(Received for publication, December 21, 1994; and in revised form, August 24, 1995)
From the
Metabolic reactivation (incubating spheroplasts with galactose and casamino acids) causes disruption of nucleosomes from the upstream regions of the yeast GAL1, GAL10, and GAL80 genes. The disruption is specific. It depends on the transcription activator Gal4; it only occurs in galactose-reactivated chromatin from galactose-grown cells; it only affects upstream region, gene-proximal nucleosomes. Due to this specificity and because some of the same regions have shown induction-dependent changes by in vivo analyses, we suggest that the nucleosome-disrupted structure produced by reactivation is the authentic chromatin structure for these regions under conditions of galactose-induced GAL1-10 and GAL80 expression. It is necessary to carry out a spheroplast reactivation treatment in order to observe this disrupted structure in nuclear chromatin because nucleosomes are redeposited onto these regions during the preliminary steps of nuclear isolation (cell harvest/spheroplast preparation) probably in response to the nonphysiological conditions associated with these steps. However, during the same isolation procedures in cells lacking Gal80 protein, there is no nucleosome deposition on these regions, and the in vivo disrupted structure remains present in the nuclear chromatin. Therefore, the nucleosome deposition process that operates in wild-type cells is dependent on Gal80 protein, defining another activity of this negative regulator.
GAL structural gene expression is tightly regulated at
the transcriptional level by carbon source(1) . GAL1-10, -7, and -2 are induced to
very high levels of expression in galactose, via the activator Gal4p, ()but are completely inactive in other carbon sources. The GAL4 gene and GAL80, the gene that encodes the GAL-specific negative regulator, are expressed in all carbon
sources (1) as might be expected of regulatory genes, but their
expression levels do vary with carbon source. For example, GAL80 expression occurs at a low basal level in glycerol but is induced
5-10-fold higher, via Gal4p, in galactose(2) .
This
gene family provides excellent opportunities to study the relationship
of chromosome structure to gene regulation. Such studies have shown,
for example, that under conditions of induced expression (galactose)
Gal4p strongly protects the major promoter element, the
UAS, on GAL1-10(3, 4, 5) and on GAL80(6) . Gal4p protection of the GAL1-10 UAS
is also strong in
glycerol(3, 4, 5) , even though the genes are
completely inactive. This reflects the distinctive
poised-for-expression status of the structural genes in this carbon
source(7) . Gal4p protection of the single UAS
on GAL80 is weaker in glycerol(6) , perhaps to minimize
interference with the basal promoter that drives GAL80 expression in glycerol(7) .
The UAS elements on GAL1-10 and GAL80 lie within
constitutively nonnucleosomal, chromatin hypersensitive
regions(4, 6, 8) . In the uninduced
expression state (glycerol, glucose), positioned nucleosomes are
located between these hypersensitive regions and the genes, in the
chromosomal copy (6, 8, 9, 10) as
well as in GAL-containing plasmids(10, 11) .
The GAL10 TATA, the GAL1 TATA/transcription start
site, and the GAL80 TATA/transcription start site are
contained in these nucleosomes. In vivo analysis detected
structural changes in two of the GAL1-10 intergenic
nucleosomal regions upon galactose-induction of expression(5) .
However, no such changes were detectable in nuclear chromatin isolated
from induced cells(9) . In this paper, we describe experiments
that allow us to detect intergenic nucleosome changes in nuclear
chromatin, thus reconciling the in vivo and nuclear results.
In addition, these experiments yield some unexpected insights on
nucleosome disruption/deposition processes taking place on gene control
regions.
Strain 21R yeast cells were grown as described previously (9) in glucose/glycerol/ethanol (D); galactose/glycerol/ethanol
(G); glycerol/ethanol (g). These media allow growth of any cells in any
medium, even 4 cells (disrupted GAL4) in
galactose, and thus allow the most controlled comparison of the various
strains. Yeast spheroplasts were made as described in (12) .
