Department of Biochemistry and Molecular
Biology and Center for Gene Regulation, The Pennsylvania State
University, University Park, Pennsylvania 16802-4500
Received 3 July 1997/Returned for modification 12 August
1997/Accepted 2 December 1997
Biochemical studies have demonstrated decreased binding of various
proteins to DNA in nucleosome cores as their cognate sites are moved
from the edge of the nucleosome to the pseudodyad (center). However, to
date no study has addressed whether this structural characteristic of
nucleosomes modulates the function of a transcription factor in living
cells, where processes of DNA replication and chromatin modification or
remodeling could significantly affect factor binding. Using a
sensitive, high-resolution methyltransferase assay, we have monitored
the ability of Gal4p in vivo to interact with a nucleosome at positions
that are known to be inaccessible in nucleosome cores in vitro. Gal4p
efficiently bound a single cognate site (UASG) centered at
41 bp from the edge of a positioned nucleosome, perturbing chromatin
structure and inducing transcription. DNA binding and chromatin
perturbation accompanying this interaction also occurred in the
presence of hydroxyurea, indicating that DNA replication is not
necessary for Gal4p-mediated nucleosome disruption. These data extend
previous studies, which demonstrated DNA replication-independent
chromatin remodeling, by showing that a single dimer of Gal4p, without
the benefit of cooperative interactions that occur at complex wild-type
promoters, is competent for invasion of a preestablished nucleosome.
When the UASG was localized at the nucleosomal pseudodyad,
relative occupancy by Gal4p, nucleosome disruption, and transcriptional
activation were substantially compromised. Therefore, despite the
increased nucleosome binding capability of Gal4p in cells, the precise
translational position of a factor binding site in one nucleosome in an
array can affect the ability of a transcriptional regulator to overcome
the repressive influence of chromatin.
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INTRODUCTION |
The interaction of transcription
factors with chromatin is an important issue in understanding gene
regulation. Transcriptional activation upon factor binding often
results in the disruption or displacement of one or more nucleosomes
within promoter regulatory regions (1, 53). However, the
mechanisms by which transcriptional regulators gain access to their
target DNA sequences in chromatin remain unclear. Biochemical studies
in vitro have disclosed that factor binding to nucleosome cores depends
on variables such as the type of factor, the number and location of
binding sites, and the status of the histone amino termini (reviewed in
reference 44). For example, Gal4p derivatives (e.g.,
Gal4-AH) can bind to reconstituted nucleosomes containing single or
multiple GAL4 binding sites to form a ternary complex (15, 68, 72,
78). Binding to multiple nucleosomal GAL4 binding sites initiates
at the edge of nucleosome cores and also occurs when a single binding site is centered at 21 bp from the edge, whereas single sites centered
at 40 or 74 bp (at the pseudodyad) from the edge were not accessible in
unmodified nucleosomes (72). These data and other physical
studies (34, 41, 60, 75) indicate that the two helical turns
of DNA at each end of the nucleosome core are not associated as
strongly with the histone octamer as sequences more internal. Because
nucleosomes occur within arrays in cells, whether a similar structure
pertains in vivo and its potential relevance to gene activation are
highly controversial (19, 47) and have not been addressed.
A previous structural study suggested that Gal4p led to perturbation of
chromatin when UASG was localized near the pseudodyad of a
positioned nucleosome in yeast (40). The fractional
occupancy of UASG by Gal4p and the degree of chromatin
reconfiguration were not determined. The mechanism by which Gal4p
gained access to and perturbed this nucleosome is also currently
unknown. Finally, nucleosome positioning is known to occur by several
different mechanisms, making generalizations from study of a single
positioned nucleosome hazardous.
One model to explain how transcription factors achieve increased access
to their regulatory sequences in vivo suggests that removal of
histones, presumed to occur transiently during replication, provides a
window of opportunity that enables factor binding (6, 65,
74). For instance, it was reported that Gal4-VP16 can potentiate
transcription of a template assembled into chromatin in vitro if added
during, but not after, DNA replication (27). Likewise, DNA
replication in the presence of specific erythroid factors is also
required for transcriptional activation of nucleosome-repressed 
-globin templates in synthetic nuclei (4). On the other
hand, Gal4p derivatives can potentiate replication-independent
transcription in vitro from preassembled chromatin templates (11,
48). DNA replication is also not required for chromatin
reconfiguration of the rat tyrosine aminotransferase, mouse mammary
tumor virus, or yeast PHO5 promoters in vivo (2, 52,
57). As these promoters are complex, cooperative binding of
multiple upstream factors and the basal transcription machinery could
be directly responsible for relieving the requirement for DNA
replication in chromatin remodeling or could be indirectly responsible
through recruitment of a chromatin remodeling-modification activity
(8, 28). The absence of such an activity in protein extracts
employed for in vitro studies could affect conclusions regarding the
role of DNA replication in effecting factor access to chromatin in
vivo.
To gain insight into the mechanism by which transcription factor
binding and chromatin remodeling occur in vivo, we have employed a
simple minichromosome system in which the interaction of a single factor with a template having a well-defined chromatin structure can be
studied (40, 42, 61). We have monitored factor binding and
changes in chromatin structure in this system by a novel in vivo
footprinting strategy utilizing SssI DNA methyltransferase (MTase) (31). Insertion of a single GAL4 binding site at 41 bp from either edge of a positioned nucleosome, a location that is
refractory to binding in nucleosomes cores in vitro (72), and growth under activating conditions led to substantial binding of
Gal4p and perturbation of chromatin structure in both replicating and
nonreplicating cells. In contrast, placing UASG at the
nucleosomal pseudodyad significantly inhibited chromatin
disruption and transcriptional activation, as only partial occupancy
and nucleosome perturbation were seen, even under conditions of Gal4p
overexpression. These data demonstrate that the ability of endogenous
Gal4p to invade a positioned nucleosome in vivo does not derive from
transient disruption of DNA-histone contacts by the DNA replication
machinery and may involve chromatin modification or remodeling
activities. Despite a potential role of such activities in
Gal4p-mediated chromatin disruption and their abundance in cells, our
results also demonstrate that the precise translational positioning of a regulatory element within a single nucleosome contained in an array
influences factor accessibility. Thus, the influence of translational
positioning on factor accessibility is not unique to nucleosome cores
and appears to be a bona fide physical property of nucleosomes in vivo
which exerts biological consequences.
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MATERIALS AND METHODS |
Plasmid construction and yeast strains.
Yeast plasmid TALS4
was created by modifying pTALS (55) (containing TALS
inserted into the HindIII site of pUC19) by PCR site-directed mutagenesis (26) as described by Kladde et al. (31); five additional CG reporter sites were inserted, and
UASgl (5'-CGGTCCACTGTGTGCCG-3'), centered at map
unit (m.u.) 1415 within GAL3 sequences (3), was
eliminated by changing its sequence to 5'-CGtTCCACgaTcTGaCG-3'
(altered positions are indicated in lowercase). In the present
study, site-directed mutagenesis by PCR (26) was used to
change sequences of the parent pTALS4 (31) from m.u. 1400 to
1431, 1432 to 1463, and 1464 to 1495 to
5'-CGATCACCGGAAGACTCTCCTCCGCGAGCTCG-3' (the
near-consensus UASG is underlined) (22),
creating pTALS4-17L, pTALS4-17C, and pTALS4-17R, respectively. The
positions of the C residues in each CG on the lower strand relative to
the operator-distal or left edge of the nucleosome in this invariant
sequence in the resulting minichromosomes (see below) are as follows:
TALS4-17L, 27, 34, 49, 51, and 57 bp; TALS4-17C, 59, 66, 81, 83, and 89 bp; and TALS4-17R, 91, 98, 113, 115, and 121 bp. The accuracy of all base substitutions was verified by double-stranded DNA sequencing. TALS4 and its UASG-containing derivatives were excised from
pUC19 by digestion with HindIII and religated prior to
transformation by electroporation (54) into
Saccharomyces cerevisiae YPH500
L.19-2 (MAT
ade2-101ochre his3-200 leu2-1 trp1-63
ura3-52 lys2-1::LYS2-GAL1
promoter/SssI) and the isogenic MATa
strain, YPH499L.19-2 (31). Propagation of each
minichromosome as a monomeric species and the absence of genetic
rearrangements were verified by Southern blotting. Where indicated,
cells were also transformed with the 2µm-based shuttle vector pRS426
(13) or pRS426-GAL4, which comprises the 3.6-kbp
BamHI fragment from pG525 (33), encompassing the
entire GAL4 promoter and coding sequence, inserted into the
BamHI site of pRS426.
