From the Department of Biochemistry and Molecular
Biology and § Program in Genes and Development, The
University of Texas M. D. Anderson Cancer Center, Houston, Texas
77030
Received for publication, September 21, 2000
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ABSTRACT |
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Posttranslational acetylation of histones is an
important element of transcriptional regulation. The yeast Tup1p
repressor is one of only a few non-enzyme proteins known to interact
directly with the amino-terminal tail domains of histones H3 and H4
that are subject to acetylation. We demonstrated previously that Tup1p interacts poorly with more highly acetylated isoforms of these histones
in vitro. Here we show that two separate classes of
promoters repressed by Tup1p are associated with underacetylated
histones in vivo. This decreased histone acetylation is
dependent upon Tup1p and its partner Ssn6p and is localized to
sequences near the point of Tup1p-Ssn6p recruitment. Increased
acetylation of histones H3 and H4 is observed upon activation of these
genes, but this increase is not dependent on transcription per
se. Direct recruitment of Tup1p-Ssn6p complexes via fusion of
Tup1p to the lexA DNA binding domain is sufficient to confer repression
and induce decreased acetylation of H3 and H4 at a target promoter. Taken together, our results suggest that stable decreases in histone acetylation levels are directed and/or maintained by the Tup1p-Ssn6p repressor complex.
The yeast Tup1p-Ssn6p repressor complex provides a novel paradigm
for transcriptional repression and for the role of chromatin in this
process. TUP1 and SSN6 are required for
the repression of several diverse families of genes in yeast, including
cell type-specific genes (regulated by the The ability of Tup1p-Ssn6p to regulate many diverse genes indicates
that these complexes may interact with some common promoter component,
such as a basal transcription factor or a component of chromatin.
Indeed, two models have been proposed to explain how Tup1p-Ssn6p
complexes confer repression. The first is based on studies that
indicate that a number of other factors necessary for repression,
including Sin4p (7, 8), Sin3p/Rpd1p (9), Rpd3p (10),
Srb10p/Are1p/Ssn3p, Srb11p/Ssn8p (11-13), and Srb8p/Are2p (14), are
associated with subcomplexes within the RNA polymerase II holoenzyme
(13). Thus, Tup1p-Ssn6p may inhibit transcription through direct
interactions with one or more of these transcription factors.
Accordingly, partial repression (2-4-fold) can be achieved in
vitro in the presence of just the basal transcription machinery, Ssn6p, and Tup1p (15, 16).
An alternative model suggests that Tup1p-Ssn6p directs repression
through modulation of chromatin structure. Tup1p interacts directly
with the amino-terminal tail domains of histones H3 and H4 in
vitro (17), and mutations in these histone domains synergistically reduce repression of multiple classes of Tup1p-regulated genes in
vivo (17, 18). Moreover, the domain in Tup1p that is required for
interaction with the histones overlaps independently defined repression
domains (6). Thus, Tup1p-Ssn6p may affect repression through
interactions with components of chromatin that lead to decreased
accessibility of promoter regions.
These two models for Tup1p-Ssn6p repression may coincide. Complete
repression could require interactions with both the basal transcription
machinery and the histones. For example, Tup1p-Ssn6p complexes might
first halt transcription by altering the activity of the basal
apparatus and then maintain the repressed state through organization of chromatin.
One aspect of chromatin that is often modulated in active and repressed
chromatin domains is histone acetylation. Lysine residues in the
amino-terminal domains of all four core histones are subject to this
posttranslational modification. Increased histone acetylation is often
correlated with increased gene expression, whereas decreased acetylation is correlated with decreased transcription. The
identification of several transcriptional regulators as histone
acetylases or deacetylases in the last few years has provided a
molecular basis for this correlation (see Refs. 19-23 for recent reviews).