Metabolic reactivation of spheroplasts consists of adding a carbon
source to 2%, usually galactose in these studies, and casamino acids
(to 0.5%) to the spheroplasts during the last few minutes of the
25-min incubation with the cell wall lytic enzyme
Oxalyticase(12) . For our strains, the typical time exposed to
reactivation conditions is 10-12 min. If reactivation media are
added too early in the incubation with Oxalyticase, spheroplasting
efficiency is affected. If the total incubation with Oxalyticase is
allowed to proceed too long, spheroplasts begin to lyse. (
)The choice of typical conditions (10 min of reactivation,
25 min of total incubation with Oxalyticase) for this set of strains is
sufficient for producing striking effects on chromatin (see below)
while avoiding significant spheroplast lysis.
Nuclei were isolated
by method I(12) , and DNase I and MNase digestions were
performed on the nuclei as described therein. DNase I and MNase
cleavage sites were located by an indirect end label
approach(13, 14) . The GAL1-10 patterns
in Fig. 1, Fig. 2, and Fig. 5were obtained by
mapping from the essentially coincident TaqI (see Fig. 1) or EcoRI (see Fig. 2and Fig. 5)
sites, which lie within the coding sequence of GAL10, 170
bp downstream of the GAL10 transcription start site (cf. (9) ). The patterns in Fig. 4were
obtained by mapping from the EcoRI site within the coding
sequence of GAL1, which lies
1100 bp downstream from the GAL1 transcription start site (cf. (8) ). The
patterns in Fig. 6were obtained by mapping from the EcoRI site within the coding sequence of GAL80, which
lies
400 bp downstream of the GAL80 transcription start
site (cf. (6) ). DNA from the nuclear digests was
purified (cf. (9) ). Samples with similar and suitable
digestion extents were chosen by analysis of the bulk patterns on
mini-gels, cut with the appropriate restriction enzyme, and
electrophoresed on denaturing gels (DNase I nuclear footprints) or
native gels (MNase profiles) as described previously (9) . DNA
was electrophoretically transferred to DBM paper using a homemade
electroblot apparatus. For all hybridizations, small (50-115 bp)
probes abutting the mapping restriction sites were used so that probe
homology ends below the smallest DNA bands on any of the patterns shown
in this work. The probe was radiolabeled by random priming (15) so both strand patterns are detected in all profiles. The
DBM paper was washed and exposed to Kodak XAR film as described
previously(9) .
Figure 1:
DNase I digestion
patterns from the GAL1-10 intergenic region. DNase I
cleavage sites were mapped from the EcoRI site within GAL10 by indirect end label analysis on 4.7% polyacrylamide,
0.6% agarose, 7 M urea denaturing gels, as described under
``Materials and Methods.'' The tracks show from left to right: track 1, a naked DNA digest; track
2, a digest from glycerol/ethanol-grown wild-type cells; tracks 3 and 4, two digests (of differing digestion
extents) from wild-type galactose-grown cells; track 5, a
digest from 80 (disrupted GAL80) galactose-grown
cells; tracks 6 and 7, two digests (of differing
digestion extents) from wild-type galactose-grown cells in which
spheroplasts were reactivated with galactose (G
); track 8, a higher exposure
of the lower part of the naked DNA profile shown in track 1.
DNA sizes were determined by comparison to the mobilities of
pBR322/MspI marker fragments run on the gel (numbers to the right of track 8): 1, 622
nucleotides; 2, 527 nucleotides; 3, 404 nucleotides; 4, 307 nucleotides; 5, 242 nucleotides). The map to the left shows the DNA sequence organization on the GAL1-10 intergenic region: UAS
elements (brackets); the TATA boxes (T); GAL1 and GAL10 transcription start sites (wavy lines). The lower case t to the right of track 6 are
TATA-like motifs. The location of the hypersensitive region (HR) is shown to the left of track 2. Other
symbols are discussed in the text.