The corresponding series of -galactosidase reporter plasmids,
YCpTALS4 and its derivatives (see Fig. 6), were constructed by using
primers 5'-GCATCGCTGCAGGTCG-3' and
5'-GAAGATCTCGAGCGTTGCCTCATCATCAATGC-3' to PCR amplify the
region from m.u. 1347 to 1619 (see Fig. 1) in TALS4 and its versions
harboring UASG. The PCR product was digested with
PstI and XhoI, and the resulting 261-bp fragment was ligated into the same sites upstream of the minimal CYC1
promoter driving expression of lacIZ in YCpCSG1
(66). The cloned region in each construct was verified by
double-stranded DNA sequencing. YCp reporter plasmids were directly
transformed into isogenic strains YPH500L.19-2 and YPH499L.19-2,
which express wild-type levels of Gal4p.
Primer extension analysis of chromatin.
High-resolution
mapping of micrococcal nuclease (MNase) cleavage sites was performed as
described by Shimizu et al. (59) and modified by Weiss and
Simpson (76). Briefly, yeast nuclei, prepared from cells
grown in 1 liter of either 2% glucose or 2% galactose complete
synthetic medium (CSM) (Bio101, La Jolla, Calif.) lacking tryptophan
(to select for TALS4-17L) and uracil (to select for
pRS426-GAL4), were treated with various concentrations of MNase. Following termination of digestion, DNA was purified and MNase-cut sites were detected by multiple rounds of primer extension with 32P-end-labeled primer
5'-CTCAAGTCGTCAAGTAAAGATTTCGTGTTC-3' followed by
electrophoresis on a 6% polyacrylamide (acrylamide-bisacrylamide, 19:1)-50% urea gel buffered by an electrolyte gradient
(58). DNA was also isolated from nuclei, deproteinized, and
subsequently treated with MNase to determine cleavage preferences in
naked DNA.
Mobility shift assay and in vitro SssI
footprinting.
Primers 5'-GCATAAACACCATCAGCCTC-3' and
5'-CAGATATCAAAACTGTTGCATTATT-3' were used to PCR amplify a
242-bp probe (m.u. 1267 to 1508) from pTALS4-17L containing a single
UASG which was subsequently gel purified.
The Gal4p derivative Gal4-AH was purified from Escherichia
coli as described by Lin et al. (35). Serial dilutions
of the Gal4-AH stock (monomer concentration of 5 µM) were made in G4D buffer (100 mM KCl, 10 mM HEPES [pH 7.4], 10 µM ZnCl2,
0.2 mM phenylmethylsulfonyl fluoride). Dilutions of Gal4-AH (2 µl)
were added to duplicate 18-µl reaction mixtures containing the
following: 1× SW binding buffer (2.5 mM HEPES [pH 7.9], 0.05 mM
EDTA, 5% glycerol, 0.01 mg of bovine serum albumin per ml, 0.1 mM
dithiothreitol, 0.02 mM phenylmethylsulfonyl fluoride), 55.6 mM NaCl,
3.33 mM MgCl2, 5.6 ng of sheared calf thymus DNA per µl,
and 0.18 mM S-adenosylmethionine. In addition, reaction
mixtures contained 1 ng of either 32P-end-labeled (7,500 cpm; for mobility shift gel) or nonradioactive (for SssI
footprinting) probe (final concentration of 0.056 ng/µl).
The radioactive binding reaction mixtures were incubated at 30°C for
42 min and then electrophoresed on a mobility shift gel as described
previously (72). Following incubation for 40 min at 30°C,
2 µl of purified SssI (New England Biolabs), diluted from
a concentration of 2 U/µl to 0.5 U/µl with 1× SW binding buffer,
was added to each duplicate, nonradioactive sample. After an additional
2 min at 30°C, 10 µl of freshly made deamination denaturation
buffer (0.9 N NaOH [dissolved from solid], 25 mM EDTA, 0.2 mg of
sheared calf thymus DNA per ml) was added. Following 5 min at 98°C,
deamination was initiated by adding 200 µl of saturated sodium
metabisulfite, and the samples were subsequently processed as described
by Kladde et al. (31). The primers used to amplify from the
purified deaminated probe DNA were MK1b1T4 (31) and MK1b2T4
(5'-GAAAATTGTTGTATTATTGTGTTTTGTATTTT-3').
Cell culturing and in vivo SssI footprinting.
Yeast cells (10 ml) for Fig. 4 and 5 were grown with shaking at 30°C
in 2% galactose CSM lacking tryptophan (to select for TALS4 and
derivatives) and uracil (to select for pRS426 or
pRS426-GAL4) to an optical density at 600 nm
(OD600) of approximately 1. The cells were then
centrifuged, resuspended in fresh selective medium containing 2 to 4%
galactose, grown for an additional 16 h, and then treated to
identify modified cytosines on the lower DNA strand as described
previously (14, 20, 31). The primers used were MK1b1T4 and
MS6b2T4 (31) for TALS4, TALS4-17L, TALS4-17C, and TALS4-17R
and YCpb1T4 (5'-TATATATATCAACACTAAAATTCCTAACCATCC-3') and
YCpb2T4 (5'-TAATTTTTATTAAAGGGAATAAAAGTTGGG-3') for the YCp plasmids used for Fig. 7.
For experiments in which DNA replication was inhibited (see Fig. 9),
initially, to repress Gal4p and SssI expression (i.e., SssI is transcribed from the GAL1 promoter),
yeast cells (10 ml) were grown in 2% glucose CSM lacking tryptophan
and uracil until they reached stationary phase (OD600,
~4). The cells were then centrifuged, washed once with medium
containing 2% galactose, resuspended in 2% galactose selective medium
plus 200 mM hydroxyurea, and incubated with shaking for another 12 h at 30°C to achieve synthesis of Gal4p and SssI. In
vivo-methylated cytosines were identified as described previously
(14, 20, 31) by using primers MK1b1T4 and MS6b2T4
(31). Under the conditions employed, as reported previously
(23, 62), the cell number did not increase throughout the
time course of the experiment. In other studies, to ensure that cells
had stopped replicating far in advance of accumulating significant
SssI activity, we determined that a minimum of 8 to 10 h of incubation is required to accumulate detectable methylation
following a shift from glucose to galactose. An additional control was
performed in which logarithmically growing cells were first
synchronized in late G1 phase by addition of 6 µg of
-factor per ml. The cells were then released from the cell cycle
block by washing once with yeast extract-peptone-dextrose (YPD) medium (54) and preincubation in YPD for 30 min at 30°C.
Following preincubation in YPD, one-half of the cells were centrifuged
and resuspended in YPD containing 200 mM hydroxyurea. No budding was observed, even after 3 h at 30°C, indicating that hydroxyurea had completely inhibited DNA replication and arrested cells in early S
phase. The remaining cells, resuspended in YPD lacking hydroxyurea,
exhibited significant budding after 30 min.
-Galactosidase expression assays.
Cells for Fig. 6 were
grown at 30°C in CSM lacking leucine and containing 2% glucose or
2% galactose for 16 to 20 h to an OD600 of about 2 to
3 and were assayed for -galactosidase activity (39, 54),
which is expressed as 1,000 × A420/(OD600 × time [minutes] × volume [milliliters]), at 30°C. For Fig. 8, following growth in
200 ml of 2% glucose CSM lacking leucine to an OD600 of
0.2, cells were washed extensively with sterile, distilled water to
remove the glucose, resuspended in 200 ml of 2% galactose CSM lacking
leucine, and incubated at 30°C with shaking. Aliquots of cells (6 OD600 units, 10 to 20 ml) were removed at 1-h intervals following resuspension in galactose-containing medium and assayed for
-galactosidase activity.
