binding of transcription
factors to nucleosomal sites in vitro (13, 81), Gal4p binds
to its site in TA17
80 in swi1
yeast cells, indicating that interactions with factors other than SWI-SNF may assist its binding in vivo (61). To determine whether Bicoid could also bind to a nucleosomal site in yeast cells lacking a functional SWI-SNF
complex, we introduced TABic4
80 along with the expression vectors
for Bicoid and GAL4-ER-VP16 into the yeast strain CY297b, which is a
swi1
strain congenic with CY296, the strain used in the
experiment of Fig. 6A. Activation of the Bicoid-lacZ
reporter gene in these swi1
cells was only about half
that seen in SWI+ cells (4), but
despite this lower activity or expression of Bicoid, perturbation of
TABic4
80 was still readily seen (Fig. 6B). Thus, Bicoid, like Gal4p,
is able to bind to a nucleosomal site in yeast in both
SWI+ and swi cells with concomitant
perturbation of chromatin.
To determine whether Bicoid could bind to nucleosomal sites in
TABic4
80 in the absence of replication, we introduced this episome
and the Bicoid expression system into YJ0bar1
yeast
cells. We arrested these cells with
-factor for 3 h and then
induced Bicoid expression for 4.25 h. Bicoid induction in
-factor-arrested cells resulted in perturbation of TABic4
80
chromatin similar to that seen in cycling cells (Fig.
7). Thus, two transcriptional activators
with different kinds of DNA-binding and activation domains are both
able to bind to nucleosomal sites in yeast in the absence of
replication.

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FIG. 7.
Remodeling of TABic4 80 chromatin by Bicoid in
arrested cells assayed by indirect end-label analysis of MNase
cleavage sites. Yeast cells (YJ0bar1 ) harboring
TABic4 80 and the Bicoid expression system were first arrested with
-factor or not, as indicated. Hormone was then added (+Bicoid lanes)
or not ( Bicoid lanes), cells were incubated an additional 4.25 h, and chromatin was isolated for MNase digestion. MNase cleavage sites
were mapped counterclockwise from the EcoRV site. Lanes C
contain chromatin; lanes D contain naked DNA controls. The locations of
nucleosomes I and II are indicated schematically at the sides, with the
small rectangle in nucleosome I corresponding to the four Bicoid
binding sites. The asterisks indicate cleavage sites induced by Bicoid
expression. MNase was used at 0 (lanes 3 and 8), 20 (lanes 9 and 10),
50 (lanes 4 and 7), and (lanes 5 and 6) 200 U/ml for chromatin samples
and at 4 (lane 1) and 10 (lane 2) U/ml for naked DNA. Lane 11 contains
X DNA digested with HaeIII. A shorter exposure was used
for lanes 9 to 11 than for lanes 1 to 8.
|
|
 |
DISCUSSION |
The mechanism by which transcriptional activators bind to
nucleosomal sites in vivo is presently unknown. One possibility is that
an activator binds to sites in chromatin during replication, when
histones are transiently removed from the DNA template (18, 70). Several previous studies have shown that chromatin
remodeling by transcriptional activators can occur in vivo in the
absence of replication (58, 62, 65, 74, 78, 83, 84). These studies, however, have generally examined promoters containing binding
sites for multiple factors that could contribute to chromatin remodeling, some of which are likely to interact with nonnucleosomal sites (58, 62, 74, 78, 84). Xu et al. (83)
recently examined binding of Gal4p in nonreplicating yeast in a more
isolated context by using a derivative of TALS in which a
near-consensus Gal4p binding site replaced the
UASGAL3 (Fig. 1A) and found no inhibition of Gal4p binding
in cells arrested with hydroxyurea for 12 h. However, in this
experiment, hydroxyurea arrest was initiated simultaneously with the
shift into galactose medium, leaving open the possibility that Gal4p
binding initiated before arrest was achieved.