We have shown previously that Tup1p binding to the amino-terminal tails
of histones H3 and H4 in vitro is inhibited by high levels
of acetylation (17, 24). These findings suggest that Tup1p binding, and
thus repression, may be modulated by changes in histone acetylation
in vivo. If so, then genes regulated by Tup1p should be
associated with different levels of acetylated histones under
conditions of repression and activation. Consistent with this idea, we
report here that genes repressed by Tup1p are associated with
underacetylated forms of histones H3 and H4 in vivo. These
same genes are associated with more highly acetylated histones when
repression is relieved, and the genes are activated. The difference in
these acetylation states is not dependent upon transcription per
se but does require Tup1p. We have also found that deacetylation
of histones H3 and H4 accompanies reporter gene repression upon
targeted recruitment of Tup1p. These data indicate that Tup1p
repression is linked mechanistically to changes in histone acetylation.
Yeast Strains and Genetic Methods--
The strains FY23, FY24,
FY716, FY24
The MFA2.1 integrated lacZ reporter strain was created by
transformation of linearized plasmid pMFA2.1 into strain TY3
(MATa ura3-52 his3-
The MFA2.1 strain and its DNA Constructs--
The Tup1p-lexA expression vector was created
by insertion of the Tup1 coding sequence into plasmid pSH2.1 at the
SalI site. The Ssn6p-lexA expression vector was created by
insertion of a fragment of the SSN6 gene encoding the
carboxyl-terminal 335 amino acids containing the TPR repeat region into
plasmid pBTM116. The MFA2.1 plasmid was created by insertion of an
oligonucleotide containing the bipartite lexA operator consensus
sequence into the plasmid pSM38 (31) at the SphI site.
RNA Preparation and S1 Nuclease Analysis of SUC2
Transcripts--
Total yeast RNA was prepared as described previously
(27), and specific transcripts were quantitated using an S1 nuclease protection protocol.1 A
32P-labeled antisense SUC2 oligonucleotide
(corresponding to reverse complement of +90 to +145 of the
SUC2 open reading frame) was radioactively labeled and
hybridized overnight at 55 °C in a 50-µl reaction containing 50 µg of total yeast RNA, 38 mM HEPES, pH 7.0, 300 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100.
Each reaction contained 0.1 pmol of the anti-SUC2
oligonucleotide as well as 0.1 pmol of an antisense ACT1
oligonucleotide (corresponding to the reverse complement of +356 to
+392 of the ACT1 open reading frame) as an internal control.
After hybridization, S1 digestion was carried out for 30 min at
37 °C by adding 450 µl of S1 digest buffer to the hybridization
reaction. S1 digest buffer contains 50 units of S1 nuclease (Life
Technologies, Inc.), 330 mM NaCl, 66 mM sodium
acetate, pH 4.6, 2.2 mM ZnCl2, and 0.01%
Triton X-100. The reaction was halted by the addition of 5 µl of 0.5 M EDTA and 5 µl of 10 mg/ml yeast tRNA. Protected
fragments were ethanol-precipitated and separated on an 8% denaturing
acrylamide gel. The protected band corresponding to the SUC2
transcript was quantitated with a Storm 840 PhosphorImager
(Molecular Dynamics) and normalized relative to the levels of
ACT1 RNA.
Chromatin Immunoprecipitation--
Chromatin
immunoprecipitations were performed essentially as described previously
(32). Antibodies specific to H3 Ac9,14 were kindly provided by C. D. Allis (University of Virginia, Charlottesville, VA) (33).
Antibodies specific to H3 Ac9,18 and unacetylated H3 have been
described previously (17). Antibodies specific for unacetylated histone
H4 are described in Ref. 34. Antibodies recognizing acetylated histone
H4 were purchased from Upstate Biotechnology. Chromatin was sheared to
an average size of 150-300 base pairs by sonication. TALS chromatin
was reduced to an approximate average size of 180 base pairs by
digestion with 400 units of micrococcal nuclease (Worthington) for 5 min at 37 °C. Chromatin immunoprecipitation experiments were
performed at least twice, and representative experiments are depicted
in the figures.
Whole cell extracts were generated from 250 ml of log-phase culture.