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RESULTS |
Strategy for assessing Gal4p-mediated chromatin disruption in
living cells.
We have employed derivatives of a yeast
minichromosome, TALS4 (31), to study the interaction of
Gal4p within two different chromatin environments that are well
characterized. In -cells, we assessed the ability of Gal4p to
compete with histones within a nucleosome that is precisely positioned
by the Mcm1p-Mat2p complex (Fig. 1).
The assignment of a nucleosome and its position adjacent to the 2
operator in -cells is based on a considerable body of earlier
mapping studies utilizing MNase (21, 46, 55, 56, 59, 76),
MTases (Dam, Sau3A1, and SssI)
(29-31), and DNase I (21, 46, 56, 59). In sum,
these studies demonstrate that, abutting the 2 operator, a
nucleosome is positioned by Mcm1p-Mat2p over the promoters of
a-cell-specific genes (21, 56), the recombination
enhancer (76), minichromosomes (29, 55, 59), and
heterologous random sequence in a lacZ-containing reporter
plasmid (46). Nucleosome positioning over such disparate sequences reinforces the likelihood that the chromatin structures of
closely related minichromosomes would be the same. Indeed, protection
against MNase (29) (see Fig. 2) and MTases
(29-31) of an extended region next to the operator,
inferred to be a nucleosome, persists in -cells when the original
TALS plasmid (55) is modified by the introduction of MTase
target sites in the nucleosome (e.g., TALS4). Most conclusively,
mutations in the globular region of histone H4 which produce
Sin
phenotypes lead to increases in accessibility
adjacent to the 2 operator in TALS4 in -cells (73),
demonstrating that a nucleosome is responsible for the observed
protection. In a-cells, which do not synthesize Mat2p,
Mcm1p remains bound and nucleosomes are present adjacent to the 2
operator but are relatively disordered. The minichromosomes offer an
additional advantage in that the interaction of a single factor within
a well-defined chromatin context can be monitored and changes in
chromatin structure are unlikely to be due to transcription. This
derives from the fact that the minichromosomes lack a natural promoter
(i.e., even the promoter of the TRP1 selectable marker is
defective such that only a low percentage of the templates are
transcribed, which is sufficient to confer growth in medium lacking
tryptophan [16]).
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FIG. 1.
Chromatin structure of the TALS4 minichromosome and its
derivatives in -cells. The lower diagram shows the locations of
preferential cleavages by MNase (arrowheads), in m.u., as determined at
low resolution (10 to 20 bp) by indirect end labeling, and inferred
positions of nucleosomes (circles) in TALS minichromosomes
(55). The 2 operator is indicated by the open rectangle.
The upper diagram shows a to-scale enlargement of the region from m.u.
1347 to 1619 (left to right) from the minichromosome TALS4 and its
derivatives. This region was amplified by PCR and cloned in the reverse
orientation to construct the lacIZ reporter plasmids of Fig.
6 and 7. The position of nucleosome IV (ellipse) (m.u. 1375 to 1520),
determined by mapping of MNase cutting sites at high resolution
(59) (Fig. 2), and locations of target CG sites (arrows)
relative to the left edge (arbitrarily assigned position 1) of the
nucleosome are given. A 32-bp sequence (see Materials and Methods)
containing a near-consensus 17-bp binding site for Gal4p
(UASG; hatched bar) was inserted at three different
positions into TALS4 (no UASG) to create the three
UASG-containing plasmids, TALS4-17L, TALS4-17C, and
TALS4-17R. In -cells, nucleosome IV is precisely positioned next to
the 2 operator, incorporating UASG at three different
translational positions, centered 41 bp from the left (17L) and right
(17R) edges or at the center (17C; pseudodyad), of the nucleosome.
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In this study, a near-consensus 17-mer binding site for Gal4p was
introduced at three different translational positions within the region
occupied by nucleosome IV of TALS4 (Fig. 1). Plasmids TALS4-17C,
TALS4-17L, and TALS4-17R contain UASG at the pseudodyad (center) and 41 bp from the left and right edges of the nucleosome, respectively. Although Gal4p is apparently insensitive to the rotational orientation of its binding site on the surface of a nucleosome (68), we kept the rotational setting of
UASG virtually identical and 7 bp flanking each element
constant to permit an accurate test of the variable of translational
position.
In view of the above-described results regarding the dominance of
nucleosome positioning mediated by Mcm1p-Mat2p, it was unlikely that
insertion of a Gal4p binding site would simply destabilize the
nucleosome in -cells during growth in glucose, where Gal4p synthesis
is repressed and Gal4p does not bind nucleosomal DNA (40,
63). For example, TALS, which contains UASgl from the GAL3 promoter at the identical location as the
near-consensus site in TALS4-17L, positions a nucleosome in -cells
grown in glucose (55, 59, 63). To control for other minor
sequence differences between TALS and TALS4-17L that formally could
contribute to nucleosome destabilization, we analyzed the chromatin
structure of the region next to the 2 operator in TALS4-17L in cells
grown in glucose versus those grown in galactose (Fig.
2). Nuclei were isolated from cells and
digested with various concentrations of MNase, and nuclease cleavages
were mapped by primer extension (59). Relative to
deproteinized DNA (Fig. 2, lanes 1 to 4), chromatin in nuclei isolated
from cells grown in glucose (lanes 5 to 8) exhibited protection against
and enhancement of MNase cleavage at several sites, indicating the
presence of a positioned nucleosome flanked by histone-free linker
regions that are nuclease hypersensitive. Compared to when cells were
grown in glucose, in galactose (Fig. 2, lanes 9 to 12) the pattern of
MNase cleavage in TALS4-17L chromatin was very similar to that observed
for protein-free DNA, consistent with disruption of the nucleosome
concomitant with binding of activated Gal4p (40, 63). While
this high-resolution experiment yields information about the status of
DNA-histone contacts in cells grown in galactose, it yields no
indication of the degree of Gal4p binding.
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FIG. 2.
Primer extension analysis of MNase-cut sites in
TALS4-17L. Nuclei (lanes 5 to 12) and protein-free DNA (D) (lanes 1 to
4) from -cells containing TALS4-17L and pRS426-GAL4 grown
in glucose (Glu) (lanes 5 to 8) or galactose (Gal) (lanes 9 to 12) were
isolated and treated with increasing concentrations of MNase as
follows: 0.05, 0.25, 0.5, and 1 U/ml, respectively, in lanes 1 to 4; 0, 2.5, 5, and 10 U/ml in lanes 5 to 8; and 0, 1.25, 2.5, and 5 U/ml in
lanes 9 to 12. The higher concentrations used to digest cells from
glucose were to compensate for increased cell numbers. The dots mark
cleavage sites that are enhanced in glucose-grown cells, and the
bracket delineates those sites that are protected relative to control
DNA. MNase cleavages and the inferred nucleosome position (ellipse) are
as those observed in the propositus TALS plasmid by Shimizu et al.
(59). Loss of protection due to binding of activated Gal4p,
indicating disruption of the nucleosome, is observed when cells are
grown in galactose (40, 63).
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As a probe for chromatin structure in living cells, we utilized the
SssI DNA MTase, stably integrated as a single copy under GAL1 control (31). This sensitive strategy offers
the advantage over conventional methodologies that it can
simultaneously map both DNA-histone and -non-histone protein
interactions. In addition, as lengthy or harsh treatments (cell
permeabilization, preparation of nuclei, or DNA alkylation damage) are
not employed, it avoids potential rearrangement or loss of chromatin
constituents (as seen above for Gal4p), allowing an accurate assessment
of in vivo states. The MTase modifies accessible CG sites at a low
level without altering the growth characteristics of cells. Following rapid isolation of DNA from cells, 5-methylcytosines are identified (14, 20), and footprints are visualized by comparison of
modifications in chromatin with those in control, protein-free DNA,
methylated in vitro (31).