Figure 2:
MNase digestion patterns from the GAL1-10 intergenic region. MNase cleavage sites were
mapped from the EcoRI site within GAL10 by indirect
end label analysis on 2.8% polyacrylamide, 0.6% agarose nondenaturing
gels, as described under ``Materials and Methods.'' In A, the tracks show from left to right: track 1, a naked DNA digest; tracks 2 and 3,
digests from wild-type galactose-grown cells minus (track 2)
or plus (track 3) spheroplast reactivation with galactose (G); track 4, a digest from
4
/80
(disrupted GAL4/disrupted GAL80) cells grown in galactose and reactivated with
galactose. In B, the tracks show from left to right: track 1, a naked DNA digest; tracks
2-4, digests from wild-type, galactose-grown cells without (track 2) or with spheroplast reactivation with galactose for
3 min (track 3) or 9 min (track 4); track 5,
a digest from galactose-grown, galactose-reactivated 4
cells; tracks 6 and 7, digests from wild-type
glycerol/ethanol-grown (track 8) or galactose-grown (track
9) cells, both reactivated with galactose. DNA sizes were
determined by comparison to the mobilities of
X174/HaeIII marker fragments run on the gel (some of
which are shown to the right of track 4: b,
1078 bp; c, 872 bp; d, 603 bp; e, 310 bp; h, 234 bp). The UAS
, the major TATA boxes (T), and the GAL1 and 10 transcription start
sites (wavy lines) are shown to the left of track
1. The location of the hypersensitive region (HR) is
shown to the right of track 3 in A. Other
symbols are described in the text. Note that the intensity response in
these blots is size dependent(19) . This causes
-
region intensity to be generally diminished relative to the other
interband regions so that longer exposures are required to visualize
-
interband intensities in the reactivated
profiles.
Figure 5:
MNase
digestion patterns in chromatin from 80 cells. MNase
cleavage sites were mapped from the EcoRI site within GAL10 by indirect end label analysis on 2.8% polyacrylamide,
0.6% agarose nondenaturing gels, as described under ``Materials
and Methods.'' The tracks show from left to right: track 1, a naked DNA digest; tracks 2 and 3, digests from wild-type galactose-grown cells minus (track 2) or plus (track 3) spheroplast reactivation
with galactose; tracks 4 and 5, digests from 80
(disrupted for GAL80) galactose-grown cells plus (track 4) or minus (track 5) spheroplast reactivation
with galactose. DNA sizes were determined by comparison to the
mobilities of
X174/HaeIII marker fragments run on the
gel (some of which are shown to the right of track 5: b, 1078 bp; c, 872 bp; d, 603 bp; e, 310 bp; h, 234 bp). The UAS
, the major
TATA boxes (T), and the GAL1 and 10 transcription start sites (wavy lines) are shown to the left of track 1. Other symbols are described in the
text.
Figure 4:
MNase
digestive patterns on the GAL1 intragenic region. MNase
cleavage sites were mapped from the EcoRI site within GAL1, by indirect end label analysis as described under
``Materials and Methods.'' The tracks show from left to right: tracks 1 and 4, two different
exposures of a digest from 4 galactose-grown cells,
reactivated with galactose; tracks 2 and 5, two
different exposures of a digest from wild-type galactose-grown cells,
not reactivated; tracks 3 and 6, two different
exposures of a digest from wild-type galactose-grown cells, reactivated
with galactose; track 7, a naked DNA digest. The fainter
exposure in tracks 1-3 show the intergenic region
chromatin pattern more clearly. The alignment is the same in the two
sets of exposures. DNA sizes were determined by comparison to the
mobilities of
X174/HaeIII marker fragments run on the
gel (some of which are shown to the right of track 6: a, 1353 bp; b, 1078 bp; c, 872 bp; d, 603 bp; e, 310 bp). The UAS region, the major TATA
boxes (T), and the GAL1 transcription start site (wavy line) are shown to the right of track
6. The arrowheads to the left of track 4 denote the regular nucleosome cutting sites on this region in the
inactive state(8) . Other symbols are described in the
text.
Figure 6:
MNase
digestion patterns on the GAL80 upstream region. MNase
cleavage sites were mapped from the EcoRI site within GAL80 by indirect end label analysis, as discussed under
``Materials and Methods.'' The tracks show from left to right: 0, a naked DNA digest; tracks 1 and 3, digests from wild-type galactose-grown cells,
unreactivated; tracks 2, 5, and 8, digests
from wild-type galactose-grown cells, reactivated with galactose (G); track 4, a digest from
wild-type glucose-grown cells; track 6, a digest from
wild-type glycerol/ethanol-grown cells, reactivated with galactose; track 7, a digest from 4
(disrupted for GAL4) galactose-grown cells, reactivated with galactose. DNA
sizes were determined by comparison to the mobilities of
X174/HaeIII marker fragments run on the gel (some of
which are shown to the right of track 8: c,
872 bp; d, 603 bp; e, 310 bp; f, 281 bp).