We have considerably extended previous work on the role of replication
in allowing binding of activators to nucleosomal sites in vivo by
examining binding of Gal4p to nucleosomal sites in two distinct
environments (TALS and TA17
80), at two stages of the cell cycle, and
at shorter intervals following cell cycle arrest. Our results indicate
that perturbation of chromatin by Gal4p via nucleosomal binding sites
can occur in nonreplicating yeast cells. First, changes in
TALS topology (Fig. 3) and MNase cleavage pattern (4)
induced by Gal4p are unaltered in cells arrested by nocodazole
(
-factor arrest could not be used in these experiments, because
strong nucleosome positioning in TALS depends on the yeast
2
protein). Second, changes in the MNase cleavage pattern, as well
as PstI accessibility, induced by Gal4p in TA17
80 are
unchanged in arrested cells (Fig. 4 and 5). The perturbation of
chromatin structure in TALS and TA17
80 requires both Gal4p and its
binding site to be present (48, 65), and so we infer that it
is caused by histone displacement or rearrangement which is a
consequence of transcription factor binding. This interpretation is
consistent with previous studies of transcription factor binding and
chromatin remodeling (73, 83). We have also examined
binding of the D. melanogaster activator protein Bicoid to a
nucleosomal site in a nearly identical context as the Gal4p binding
site in TA17
80 and find that induction of Bicoid protein elicits
similar perturbation in this context as Gal4p does in TA17
80, that
it can do so without assistance from the SWI-SNF complex, and that it
can do so in
-factor-arrested yeast cells.
Nucleosomal sites similar in their location to those used in this study
are inaccessible to Gal4p in vitro (75). There are no
published reports of Bicoid binding to nucleosomal sites in vitro.
However, the full array of contacts made between the closely related
homeodomain protein, Antennapedia, and its binding site appears
incompatible with nucleosome structure (19), and the MNase
cleavages seen in the region of nucleosomes I and II of TABic4
80
upon induction of Bicoid indicate nucleosome perturbation. It therefore
appears likely that Bicoid, like Gal4p and most other DNA-binding
proteins examined to date, cannot access sites near the center of a
nucleosome in vitro. Our results imply that some mechanism other
than replication must allow Gal4p, and probably Bicoid, to access
nucleosomal sites in vivo. One possibility is that activators
recruit a chromatin remodeling complex(es) that assists in their
binding to nucleosomal sites. We have ruled out a requirement for
SWI-SNF (61) or GCN5 (67) in this role, but other
candidate activities could be involved (10, 81). Alternatively, binding sites might be in dynamic equilibrium between histone-bound and accessible states (46, 56), thereby
allowing activator binding at sufficient concentrations.
However, this mechanism would not explain the examples of
DNA-binding proteins which do not access nucleosomal sites in vivo
(39, 73), nor would it account for a role for
activation domains in assisting chromatin perturbation (48, 65,
71).
Interestingly, remodeling of the PHO5 promoter by Pho4p in
the absence of replication requires glucose, suggesting an
energy-dependent process (62), whereas we observe remodeling
of TALS and TA17
80 chromatin by Gal4p in arrested cells in both
raffinose medium (Fig. 3A) and galactose medium (Fig. 3B and 4). We
also observe strong induction of the GAL1 message, which is
normally accompanied by chromatin remodeling (2), in
arrested cells in both raffinose and galactose (Fig. 2). Perhaps the
requirement for glucose in the PHO5 system has to do with
changes in protein phosphorylation or some other event specifically
needed for activation of PHO5 (72).
Replication has been shown to allow repression of transcription by
chromatin to be overcome in vitro (5, 37). An in vivo correlate to these findings has not yet been discovered. In some cases
factor binding is inhibited by chromatin, even in replicating cells
(24, 73, 83), whereas in other examples in which factor binding does occur in chromatin, replication is not required
(references 58, 62, 74, 78, 83, and
84 and this work). The discrepancy between the in
vitro and in vivo findings suggests that replication may be important
for factor binding to chromatin only under very specific conditions in
vivo. Consistent with this notion, the specialized chromatin structure
present at yeast telomeres allows transactivation of a URA3
reporter gene in G2/M phase but not in cells arrested in
G0 or G1 (1). Further work
will be required to determine whether other specific chromatin
structures, perhaps involving linker histones (6, 68),
resist factor binding in a cell cycle-dependent manner.
 |
ACKNOWLEDGMENTS |
We thank Kellie Cummings for initiating construction and
characterization of TABic4
80; David Gross, Steve Hanes, Dave Burz, Stephen Johnston, Mark Johnston, and Joan Curcio for gifts of yeast
strains and plasmids; Michael Kladde and Robert Simpson for helpful
discussions; and the Wadsworth Center Molecular Genetics Core for
oligonucleotide synthesis and DNA sequencing.
This work was supported by grant GM51993 from the National Institutes
of Health.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Wadsworth
Center, Albany, NY 12201-2002. Phone: (518) 486-3116. Fax (518)
474-3181. E-mail: Randall.Morse{at}wadsworth.org.
 |
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