Nucleosome translational positioning affects Gal4p-mediated
chromatin reconfiguration in vivo.
We first analyzed the
interaction of the Gal4p derivative Gal4-AH by footprinting with
SssI in vitro to provide a benchmark for comparison with in
vivo experiments in chromatin (Fig. 3). A
242-bp fragment was amplified by PCR from TALS4-17L DNA (Fig. 1)
containing a single UASG. The mobility shift gel of this
fragment in the presence of increasing concentrations of Gal4-AH is
shown in Fig. 3A. In parallel, identical binding reactions, purified SssI enzyme was added to footprint the interaction of
Gal4-AH on protein-free DNA. As more Gal4-AH was added to the binding reactions, a footprint became apparent as evidenced by strong protection of cytosines (sites 34 and 49) within the major groove of
each half-site of UASG. In addition, adjacent to
UASG, there was protection at sites 27 and 51 and striking
enhancement of methylation (sites 57 and 59). Accessibility at other CG
sites was relatively unaffected by bound Gal4-AH. A change in the ratio of the modification at the hypermethylated cytosine to that at either
of those protected against methylation in each major groove of the
binding site could be detected at only 10% occupancy of the probe
(Fig. 3, lanes 3). This ratio increased dramatically as additional
Gal4-AH was included in the binding reaction. Thus, SssI
footprinting should be a sensitive and effective means for detecting
Gal4p binding in vivo. As each 17-bp UASG (17L, 17C, and
17R) is flanked by identical nearest-neighbor sequences (i.e., the
UASG was contained within a constant 32-bp sequence that
was inserted into the original TALS plasmid), the same characteristic protections and enhancements would be expected to occur upon occupancy of UASG in each plasmid.
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FIG. 3.
In vitro SssI footprinting of Gal4-AH bound
to a single site. (A) Mobility shift gel showing resolution of naked
DNA probe containing sequences from m.u. 1267 to 1508 of TALS4-17L
(DNA) from the complex of probe bound by Gal4-AH (Gal4-AH · DNA). The concentrations of Gal4-AH monomer added to each sample and
the resultant percent occupancies (quantified with a phosphorimager) of
the probe are as follows: lane 1, 0 nM; lane 2, 0.21 nM, 7.1%; lane 3, 0.42 nM, 10%; lane 4, 0.85 nM, 17%; lane 5, 1.7 nM, 59%; lane 6, 3.3 nM, 70%; lane 7, 6.6 nM, 90%; lane 8, 13.3 nM, 90%; and lane 9, 27.5 nM, 93%. (B) Duplicate binding reaction mixtures were incubated with
purified SssI DNA MTase, and selected reaction mixtures
(numerals indicated above gel correspond to samples in panel A) were
treated to identify methylated cytosines as described previously
(31). The signal intensity of a given band corresponds
directly to the amount of methylation at that cytosine.
UASG is indicated by the bar, and the location of each CG
site in bases from the left edge of the positioned nucleosome in
-cells is given to facilitate comparison to data in Fig. 4, 5, and
9. Protection against SssI of two CG sites, one in each
UASG half-site (sites 34 and 49), as well as sites just
outside UASG (sites 27 and 51), and an accompanying
enhancement of methylation (marked by dot) occur with increasing
concentrations of Gal4-AH. As the 17-bp UASG is contained
in the same 32-bp sequence within each of the plasmids, these sites can
be used to assess protections and enhancement of SssI
methylation upon Gal4p binding within chromatin in vivo. The
corresponding positions of CG sites from the left edge of nucleosome IV
within TALS4-17C and TALS4-17R can be obtained by adding 32 and 64 bp,
respectively, to each of the above locations.
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To analyze the chromatin structure of each multicopy minichromosome,
the TALS4 series of plasmids was transformed into
SssI-producing a- and -cells which also
overexpress Gal4p (Fig. 4). All samples
were cultured in galactose to induce synthesis of Gal4p and
SssI and subsequently processed to identify methylated
cytosine residues. TALS4, which lacks a UASG, was
methylated by SssI in -cells in the linker (bottom of
gel) between two positioned nucleosomes and the adjacent 20-bp DNA in
the nucleosome edge; methylation was inhibited substantially by
DNA-histone contacts nearer to the nucleosomal pseudodyad (Fig. 4,
compare lanes 2 and 3). This pattern of modification, as demonstrated
previously with SssI (31) as well as Dam
(29) and Sau3A1 (30) MTases, is
diagnostic of a positioned nucleosome. We have further verified by
indirect end labeling that nucleosome IV in TALS4 protects against
cleavage by MNase (data not shown). Levels of SssI
methylation in TALS4 in a-cells (Fig. 4, lane 1),
particularly at sites 59 to 107, were significantly greater than those
in -cell chromatin (lane 2) but less than those of naked DNA (lane
3), suggesting the presence of nucleosomes that are positioned less
precisely (31, 55, 59). This conclusion is supported further
by the reproducible decrease in modification at sites 7 to 
9, the
linker region in -cells, indicating that the central, inaccessible
region of some nucleosomes occupies these sites in a significant
fraction of a-cell minichromosomes.
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FIG. 4.
Remodeling of a positioned nucleosome by Gal4p binding
is dependent on the position of UASG. The chromatin
structures in living yeast cells grown in galactose of the four
different plasmids depicted in Fig. 1, TALS4 (lanes 1 to 3), TALS4-17L
(lanes 4 to 6), TALS4-17C (lanes 7 to 9), and TALS4-17R (lanes 10 to
12), were analyzed in the presence of pRS426-GAL4, which
overexpresses Gal4p from the wild-type GAL4 promoter.
Protein-free DNA for each plasmid (D) was methylated by SssI
in vitro to identify target CG sequences and site preferences of the
enzyme (lanes 3, 6, 9, and 12). For analysis of chromatin, DNA was
rapidly isolated from isogenic, SssI-expressing
a-cells (lanes 1, 4, 7, and 10) and -cells (lanes 2, 5, 8, and 11), and modified cytosines were identified as described
previously (31). The number of nucleotides that the C
residue in each target CG site is away from the operator-distal edge
(i.e., left edge) of the positioned nucleosome in TALS4 in -cells is
indicated. Where present, each UASG is marked by a bar at
the immediate right of the samples that were methylated in vitro (D),
and the position of the hypermethylated residue next to each binding
site is indicated by a dot.
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The patterns of SssI methylation in the region of nucleosome
IV were changed to different extents depending on the translational setting of UASG and the cell type (Fig. 4). There are four
possible means by which methylation at a particular CG site may be
altered in this experiment: protection due to bound Gal4p or histones and increases in methylation either directly due to Gal4p binding (i.e., hypermethylation adjacent to UASG as seen in Fig.
3B) or indirectly through disruption of DNA-histone interactions.
First, we consider changes in methylation at CG targets which are more remote from UASG and thus are not likely to be influenced
by bound Gal4p (Fig. 3B). In a-cells, where the nucleosomes
are less positioned than in -cells, substantial changes in
methylation were seen at several CG sites in TALS4-17L (sites 70 to
107), TALS4-17C (sites 30 to 49 and 107), and TALS4-17R (sites 30 to 83) relative to the control TALS4 plasmid lacking UASG
(Fig. 4, compare lanes 4, 7, and 10 to lane 1; for normalization of
methylation intensity, it is recommended to use site 7 because it is
localized within the accessible periphery of the nucleosome in
-cells and is distant from each UASG). Since Gal4p can
bind nucleosomes in vivo (40, 63) (Fig. 2) and
SssI detects differences in chromatin structure (e.g.,
between a- and -cells [Fig. 4, lanes 1 and 2]
[31]), we conclude that in a-cells,
overexpressed levels of Gal4p can efficiently gain access to
UASG 17L, 17C, and 17R and lead to disruption of chromatin.