Chromatin corresponding to 1 × 108 cell equivalents
of whole cell extract was combined in a final volume of 0.25 ml with
the following volumes of antisera: 20 µl of anti-H3; 2.5 µl of anti
H3 Ac9,14; 20 µl of anti-H3 Ac9,18; 15 µl of anti-H4; and 15 µl
of anti-acetylated H4 (Upstate Biotechnology). Antibody incubations
were carried out overnight at 4 °C with rotation. Immune complexes
were purified after a 1-h incubation (4 °C with rotation) with 60 µl of protein A-Sephadex beads (1:1 slurry; Pharmacia CL4B). Washes
and DNA purification were performed as described previously (32).
Quantitation of Immunoprecipitated DNA--
DNA purified from
immunoprecipitated complexes was slot-blotted onto nylon membrane
(GeneScreen Plus; DuPont) according to the manufacturer's instructions
and hybridized with gene-specific probes (35). Probes were generated by
polymerase chain reaction (PCR)2 from yeast strain
FY24T genomic DNA using primer sets specific for each gene being
analyzed (the following probes were generated by PCR amplification of
the indicated nucleotides: TALS A, nucleotides 1609 to 59 of TALS
plasmid; TALS B, 121-370; TALS C, 380-623; TALS D, 662-932; TALS E,
942-1126; TALS F, 1194-1431; ACT1 probe,
Quantitation of MFA2.1 DNA was carried out by PCR
essentially as described in Ref. 32. The promoter region of
MFA2.1 (nucleotides Acetylation of TALS Chromatin Is Markedly Lower under Conditions of
Tup1p-Ssn6p Repression--
We examined histone acetylation levels
associated with Tup1p-Ssn6p-regulated genes under conditions of
repression and activation using chromatin immunoprecipitation (32,
37-39). Cells were grown under conditions of repression or activation
specific for each gene and then fixed with formaldehyde to cross-link
proteins to DNA. The cross-linked chromatin was reduced to an average
length of 150-300 base pairs. The protein-DNA complexes were then
immunoprecipitated using antibodies recognizing specific acetylated
isoforms of histone H3, unacetylated H3, acetylated H4, or unacetylated
H4. Immunoprecipitated DNA was analyzed by slot blot hybridization
using gene-specific probes. This method allows quantitation of small
changes in histone acetylation at a specific locus that accompany
alterations in transcriptional competence. Our initial experiments
involved analysis of the acetylation state of the TALS plasmid, a yeast
episome containing the
Immunoprecipitations were performed using chromatin fragments isolated
from a cells (condition of activation),
To determine the extent of the region of altered H3 acetylation
relative to the location of the
Deacetylation of histone H4 has been observed concomitant with
transcriptional repression mediated via other yeast repressor complexes
(43, 44). Using chromatin immunoprecipitation, we examined the
acetylation state of histone H4 in TALS chromatin purified from either
a cells, SUC2 Exhibits Transcription-independent Changes in Acetylation of
Histone H3--
To further examine the link between Tup1p-Ssn6p
repression and decreased histone acetylation, we examined a non-cell
type-specific gene regulated by this corepressor. SUC2
encodes the enzyme invertase, which catalyzes the hydrolysis of sucrose
and is repressed when yeast are grown in high concentrations of glucose
(Fig. 2A, lanes 1 and 3; Refs. 45 and 46).
Glucose repression of SUC2 is mediated by a
sequence-specific DNA-binding protein, Mig1p, which recruits Tup1p and
Ssn6p to the SUC2 promoter (47). SUC2 is
expressed constitutively in strains lacking either TUP1 or
SSN6, even when strains are grown under repressing
conditions (Fig. 2A, lanes 5 and 6; Refs. 48-53).
Consistent with the results described above, decreased levels of H3
acetylation are associated with SUC2 repression. Chromatin immunoprecipitation experiments demonstrate that a fragment of the
SUC2 promoter adjacent to the Mig1p binding site is
associated with decreased acetylation of H3 (approximately 2-fold
lower) under repressing conditions (high glucose) as compared with
derepressing conditions (low glucose), coincident with activation of
SUC2 (Fig. 2B).