The UAS region (U), the TATA box (T), and the GAL80 transcription start site (wavy line) are shown
to the left of track 1. The location of the
hypersensitive region (HR) is shown to the right of track 1. Other symbols are described in the
text.
As reported
previously(9) , the intergenic region nuclear chromatin
profiles from induced cells, which actively express the GAL1-10 genes, are basically the same as profiles from
cells in which GAL1-10 are inactive, whether the
inactivity is due to growth in a noninducing carbon source (Fig. 1, compare tracks 2 and 3) or to the
absence of the transcription activator Gal4p (Fig. 2A,
compare tracks 4 and 2). The single difference around
the GAL1 5` end in MNase profiles will be discussed below.
This basic similarity would suggest that the chromatin structure of the
intergenic region is insensitive to gene activity(9) . However, in vivo analysis (5) detected induction-dependent
structural changes within two nucleosomal portions of the intergenic
region ( to the right of Fig. 1, track 5, and Fig. 2A, track 3). Since nucleosome structure
on upstream regions may be an aspect of gene
control(16, 17) , we wanted to determine why these
nucleosomes do not seem to be sensitive to gene activity in our nuclear
chromatin. This issue was particularly perplexing because intergenic
nucleosome changes were observed in a previous nuclear chromatin
analysis(8) , which used a yeast strain of uncharacterized GAL genotype. The strains used in (9) (and below)
comprise an isogenic series with a well characterized GAL pedigree and are widely used in GAL analysis. Note that in vivo and nuclear chromatin approaches only differ for the
nucleosomal regions; the two approaches give similar results concerning
UAS
protection and the absence of induction-dependent
change in the nonnucleosomal hypersensitive region.
Including a spheroplast
reactivation step in the isolation of nuclei from galactose-induced
cells results in exposure of the protected chromatin regions to
DNase I cleavage (Fig. 1, tracks 6 and 7), and
of the
-
,
-
, and
-
interband regions to
MNase cleavage (Fig. 2A, track 3). The
exposure of DNA that is nucleosome-protected in unreactivated chromatin
indicates that reactivation causes a significant nucleosome structural
alteration, either unfolding or nucleosome loss. Reactivation also
causes a loss of the high intensity MNase ladder pattern, due to
reduced cleavage at sites
,
, and
(Fig. 2B, tracks 2-4 and 7).
All of the reactivation-induced changes depend on functional Gal4p (Fig. 2B, tracks 4 and 5) and can
only be triggered when cells are grown and spheroplasts are reactivated
in galactose. Even spheroplasts from glycerol-grown cells show no
evidence of nucleosome alterations (Fig. 2B, track
6), even if reactivated with galactose for up to 25 min (not
shown), a period of time that is longer than the time required for
galactose to induce full GAL1-10 expression in cells
growing in glycerol. Reactivating spheroplasts from induced cells with
nongalactose carbon sources also fails to produce any GAL1-10 chromatin changes (not shown). Thus, when isolated from
reactivated spheroplasts, nuclear chromatin demonstrates galactose- and GAL4-dependent change in the GAL1-10 intergenic
nucleosome regions.
We can exclude a number of artifactual
explanations for the DNA exposure produced by reactivation. Endogenous
nuclease activity is negligible in both types of nuclei (not shown) and
thus cannot explain the results. Spheroplast preparation can cause a
heat shock-like response, which can be prevented by azide treatment of
cells prior to harvest; reactivation effects are the same
with or without this treatment (not shown). Treatment with
cycloheximide just prior to harvest does not alter the reactivation
effect (not shown), so protein synthesis is apparently not required.
Most importantly, histone profiles of nuclei from reactivated or
unreactivated spheroplasts are very similar (Fig. 3, tracks
1 and 2). Thus, the reactivation-induced DNA exposure
does not reflect a proteolysis artifact.