Increased accessibility to SssI was particularly striking in
the region between the 2 operator and UASG, likely
reflecting exclusion of nucleosomes from this region with simultaneous
binding of Gal4p and Mcm1p (or Mcm1p-Mat2p in -cells [see
below]). Hence, assessment of methylation between the 2 operator
and UASG appears to provide the most informative estimate
of the degree of Gal4p-mediated chromatin disruption. The term
disruption is used only to refer to the apparent changes in DNA-histone
contacts sensed by SssI, and no mechanism regarding
displacement of histones in trans is implied.
Evaluation of the changes in methylation within and in the vicinity of
UASG in a-cells allows an estimate of
UASG occupancy. As the 17-bp UASG is embedded
within the same 32-nucleotide sequence in each construct, we can
compare the methylation in chromatin in each plasmid to that seen in
vitro in the presence of bound Gal4-AH (Fig. 3B). In contrast to the
increased accessibility of SssI to sites remote from Gal4p,
relative to protein-free DNA (Fig. 4, compare lanes 4 and 6, lanes 7 and 9, and lanes 10 and 12), there was clear protection against
SssI of the CG in each half-site of UASG (17L,
sites 34 and 49; 17C, sites 66 and 81; 17R, sites 98 and 113) and of
sites near the binding site (17L, sites 27 and 51; 17C, sites 59 and
83; 17R, sites 91 and 115). Although sites within each UASG
are strongly protected in the classical sense of a footprint (comparing
methylation in chromatin to that in protein-free DNA), by themselves
these data do not formally allow protection due to bound Gal4p versus
histones to be ascribed. Nonetheless, given that Gal4p is overexpressed
in this experiment and the significant disruption of DNA-histone interactions flanking each UASG, it is likely that most of
protection within the binding site can be attributed to Gal4p. In
addition, as in naked DNA occupied by Gal4-AH (Fig. 3B), the
hypermethylation near UASG was also evident in each
construct (Fig. 4) (17L, sites 57 and 59; 17C, site 89; 17R, site 121).
Since complete loss of nucleosomes would lead to the methylation levels
of naked DNA, it is probable that the pronounced hypermethylation
indicates substantial occupancy of UASG by Gal4p in
a-cells. In addition, we are confident that the local
enhancements in methylation are due to Gal4p binding because (i) they
are absent from the control (TALS4) which does not contain
UASG, (ii) they are seen when Gal4-AH is bound to DNA in
vitro (Fig. 3B), (iii) Gal4p can access nucleosomal DNA in vivo
(40, 63) (Fig. 2), and (iv) they vary with the level of
expression of Gal4p (i.e., endogenous versus overexpression) (see Fig.
5).
To determine the influence of translational location of
UASG in a nucleosome on Gal4p binding and chromatin
disruption, we analyzed SssI accessibility of the same
series of multicopy plasmids in -cells in the presence of Gal4p
overexpression. Relative to that of TALS4, increases in methylation
occurred in TALS4-17L, and to a lesser extent in TALS4-17R,
concomitant with protection of UASG and hypermethylation at
sites 57 to 59 and 121, respectively, suggesting that Gal4p binding
occurred at a site centered 41 bp from either edge of the nucleosome
and led to disruption (Fig. 4, compare lanes 5 and 11 to lane 2).
Again, as observed above in a-cell chromatin, the most
pronounced changes in methylation occurred between UASG and
the 2 operator. The absence of such sites in TALS4-17R might
contribute to the seemingly lesser disruption compared to that in
TALS4-17L; however, the similar extents of methylation of TALS4-17R in
a- and -cells suggests that Gal4p efficiently accessed
UASG 17R (Fig. 4, compare lanes 10 and 11). When
UASG was inserted at the pseudodyad of nucleosome IV (17C),
in -cell chromatin, methylation was increased relative to that with
TALS4 at positions 107, 93, 89, and 30 (Fig. 4, compare lanes 8 and 2),
suggesting that Gal4p was able to bind UASG 17C in the
presence of a high-copy-number expression plasmid. Modification at
these CG sites, however, was reproducibly weaker than that in
a-cell chromatin (Fig. 4, compare lanes 8 and 7), suggesting that the UASG 17C was less accessible to Gal4p than
UASG 17R or 17L. Reduced hypermethylation at position 89 adjacent to UASG 17C in -cells (Fig. 4, lane 8) relative
to that in a-cells (lane 7) also suggests less binding by
Gal4p in the former.
Relation of extent of chromatin disruption to UASG
fractional occupancy.
We wanted to investigate further the
relative efficacy with which Gal4p can access different positions in a
nucleosome (Fig. 5). It should be
possible to determine this under conditions in which Gal4p is more
limiting, in the absence of the Gal4p expression vector at endogenous
or wild-type levels of Gal4p (i.e., TALS4 and its derivatives are
multicopy minichromosomes). At this reduced level of Gal4p, in contrast
to when Gal4p was overexpressed (Fig. 4), methylation in TALS4-17C in
-cells was indistinguishable from that in TALS4 (Fig. 5A, compare
lanes 3 and 5 to lane 4; Fig. 5B, compare scans 10 and 11). Strikingly,
methylation in -cells of TALS4-17L at endogenous levels of Gal4p
expression (Fig. 5B, scan 12) was significantly more than that in TALS4
(scan 10) or TALS4-17C at both endogenous (scan 11) and overexpressed (scan 14) levels of the factor. Endogenous levels of Gal4p led to
substantial methylation throughout the region of nucleosome IV in both
TALS4-17C and TALS4-17L in a-cells (Fig. 5A, compare lanes 7 and 8 to lane 10; Fig. 5B, compare scans 2 and 3 to scan 1), indicating
that chromatin in a-cells is significantly more susceptible
to disruption than that in -cells. This susceptibility is also
evident in that wild-type levels of Gal4p expression led to more
disruption of a-cell chromatin than was observed in
TALS4-17C upon overexpression of Gal4p in -cells (Fig. 5A, compare
lanes 7 and 8 to lanes 1 and 2; Fig. 5B, compare scans 2 and 14).
Overexpression of Gal4p did not alter methylation in the nucleosome IV
region in either cell type when UASG was absent (i.e., in
TALS4) (Fig. 5A, compare lane 4 to lanes 3 and 5 and compare lane 9 to
lane 10; Fig. 5B, compare scans 4 and 1 and scans 13 and 10). Thus, the
SssI methylation assay suggests that the ability of
endogenous or overexpressed levels of Gal4p to outcompete histones for
DNA occupancy in vivo is affected by the translational position of
UASG in a nucleosome and the overall chromatin organization
of a region (i.e., a- versus -cells).
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FIG. 5.
Chromatin remodeling and Gal4p occupancy in
a- and -cells at endogenous and overexpressed levels of
Gal4p. (A) TALS4-17C (+UASG) and TALS4 (UASG)
were introduced into SssI+ a- and
-cells containing endogenous levels of Gal4p
(pRS426-GAL4) (i.e., transformed with pRS426 vector only)
or overexpressing Gal4p (+pRS426-GAL4) and probed with
SssI MTase by growing the cells in galactose. Positions of
CG sites were identified in protein-free DNA (D) as indicated in the
legend to Fig. 4. Endogenous levels of Gal4p in a-cells led
to marked disruption of chromatin in the region of nucleosome IV
(compare lanes 7 and 8 to lane 10), whereas no remodeling was visible
in -cells (compare lanes 3 and 5 to lane 4). Overexpression of Gal4p
increased disruption of nucleosome IV in -cells only slightly at
sites 107, 89, and 30 (compare lanes 1 and 2 to lanes 3 and 5). Note
that overexpression of Gal4p in cells harboring TALS4 (no
UASG) had no effect on chromatin structure (compare lanes 9 and 10). (B) Scans of in vivo SssI probing data.
Phosphorimager scans from an experiment completely independent from
that presented in panel A are shown, analyzing disruption of TALS4,
TALS4-17C, and TALS4-17L in a- and -cells grown in
galactose containing endogenous (transformed with pRS426 vector) (scans
1 to 3 and 10 to 12) or overexpressed (transformed with
pRS426-GAL4) (scans 4 to 6 and 13 to 15) levels of Gal4p.