As observed in TALS chromatin, the changes in acetylation that are
observed at the SUC2 locus are localized to a region
proximal to the site of Tup1p-Ssn6p recruitment. Use of a probe
specific for a portion of the SUC2 coding sequence does not
reveal a change in acetylation of H3 in chromatin more distal to the
Mig1p binding site (Fig. 2C).
The changes in acetylation we observe might be due to an active
increase in H3 acetylation upon transcription of SUC2, to an
active decrease in acetylation upon recruitment of Tup1p-Ssn6p, or
both. To distinguish between these possibilities, we examined H3
acetylation in a strain (FY716) containing a nontranscribed allele of
SUC2, suc2-104, that contains a mutated TATA box
(Fig. 2A, lanes 2 and 4; Ref. 25). We found that
acetylation of H3 is still increased in the promoter region of this
allele under derepressing conditions (Fig. 2B,
To determine whether the decreased H3 acetylation observed upon
SUC2 repression in high glucose is dependent on Tup1p and Ssn6p, we examined acetylation of H3 associated with the
SUC2 promoter in strains lacking either TUP1 or
SSN6. We found increased acetylation of H3 in this region in
both of these mutant strains, even when strains were grown under
repressing, high glucose conditions (Fig. 2B). Again, this
increased acetylation is not caused by increased transcription because
acetylation of the nontranscribed suc2-104 allele is also
increased in the absence of Tup1p (data not shown).
Direct Recruitment of Ssn6p or Tup1p Is Sufficient to Induce
Repression of acell-specific Promoter and Decreased
Acetylation of H3 and H4--
The above-mentioned data raise the
possibility that changes in histone acetylation may be directed by the
corepressor complex. Alternatively, changes in acetylation might occur
before Tup1p-Ssn6p recruitment, perhaps directed by other proteins
required for repression, such as
Sequences corresponding to the lexA operator were inserted just
upstream of the
To test the ability of Ssn6p or Tup1p to direct changes in the
expression of MFA2.1, we expressed Ssn6p-lexA or Tup1p-lexA fusion proteins in a cells. As a control, the lexA DNA binding domain alone (lexA-DBD) was expressed separately in
these same cells. The lexA DNA binding domain could not repress
MFA2.1 under any conditions, as expected (Fig.
3B). However, Tup1p-lexA was
able to confer efficient repression of this gene even in the absence of
endogenous TUP1 or SSN6 (Fig. 3B). In
contrast, Ssn6p-lexA could only partially direct repression of
MFA2.1, and this repression required endogenous
TUP1 but not SSN6 (Fig. 3B). These
findings are consistent with previous reports by others, which indicate that repression by Ssn6p requires Tup1p, but Tup1p-mediated repression can occur independently of Ssn6p (6).
To determine whether direct recruitment of Tup1p is sufficient to
induce decreased acetylation of H3 and H4, we again performed chromatin
immunoprecipitations. Decreased acetylation of H3 and H4 associated
with MFA2.1 promoter sequences was observed upon recruitment
of Tup1p-lexA (Fig. 4, Histone acetylation is thought to alter gene expression by
altering DNA-histone interactions within single nucleosomes and by
altering nucleosome-nucleosome interactions involved in higher order
chromatin packing. Histone acetylation is likely to affect interactions
of non-histone regulatory proteins with chromatin as well, although
such effects are understudied at present. Tup1p is one of only a few
proteins determined to interact directly with the amino-terminal tails
of histones H3 and H4. Others include Sir3p and Sir4p, which are
thought to facilitate formation of heterochromatic-like structures
associated with gene silencing in yeast. Proteins like Tup1p and the
Sir proteins may be critical in establishing the architecture and
functions of particular chromatin domains.