Figure 3:
Histone profiles. Histones were extracted
from nuclei isolated from galactose-grown, galactose-reactivated (G) or unreactivated (G)
spheroplasts by methods in (12) . Chicken erythrocyte marker
histones (ch) are shown in track 3. Note that track 2 is more highly loaded than track 1, so all
protein bands, including the slight amount of possible degradation
below H2B, appear stronger in this track than in track
1.
Reactivation produces a strong MNase band at the GAL1 5`
end. This band has similar mobility to a band in the naked DNA profile
and is a doublet like the naked band (Fig. 2B, tracks 1, 3, and 7). It probably reflects
exposure of the DNA at the GAL1 5` end due to the
reactivation-induced loss of the -
nucleosome that covered
this site. The GAL1 5` end region is extensively protected in
unreactivated profiles from induced cells, over an even larger region
than the nucleosome protection seen in the inactive state (Fig. 2A, tracks 2 and 4). This
extensive protection is the one feature that differs between MNase
digests of inactive and induced (unreactivated) chromatin. It may
reflect the presence of stalled transcription-associated complexes near
the GAL1 5` end due to the nonphysiological conditions during
harvest/spheroplast pretreatment (below).
Reactivation produces no
detectable change in the UAS footprint or within the
hypersensitive region (Fig. 1, tracks 6 and 7)
and, except for the extreme 5` end, has no effect on the GAL1 intragenic chromatin pattern. As shown
previously(8, 10) , galactose-induction of expression
abolishes the very regular intragenic nucleosome pattern of inactive GAL1 (Fig. 4, track 4), resulting in more
heterogeneous nuclease cutting and new bands (
, Fig. 4, track 5). However, reactivated and unreactivated patterns from
galactose-induced cells are quite similar (Fig. 4, tracks 5 and 6). In these same profiles, the striking
reactivation-dependent changes on the GAL1-10 intergenic
region can be seen clearly (Fig. 4, tracks 2 and 3). Thus, reactivation only affects the intergenic
nucleosomes.
The chromatin regions affected by reactivation in
wild-type spheroplasts include most of the sites affected in vivo by the induction of GAL1-10 expression (, Fig. 1and Fig. 2). Because of this similarity and the
association of the reactivation changes with conditions specific to
induced GAL1-10 expression (Gal4p, galactose), we
suggest that the nucleosome-disrupted structure produced by
reactivation is the authentic and in vivo active chromatin
structure for the GAL1-10 intergenic region. Why then
are nucleosomes found on this region in unreactivated nuclear chromatin
from wild-type induced cells? The most likely explanation is that these
nucleosomes were deposited on the region during the 1-2 h of cell
harvest/spheroplast pretreatment, probably in response to the
nonphysiological (0 °C/starvation) conditions associated with these
steps. Reactivation then causes disruption of these deposited
nucleosomes, restoring the in vivo structure. On the other
hand, the nucleosome-disrupted structure is maintained through these
same steps when cells lack Gal80p. Thus, nucleosome deposition does not
occur on the intergenic regions during the isolation steps when Gal80p
is absent. Since the same isolation protocol is used for both wild-type
and 80
and they are isogenic strains, the absence of
nucleosome reprotection in 80
cells indicates that the
nucleosome deposition process that occurs in wild-type during isolation
depends on Gal80p, directly or indirectly. This explains why GAL80-dependent structural features were observed in nuclear
chromatin (9) but not in vivo(5) .
Figure 7:
Reactivation-induced changes in the GAL1-10 and GAL80 upstream regions. The
sequence organization of the GAL1-10 and GAL80 upstream regions are shown on the center lines. The transcription
start sites (arrows), the TATA (T) and UAS are located to scale on these lines. The location of upstream
nucleosomes whose presence is altered by spheroplast reactivation are
shown as stippled strips below the center lines, and labeled, A-D. The GAL1 and GAL80 start
sites lie within nucleosomes C and D, respectively.
Above the central lines, the MNase cutting sites (
,
,
,
,
) in unreactivated or inactive chromatin (i/u) are
shown by filled arrowheads. The GAL1-10 sequences exposed to DNase I (D) or MNase (M) by
reactivation are located by regions of + separated by dashes. The specific GAL80 sequences exposed to MNase
by reactivation are located by +, the sequences which become more
protected by
.