UASG is indicated by filled bars, and the site that becomes
hypermethylated upon Gal4p binding is marked by dots. The CG within the
2 operator is located at the leftmost side of each scan, and the
linker is at the right. Perturbation of control a-cell
chromatin, seen as increases in methylation at many CG sites in the
region (except sites within UASG that are protected by
bound Gal4p in the presence of pRS426-GAL4), occurs equally
well in TALS4-17C and TALS4-17L (compare scans 2 and 3 to scan 1 and
compare scans 5 and 6 to scan 4). The precisely positioned nucleosome
in TALS4 and TALS4-17C in -cells is depicted as a solid ellipse.
Perturbation of this nucleosome in -cells in TALS4-17L (compare
scans 12 and 10 and compare scans 15 and 13) is indicated by the dashed
ellipse. Endogenous levels of Gal4p efficiently remodel
UASG-containing a-cell chromatin (compare scans
2 and 3 to scan 1) but disrupt chromatin in -cells only when
UASG is removed from the pseudodyad in TALS4-17L (i.e.,
lack of chromatin perturbation in scan 11 versus scan 10 but clear
disruption in scan 12 versus scan 10 or 11). Note that the level of
disruption in TALS4-17L at endogenous levels of Gal4p (scan 12) is
greater than that in TALS4-17C even when Gal4p is overexpressed (scan
14).
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Since the intensity of SssI hypermethylation adjacent to
each UASG is a sensitive indicator of Gal4p binding (Fig.
3B), this gave us a unique opportunity to relate the degree of
chromatin disruption to the fractional occupancy of UASG in
different chromatin contexts. First, in -cells, there was excellent
concordance between the severity of chromatin disruption and the
intensity of hypermethylation adjacent to UASG (Fig. 4 and
5; see Fig. 7). For example, hypermethylation at position 89 in
TALS4-17C increased with Gal4p overexpression and was UASG
dependent (Fig. 5A, compare band marked by dot in lanes 1 and 2 to that
in lanes 3 to 5; Fig. 5B, compare peak marked by dot in scan 14 to that
in scan 11). Similar findings were also seen at positions 57 and 59 abutting UASG 17L (Fig. 5B, compare scans 15 and 12). In
addition, in -cells at endogenous levels of Gal4p, while
hypermethylation was not detectable next to UASG 17C it was
apparent at UASG 17L (Fig. 5B, compare peak indicated by
dot in scans 11 and 12). Therefore, as the intensity of SssI hypermethylation in -cells appears to reflect accurately the degree
of UASG saturation and the extent of chromatin disruption, we conclude that accessibility of Gal4p at the pseudodyad of a positioned nucleosome is substantially restricted relative to that 41 bp from the nucleosome edge.
In contrast to the differences between hypermethylation at
UASG 17C and 17L in -cells, in a-cells the
hypermethylation was greater at each level of Gal4p expression and was
observed next to both UASs (Fig. 5A, compare band marked by dot in
lanes 3 and 5 to that in lanes 7 and 8 and compare lanes 1 and 2 to lane 6; Fig. 5B, compare peak indicated with dot in scans 2 and 11, 3 and 12, 5 and 14, and 6 and 15). Interestingly, protection of cytosines
in each binding site was not readily apparent at UASG 17C
and 17L in a-cells at endogenous levels of Gal4p and could
be detected only by quantitative scanning with a phosphorimager (Fig.
5A, compare lanes 7 and 8 to lane D; Fig. 5B, compare scans 2 and 8 and
scans 3 and 9), suggesting a low level of fractional occupancy by
Gal4p. It is intriguing that the apparently low occupancy at wild-type
levels of Gal4p is sufficient to lead to extensive chromatin
perturbation in a-cells (see Discussion). From the
comparison of the scans in Fig. 5B, binding and chromatin remodeling by
Gal4p were apparently most effective in the following order:
a-cell chromatin > 41 bp from nucleosome edge in -cell chromatin > nucleosome pseudodyad in -cell chromatin.
Translational positioning of UASG affects Gal4p-induced
gene expression.
The fragment encompassing the 2 operator and
region of nucleosome IV from TALS4 and each of its derivatives (Fig. 1)
was cloned upstream of the minimal CYC1 promoter driving
expression of lacIZ to generate the corresponding series of
single-copy YCp plasmids (Fig. 6). In
each plasmid, the 2 operator was intentionally placed at a
relatively far distance from the promoter in order to abrogate both
activation by Mcm1p (46) bound at the operator in
a-cells (i.e., in the absence of MAT2p) (Fig. 6, YCpTALS4 data) and repression of RNA polymerase II transcription by MAT2p in
-cells (51). In addition, this excludes all
CYC1 TATA elements, which are constitutively bound by
TATA-binding protein (12), from the positioned nucleosome.
Although the presence of a -galactosidase reporter was previously
shown not to interfere with the positioning of a nucleosome adjacent to
the 2 operator in -cells (46), we confirmed the
presence of the nucleosome in YCpTALS4 (no UASG) by
SssI (Fig. 7). In -cells,
YCpTALS4 exhibited protection against SssI and a defined
linker (compare scans 2 and 1), demonstrating the positioning of
nucleosomes adjacent to the operator in a predominant translational
setting. In addition, Gal4p occupancy and chromatin disruption required
the presence of UASG (Fig. 7, compare scans 2 and 3).
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FIG. 6.
The location of UASG in a positioned
nucleosome modulates transcriptional activation by Gal4p. The region
indicated in Fig. 1 encompassing the 2 operator and nucleosome
(ellipse) from TALS4 and its derivatives (UASG is indicated
by hatched bar) were subcloned upstream of lacIZ to create
the four indicated YCp constructs. Reporter plasmids were transformed
into cells expressing wild-type levels of Gal4p, and levels of
-galactosidase activity were determined after growth in medium
containing glucose (Glu) or galactose (Gal). The expression levels of
-galactosidase shown are the averages obtained from two different
yeast transformants from two representative, independent experiments
that included all of the samples. In addition, although the absolute
levels of expression varied slightly between experiments, the fold
inductions from glucose to galactose were very similar for YCpTALS4-17C
versus YCpTALS4-17L and YCpTALS4-17R in -cells for two independent
transformants in two additional experiments.
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FIG. 7.
A nucleosome is positioned next to the 2 operator in
the lacIZ reporter plasmids in -cells. Linear
phosphorimager scans of SssI methylation of YCpTALS4 (no
UASG) and YCpTALS4-17C (UASG is demarcated by
the filled bar) in SssI-expressing cells which express
endogenous levels of Gal4p are shown. The open bar indicates the 2
operator. Cells were grown in galactose and then processed to identify
methylated cytosines. For YCpTALS4 (scan 2), note the protection by the
nucleosome (solid ellipse) (compare scans 2 and 1). Insertion of
UASG (i.e., YCpTALS4-17C [scan 3]) leads to partial
disruption of the nucleosome (depicted by the dashed ellipse) in
-cells (compare scans 3 and 2). The increase in signal at the
hypermethylated site (marked by the dot) also indicates partial
occupancy of UASG by Gal4p.
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As shown in Fig. 6, compared to the low level of -galactosidase
activity of cells grown in glucose, transcription was stimulated in
galactose in all cells harboring a minichromosome containing UASG. This stimulation was clearly Gal4p and
UASG dependent, because galactose did not stimulate
transcription in cells harboring YCpTALS4 which lacks UASG.