Mutations in the amino-terminal regions of H3 or H4 that disrupt
Tup1p-histone interactions synergistically compromise Tup1p repression
(17, 24). These synergistic effects support an important, if redundant,
role for these histones in Tup1p functions in vivo.
Acetylation of H3 and H4 appears to antagonize Tup1p functions. Tup1p
binds most favorably to underacetylated forms of H3 and H4 in
vitro, and loss of class I histone deacetylase activities that
alter the acetylation state of these histones leads to loss of
repression in vivo (17, 24, 54). Moreover, we show
here that underacetylation of histones H3 and H4 accompanies Tup1p
repression of both an a cell-specific gene and
SUC2. We have observed a similar decrease in acetylation of
these histones associated with repression of the DNA damage-inducible
gene RNR3 by
Tup1p-Ssn6p.3 Thus, at least
three distinct classes of Tup1p-regulated genes exhibit a change in the
acetylation state of associated histones. Our finding that direct
recruitment of Tup1p leads to decreased histone acetylation of target
promoters is also consistent with our recent discovery that the
corepressor complex is associated with multiple histone deacetylase
activities in vivo (54).
The differential acetylation we observe associated with the
nontranscribed suc2-104 allele indicates that changes in
acetylation of histones associated with Tup1p-regulated genes do not
result from a change in transcription states but rather occur at an
upstream step. The increased acetylation we observe under activating
conditions might be directed by histone acetyltransferase
activities that are recruited by transactivators that bind to
the upstream activating sequences in the SUC2
promoter even in the absence of a functional TATA box. Even so, the
Tup1p-Ssn6p-dependent decrease in histone acetylation that
we observe under repressing conditions indicates that these activities
are either limited in function by Tup1p-Ssn6p or counteracted by
opposing histone deacetylase activities recruited by the corepressor
complex. The underacetylated state might be further stabilized by
Tup1p-histone interactions, which could limit reacetylation of the
histone tails. Histone deacetylase recruitment and protection of the
tails would account for our finding that changes in acetylation are
greatest proximal to the site of Tup1p-Ssn6p recruitment.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 and a1/
2
repressors), as well as genes responsive to different physiological
conditions including SUC2 (responsive to change in carbon
source), RNR3 (responsive to DNA damage), and
ANB1 (responsive to oxygen levels), among others (see Ref. 1
for review and Ref. 2). Neither Ssn6p nor Tup1p binds directly to DNA,
but these proteins are recruited to promoters through interactions with
sequence-specific DNA binding factors such as the
2 repressor (3, 4)
and Crt1p (2). Bypass of the DNA binding factor by fusion of Ssn6p or
Tup1p to a heterologous lexA DNA binding domain demonstrates that these proteins can directly orchestrate repression. Ssn6p-lexA fusions require Tup1p (5) to confer repression of an artificial promoter containing lexA operator sequences. However, Tup1p-lexA fusions can
confer repression in the absence of Ssn6p (6), suggesting that Tup1p is
the dominant repressor moiety in the complex.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
tup1, and FY24
ssn6 have been described previously (25,
26). Strains FY23 and FY24 carrying the TALS plasmid (FY23T and FY24T)
have been described previously (26). The FY716
tup1 strain was
created by transformation of strain FY716 with the plasmid tup1
406
cut with AvrII (26) followed by selection for uracil
auxotrophy. Disruption of the TUP1 gene was confirmed by
Southern blot. All strains were grown according to standard procedures
(27) in either rich media or selective media, synthetic dextrose media
supplemented with the appropriate amino acids. Derepression of the
SUC2 gene was carried out as described previously (25).
Yeast transformations were carried out by the method of Hill and
Griffiths (28).
200 leu2-
1
trp1-
6, which is isogenic to FY251) along with
pRS306 (29). The transformants were selected for uracil prototrophy,
and colony lift assays were performed to identify strains expressing
high levels of
-galactosidase (27). Colonies positive for lacZ
expression were then screened by Southern blot to confirm insertion of
the reporter gene at the MFA2 locus. MFA2.1 derivatives
containing deletions of either the TUP1 gene or the SSN6 gene were created by the method of Güldener
et al. (30).
tup1 or
ssn6 derivatives were used to create the
strains used to test the effects of artificial recruitment of Tup1p and
Ssn6p by transformation of the respective lexA-Tup1p and lexA-Ssn6p
expression vectors.