Reactivation also results in some protection within
the hypersensitive region, near the GAL80 UAS (Fig. 6, tracks 2, 5, and 8).
This protection is GAL4-dependent (Fig. 6, tracks 7 and 8), but whether it is due to Gal4p or to other
elements of the transcription apparatus is not clear. Gal4p protects
the GAL80 UAS
strongly, with or without
reactivation, in chromatin from induced cells, ( (6) and data
not shown).
A chromatin response to spheroplast treatments is not limited to GAL genes nor to gene activation. Incubation of spheroplasts with phosphate results in specific nucleosome deposition on the PHO5 promoter region, assayed, as here, in nuclei isolated from treated spheroplasts(21) . The presence of these nucleosomes is associated with the inactive state of PH05, and phosphate is the normal inactivation signal. Thus, on PH05 as on the GAL genes, a metabolic signal can exert its gene-specific chromatin effect in spheroplasts. Such responsiveness should not be unexpected; spheroplasts are intact cells and on solid media can regenerate a cell wall and resume normal growth. Spheroplast treatment should thus be included in all studies of gene-specific yeast chromatin structure.
The GAL4-dependence of nucleosome disruption and GAL80-dependence of nucleosome deposition could reflect the direct action of these factors or indirect action mediated via other factors. For example, Gal4p might act via Swi/Snf(26) , which have been implicated in the disruption of TATA-bound nucleosomes in vitro(27) . Concerning the nucleosome target, it has been observed that removal of histone H4 N-terminal tails results in a significant reduction in the in vivo level of GAL1-induced expression(28) . This reduction might reflect involvement of the H4 tails in mediating the Gal4p-dependent nucleosome disruption associated with induction. Conversely, removal of histone H3 N-terminal tails results in an increased level of GAL1 induced expression(29) , roughly similar to that produced by removal of Gal80p function(22) . Perhaps H3 tails mediate Gal80p-dependent nucleosome deposition and loss of these tails (or of Gal80p) hinders this process, thereby affecting expression.
Nucleosome loss on upstream regions in association with gene
expression is common on yeast and other eukaryotic
genes(16, 17) . On many genes, the nucleosomes are
lost from gene-specific promoter elements. However, on GAL1-10 and GAL80, the specific promoter
elements, the UAS, lie in constitutively nonnucleosomal
regions(4, 6, 8) . The disrupted nucleosomes
come from the TATA/transcription start site regions, and their
disruption/deposition occurs without any effect on activator-UAS
interactions. Thus on GAL genes, the activator-specific
promoter interaction and disruptable nucleosome occupation probably
reflect distinct levels of control. This can allow cells to respond to
fluctuating carbon sources or growth conditions in stages. In
noninducing carbon sources like glycerol, the activator is bound to the
UAS
, but subsequent stages of expression, which involve
upstream nucleosome disruption, are not implemented. If galactose
becomes available, the inhibition at these subsequent stages can be
quickly released. Transcription-associated DNA melting occurs
20
bp downstream of the GAL1 and GAL10 TATA(30) , in regions that are exposed by the upstream
nucleosome disruption. In addition to making this DNA more accessible,
nucleosome disruption might also aid the DNA melting process by
liberating the constrained negative supercoiling residing in these
upstream nucleosomes. Negative supercoiling is generated behind a
transcribing polymerase(31) . Negative supercoiling favors
nucleosome formation and thus would favor the redeposition of
nucleosomes on these TATA/start site sequences after a polymerase
initiates transcription. Transcription initiation might therefore
involve a cycle of upstream nucleosome disruption/redisposition. Such a
cycle is consistent with the structural view presented above, of a
balanced competition of Gal4p-dependent nucleosome disruption and
Gal80p-dependent nucleosome deposition processes operating on the
upstream nucleosomes, and may play a facilitating role in the events
that take place at transcription initiation of GAL genes. We
also note that the need for metabolic reactivation in the disruption of GAL nucleosomes is consistent with the recent demonstration
that nucleosome disruption is energy requiring(32) .