In a-cells, Gal4p binding led to high levels of
-galactosidase expression from all three UASG-containing episomes. The increase in basal-level transcription in glucose may
result from minor synergism between Mcm1p, bound at the 2 operator,
and residual Gal4p binding at UASG. In contrast, when UASG was incorporated 41 bp from the edge of the positioned
nucleosome (YCpTALS4-17L and YCpTALS4-17R in -cells), Gal4p
stimulated -galactosidase expression in galactose to more than 120 U
of activity. In contrast, -cells harboring YCpTALS4-17C showed a
much lower level of transcription (26 U). In this and other
experiments, the inhibition of activated transcription exerted at the
nucleosome center was reproducibly four- to sixfold. The occurrence of
two transcriptional maxima (17L and 17R) flanking a minimum (17C)
strongly implies that repression of UASG at 17C in
-cells is due to restricted access of Gal4p mediated by a positioned
nucleosome. These data rule out the possibility that our results can be
attributed to different distances between UASG and either
the 2 operator or CYC1 promoter. The four- to sixfold
transcriptional repression is expected to be an underestimate of the
ability of a positioned nucleosome to exclude Gal4p, because the
presence of a functional promoter is known to increase fractional occupancy of a single Gal4p binding site in vivo (see Discussion) (70). Likewise, it is not known whether this repression of
Gal4p, which has a strong acidic activation domain, would be improved with a weaker activator.
As the data in Fig. 6 were obtained under conditions used for probing
with SssI, where expression of Gal4p was maximal, we hypothesized that reduced levels of Gal4p expression might lead to a
greater level of repression at 17C in -cells. Cells containing each
YCp construct were initially grown in glucose-containing medium to
repress synthesis of Gal4p. Following transfer to galactose, aliquots
of cells were removed and assayed for -galactosidase activity. At
each time assayed, the activity mediated by UASG 17L and
17R (removed from the nucleosomal dyad) was greater than that exerted
by UASG 17C located at the dyad (Fig.
8). The inset of Fig. 8 shows that the
relative activity of UASG 17C versus those of 17L and 17R
was 13.5-fold reduced immediately following the lag in induction (at
3 h) and sequentially diminished to ~5-fold as observed in Fig.
6 as levels of Gal4p expression increased. This result is consistent
with preferential binding of limiting levels of Gal4p to each off-dyad
site and subsequent occupancy at the dyad driven by mass action. In
conclusion, the functional data correlate well with the structural
results: Gal4p stimulated transcription more efficiently when
UASG was centered 41 bp from the edge of the positioned
nucleosome (-cells) or when the chromatin was not as precisely
organized (a-cells).
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FIG. 8.
Transcriptional activation of the lacIZ
reporter plasmids in -cells at limiting levels of Gal4p expression.
Cells containing YCpTALS4, YCpTALS4-17C (17C), YCpTALS4-17L (17L), and
YCpTALS4-17R (17R) were initially cultured in medium containing glucose
then transferred to galactose and assayed at 1-h intervals for
expression of -galactosidase. The activity of YCpTALS4 lacking
UASG remained below 0.1 Miller unit at all time points. The
inset shows the relative activities of 17L and 17R versus 17C at
several time points.
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Gal4p is capable of invading and reconfiguring a preexisting
nucleosome.
Considering that the UASG elements in
TALS4-17C and TALS4-17L differ in location by only 32 bp, it seemed
unlikely that transient removal of histones by DNA replication would
favor Gal4p binding in one minichromosome over the other. To
investigate the influence of replication on Gal4p binding, we checked
the methylation patterns of TALS4 and TALS4-17L when DNA replication
was inhibited by hydroxyurea. Under the experimental conditions
used, SssI was not expressed until DNA replication was
completely blocked (see Materials and Methods). As shown in Fig.
9, in the presence of hydroxyurea, protection of methylation by nucleosome IV was still evident in TALS4
chromatin in -cells (compare lanes 4 and 5 and lanes 4 and 6).
Significant increases in methylation near the pseudodyad as well as a
Gal4p footprint were observed in TALS4-17L chromatin in both cell types
(Fig. 9, compare lanes 1 and 4 and lanes 3 and 6). Therefore, without
prior removal of histones by DNA replication, Gal4p can directly access
UASG when located 41 bp from the edge of a preexisting
nucleosome and can effect destabilization or disruption.
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FIG. 9.
Gal4p binds 41 bp from the edge of a positioned
nucleosome and causes disruption in the absence of DNA replication.
Cells overexpressing Gal4p and harboring TALS4-17L (lanes 1-3;
UASG is indicated by the bar at right of lane 3) or TALS4
(lanes 4 to 6; no UASG) were initially grown in
glucose-containing medium to repress synthesis of SssI MTase
and Gal4p and were then transferred to galactose medium that also
contained hydroxyurea. Following the induction of Gal4p and
SssI expression, identification of lower-strand cytosines
methylated in chromatin in vivo was performed as described
(31). Positions of CG sites were identified in protein-free
DNA (lanes D) as indicated in the legend to Fig. 4. Note the increase
in methylation in chromatin of both cell types in the presence of
UASG (for -cells, compare lanes 1 and 4; for
a-cells, compare lanes 3 and 6), which is indicative of
chromatin remodeling.
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DISCUSSION |
We have utilized derivatives of TRP1ARS1 minichromosomes, which
have a well-characterized chromatin structure, to assess the structural
and functional consequences of introducing cis-acting elements at various locations in positioned nucleosomes (40, 42,
61, 63). In any such study in vivo, it is important to dissociate
structural changes which precede transcription from those which are
consequences of transcription. In our study, transcription should not
affect the structural results. While the TRP1 gene is
expressed, the level of expression from the 102 bp of the 5' flanking
sequence is 3 to 4% of that from the wild-type promoter (16). This absence of a natural promoter within these
minichromosomes permits study of the interaction of a single
transcription factor in chromatin with a greatly reduced, if not
eliminated, influence of the basal transcriptional machinery. In the
present study, we have introduced a single, near-consensus Gal4p
binding site, UASG, at various locations in the region
incorporated within a nucleosome positioned by Mcm1p-Mat2p, allowing
us to assess Gal4p accessibility in two different, cell-type-specific
chromatin environments (55, 59).
Location of UASG within a nucleosome affects occupancy
by Gal4p in vivo.
Evaluation of the SssI footprinting
and reporter expression studies leads us to conclude that, in vivo,
relative to binding at the pseudodyad, Gal4p disrupts a precisely
positioned nucleosome (in -cells) and activates transcription more
efficiently when UASG is centered 41 bp from a nucleosome
edge. Thus, as is the case for SssI MTase (Fig. 4 and 5)
(31), Dam MTase (29), and the DNA replication
machinery (61), the accessibility in vivo of Gal4p to DNA in
a positioned nucleosome is greater at translational positions removed
from the nucleosomal pseudodyad. In vitro, binding of the Gal4p
derivative Gal4-AH (72), elongation of transcription by
bacteriophage SP6 RNA polymerase (64), and digestion by
restriction endonucleases (49) also occur more readily
within the two helical turns entering and exiting a nucleosome, a
region where DNA-histone interactions are less stable than those at the
pseudodyad (34, 41, 60, 75). Accessibility to sites more
internal was compromised. While it is likely that the increased
repression further into the nucleosome is primarily due to more robust
DNA-histone interactions, it is also possible that Gal4p binding is
impaired by underwinding of the DNA helix and/or steric occlusion of
UASG by the neighboring gyres of DNA at the pseudodyad
(24, 25). Thus, our data provide evidence that the general
physical property of increased accessibility further from the
pseudodyad is retained in vivo and translates to functional
consequences in gene expression.
What is the mechanism by which Gal4p exhibits increased accessibility
at the periphery of nucleosomes? It is possible that transient
disruption of DNA-histone contacts near the edges of nucleosomes, a
"nucleosome breathing," exposes sites for protein binding.
According to one model (49, 50), disruption of DNA-histone contacts initiates at nucleosome termini and proceeds sequentially into
the nucleosome, leading to dissociation of the DNA helix from the
histone octamer surface. This dynamic structure of nucleosomes would
obviate the need for replication to precede factor binding and predicts
less occupancy of a target sequence when it is located at the
pseudodyad versus the nucleosome periphery, as we have observed. It
should be noted, however, that in these previous studies, the region of
relatively increased accessibility to restriction endonucleases in
nucleosome cores was confined to the terminal 20 to 25 bp of DNA. For
example, the rate of cleavage at a site 7 to 11 bp from the end of
nucleosome cores decreased at least 10-fold when the site was 43 to 52 bp from the end (49). In vitro, Gal4-AH also binds
preferentially 7 to 21 bp from the edge of nucleosome cores, but
binding was very poor when UASG was centered at 26 to 40 or
60 to 74 bp from the nucleosome core edge (the 14-bp uncertainty is due
to potential heterogeneity of the position of the reconstituted
nucleosome on the probe DNA [72]). Thus, these in
vitro studies demonstrate that, energetically, the cumulative disruption of DNA-histone contacts beyond approximately 25 bp into the
nucleosome is very unfavorable.