-Galactosidase Assays--
Yeast strains were grown to log
phase (between 2.5 and 5.0 × 107 cells/ml) in liquid
culture in synthetic dextrose media to measure expression of the MFA2.1
lacZ reporter gene. Assays were carried out by the method of Rose
(27).
289 to +66,
with +1 representing the start of translation of ACT1;
MFA2,
154 to +101; STE6,
144 to
18;
SUC2 A,
399 to
218; and SUC2 B, +76 to
+329). Gel-purified PCR products were radioactively labeled
using a modification of the random primed labeling technique (36)
substituting sequence-specific primers for random hexamers. DNA on slot
blots was quantitated with a Storm 840 PhosphorImager. Data presented
were normalized against input DNA.
286 to
68, primer sequences
are available upon request) adjacent to the lexA operator was amplified
from DNA derived from chromatin immunoprecipitation experiments. PCR
products were run on 7% acrylamide gels and stained with SYBR Green I
(Molecular Probes) at a dilution of 1:10,000 (in 1× Tris borate-EDTA
buffer, pH 7.5) for 30 min. Quantitation was carried out after scanning
the gel on a Storm 840 PhosphorImager. Multiple dilutions were
performed to ensure the linearity of the assay, and data presented were
normalized relative to input DNA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2/Mcm1 operator inserted into the TRP1/ARS1
plasmid upstream of the TRP1 gene. The
2/Mcm1
operator, which is derived from the promoter of the a
cell-specific STE6 gene, is sufficient to confer
Tup1p-Ssn6p-mediated repression to downstream genes in
cells
(40, 41). The chromatin structure of the TALS plasmid has been
extensively mapped in a and
cells (41), and its
structure and regulation are representative of natural a
cell-specific genes (42).
cells (condition
of repression), or
cells bearing a disruption of the
TUP1 gene (which abolishes repression in
cells). The
immunoprecipitated DNA was probed with specific regions of the TALS
plasmid (Fig. 1, probes A-F). Data
obtained with probe A indicate that sequences adjacent to the
2/Mcm1
operator are enriched in H3 acetylated at lysine 9 and/or 18 in
a cells relative to
cells (Fig. 1B). We
consistently observe that acetylation of histones associated with this
region of TALS is about three times higher in a cells than
in
cells (4.5 versus 1.5, Fig. 1, B and
C). The same trend and relative degree of acetylation change
were observed in immunoprecipitations using an antisera specific for H3
acetylated at lysine 9 and/or 14 (data not shown). Additionally,
deletion of TUP1 leads to hyperacetylation of this region of
TALS in
cells to an extent even greater than that observed in
wild-type a cells (increase in histone acetylation relative
to wild-type
cells = 7.3; 11 versus 1.5, Fig.
1B). A difference in acetylation of histone H3 between
a and
cells is not observed at the ACT1
locus, a gene not subject to regulation by Tup1p (data not shown). This
indicates that the changes we see at TALS do not simply reflect a
global, cell type-specific difference in acetylation levels.
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Fig. 1.
Decreased acetylation of histones H3 and H4
in TALS plasmid chromatin in cells.
A, schematic representation of the TALS plasmid. The letters
A-F indicate the DNA fragments (thin lines)
generated by PCR to probe specific regions of the plasmid in chromatin
immunoprecipitation experiments. Also indicated are the sites of the
2/MCM1 operator, the autonomous replication sequence
(ARS1), and the TRP1 gene. The filled
circles represent nucleosomes that are positioned in
cells.