Accessibility of Gal4p to nucleosomes is enhanced in vivo relative
to in vitro.
In contrast to these in vitro results, in vivo we
have observed more occupancy of single Gal4p sites that were localized
33 to 49 bp internal (i.e., centered 41 bp from the edge) to a
positioned nucleosome than of one that was positioned at the
pseudodyad. Morse and colleagues have reported disruption of a
nucleosome in vivo in a TRP1ARS1 derivative containing UASG
near the pseudodyad (40) and in TALS (63), which
has a natural Gal4p binding site centered at 41 bp from the nucleosome
edge. A comparison of the degrees of chromatin disruption and relative
occupancies of these binding sites was not made. In any case, Gal4p has
the capability to access a nucleosome in vivo in a region that is quite
refractory to binding in nucleosome cores in vitro.
The enhanced ability of Gal4p to bind at sites more internal in the
nucleosome in vivo compared to in vitro may be due to a difference from
Gal4-AH, altered nucleosome structure (e.g., nucleosome cores versus
minichromosomes [19, 47]), an influence of DNA
replication, or the presence of nucleosome modification activities,
etc. In comparison to that of Gal4p in vivo, several observations make
it unlikely that the decreased capability of Gal4-AH to access
nucleosomes in vitro can be ascribed simply to a trivial deficiency.
First, since the DNA-binding and dimerization domains of Gal4p are
contained within amino acids 1 to 94, all GAL4 derivatives containing
these residues and wild-type Gal4p, purified from yeast as a Gal80p
complex, exhibit similar specific affinities for protein-free DNA
(10, 45, 68, 78). Second, activation domains are not
required for the binding of GAL4 derivatives to nucleosomes or for
displacement of histones after binding in vitro (45, 68),
making it doubtful that wild-type Gal4p would behave differently in
such assays. Third, as mentioned above, in addition to that of Gal4-AH,
the accessibility of several different proteins to nucleosome cores is
similarly restricted to nucleosome termini (29, 31, 49, 61, 64,
72). Interestingly, the enhanced ability of Gal4p to bind
nucleosomes in cells also does not appear to be solely due to
dissimilar nucleosome structures in vivo as opposed to in vitro; in
vivo accessibility of both the Dam and SssI MTases is also
restricted beyond 20 to 29 bp (i.e., sites at 
30 bp internal are
protected from methylation) into a positioned nucleosome in both
replicating (29, 31) (Fig. 4, lane 2; Fig. 5A, lane 4) and
nonreplicating (Fig. 9, lane 4) cells. These observations suggest that
the prokaryotic enzymatic probes, foreign to the environment of
chromatin in S. cerevisiae, reflect the state of
accessibility of a particular region, whereas Gal4p has additional
means for invading chromatin structure.
Disruption of chromatin architecture accompanying passage of the DNA
replication fork could provide a greatly enhanced opportunity for
activators to access regulatory sites, with the outcome being disruption of nucleosomes through the exclusion of the core histones (6, 74, 77). It has been suggested that this is a primary mechanism by which the majority of factors access their binding sites
in chromatin (65). On the other hand,
replication-independent activation of transcription by Gal4p
derivatives has been seen in vitro in templates preassembled into
chromatin by Drosophila or Xenopus oocyte
extracts (11, 48). While these experiments utilized arrays
of physiologically spaced nucleosomes (5), the arrays in the
population are heterogeneous in that they are not precisely positioned
with respect to the underlying sequence. Since ~40% of the DNA in
any given molecule would be accessible to an activator due to
localization of a single binding site in an internucleosomal linker or
in the periphery of a nucleosome, an accurate test of the effect of
nucleosome positioning in this system is precluded. This is a
conservative estimate of the percentage of accessible DNA, as multiple
factor binding sites (48) are usually employed and most
extracts contain ATP-dependent remodeling activities (48,
69) which may facilitate factor binding in chromatin.
In some instances, particularly at genes that rapidly respond to
environmental signals, chromatin reconfiguration and transcriptional activation in vivo are replication independent (2, 52, 57). It is possible that this replication-independent activation of these
genes is accomplished through cooperativity between the multiple bound
factors and/or the basal transcription machinery (18, 44, 70,
79). At the PHO5 promoter, for example, a low-affinity
binding site for Pho4p is located between two positioned nucleosomes,
one of which (nucleosome 2) contains additional sites for Pho4p and
Pho2p (17, 71). During induction in low-phosphate medium,
these factors bind their sites and reconfigure chromatin in the absence
of replication (57). Consistent with these studies, we find
that the trans-activator Gal4p is also able to effect chromatin disruption in nonreplicating cells (Fig. 9). In studying binding to a single UASG, in the absence of a known
promoter, our data extend previous observations indicating that a
single Gal4p dimer does not require DNA replication to facilitate
binding in chromatin. This suggests a two-step process in which,
following passage of the DNA replication fork, chromatin assembly
precedes Gal4p binding (and methylation by SssI) and
subsequently the activator invades and disrupts preformed nucleosomes.
Such a model is consistent with the ability of Gal4p derivatives to
form a ternary complex with nucleosomes containing a single binding
site in vitro (15, 72) and, furthermore, is in agreement
with investigations which have revealed that an active chromatin
configuration is not inherited directly but must be continually
reestablished following each successive round of replication
(36).
Binding of Gal4p to a preformed nucleosome in vivo is striking because
the affinity of the activator for a single site in reconstituted
chromatin is approximately 100-fold lower than that in DNA
(68). Our data obtained from a-cells expressing wild-type levels of Gal4p may give an indication of how the activator overcomes this impediment in vivo. In this situation, although only a
partial occupancy of UASG by Gal4p was observed, disruption of the chromatin structure was apparently fairly extensive (Fig. 5).
The simplest interpretation of this phenomenon is that transient binding of Gal4p leads to recruitment of a nucleosome
modification-remodeling activity which irreversibly disrupts
DNA-histone contacts. For example, the activation domain of Gal4p
interacts in vitro with Ada2p (38), which is part of a
larger yeast complex that contains the histone acetyltransferase, Gcn5p
(7, 8). A mechanism in which a recruited
nucleosome-remodeling complex is only transiently required to
reconfigure chromatin can also be envisaged (43). However,
despite the presence of numerous histone modification and nucleosome
disruption activities within the cell (28), moving UASG to the pseudodyad significantly inhibited disruption
and transcriptional activation.
In conclusion, our data suggest a role for nucleosome positioning in
modulating DNA occupancy of specific upstream activators in addition to
factors within the basal transcription machinery (32, 37,
79). Our results also indicate that DNA occupancy is a primary
element in effecting chromatin disruption and fine-tuning levels of
gene expression (9, 67, 70, 80). While we have not
determined the exact nature of the Gal4p-mediated nucleosome reconfiguration, our data clearly demonstrate that the activator substantially perturbs DNA-histone contacts in intact cells without requiring DNA replication. Furthermore, our demonstration that the
surface along a nucleosome is not uniformly repressive implicates another level of regulation in factor binding that can be utilized to
achieve appropriate physiological responses.
We are particularly grateful to Randy Morse for many valuable
discussions throughout the course of this study, for editing an earlier
version of the manuscript, and for communicating results before
publication. We also thank H.-G. Patterton, J. L. Workman, and
other members of the Simpson and Workman laboratories for many helpful
suggestions, Randy Morse for providing pRS426-GAL4, and Tom
Owen-Hughes and Jerry Workman for supplying the purified Gal4-AH.
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Abstract
Introduction