B, top, slot blot data gathered from chromatin
immunoprecipitation experiments examining acetylation of histone H3 at
the region corresponding to TALS probe A. Immunoprecipitations were
carried out using chromatin isolated from strains FY23T (a),
FY24T (
), and FY24T
tup1 (
tup1), with
antisera specific for unacetylated histone H3 (H3) or
histone H3 acetylated at lysine 9 and/or 18 (acetyl H3).
Quantitation of the slot blot data is shown as a bar graph
below the blot. C, summary of data from chromatin
immunoprecipitation experiments examining the acetylation of histone H3
using probes specific for all regions of the TALS plasmid (probes A-F)
and chromatin from strain FY23 (a,
) or strain FY24 (
,
). The positions of the
2/MCM1 operator and the TRP1
gene relative to probes A-F are indicated. D, top, slot blot
data gathered from chromatin immunoprecipitation experiments examining
the acetylation state of histone H4 in TALS chromatin at the region
corresponding to probe A. Immunoprecipitations were carried out using
chromatin isolated from strains FY23T (a), FY24T (
), and
FY24T
tup1 (
tup1), with antisera specific
for an antibody recognizing isoforms of histone H4 acetylated at any
one of lysines 5, 8,12, or 16 (acetyl H4) or unacetylated
histone H4. Quantitation of the slot blot data is shown as a bar graph
below the blot.
2/Mcm1 operator (the point of
Tup1p-Ssn6p recruitment), immunoprecipitated DNA was probed with
sequences specific for other regions of the TALS plasmid (Fig.
1C). In
cells (Fig. 1C,
), the lowest
levels of H3 acetylation are associated with sequences adjacent to
either side of the
2/MCM1 operator (probes A, F, and E), whereas
increased acetylation is associated with more distal sequences (probes
B, C, and D). Increased acetylation is observed for all probes (with
the exception of probe E) in chromatin isolated from a
cells, with the highest levels occurring in the region of the
TRP1 gene (probes A-C). The largest changes in relative
acetylation between a and
cells occur at regions of TALS
directly adjacent to the
2/MCM1 operator (probes A and F, Fig.
1C). We have also observed similar decreases in acetylation
of histone H3 associated with sequences proximal to the
2/Mcm1
operator in the promoters of the endogenous STE6 and
MFA2 genes (data not shown). Thus,
Tup1p-Ssn6p-dependent repression of cell type-specific
genes is associated with localized decreases in histone H3 acetylation.
cells, or
cells containing a deletion of
the TUP1 gene. Similar to the loss of H3 acetylation observed above, we detected a reproducible 2-fold decrease in acetylation of histone H4 in TALS chromatin (region A) from
cells
as compared with a cells (Fig. 1D). Again, as
observed for H3, this change in acetylation was contingent upon the
TUP1 gene (Fig. 1D). These data suggest that
recruitment of Tup1p-Ssn6p in
cells leads to localized decreases in
both H3 and H4 acetylation levels.
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Fig. 2.
Decreased acetylation of SUC2
in glucose is dependent on TUP1.
A, S1 analysis of SUC2 RNA levels in strains FY24
(lanes 1 and 3), FY716 (lanes 2 and
4), FY24 tup1 (lane 5), or FY24
ssn6
(lane 6) grown under repressing conditions (2.0%
glucose, lanes 1, 2, 5, and 6) or switched
to derepressing conditions ((0.05% glucose) for 2.75 h
(lanes 3 and 4). The amount of SUC2
RNA detected relative to ACT1 RNA levels (ACT1
data are not shown) is shown below each lane. B,
summary of data from chromatin immunoprecipitation experiments
analyzing acetylation of histone H3 at the SUC2 promoter
proximal to the Mig1p binding site. Chromatin was isolated from strains
FY24 (WT), FY716 (
TATA),
FY24
tup1 (
tup1), and FY24
ssn6 (
ssn6)
grown under derepressing conditions (low glucose,
) or repressing
conditions (high glucose,
). C, summary of data from
chromatin immunoprecipitation experiments analyzing acetylation of
histone H3 at the SUC2 promoter distal to the Mig1p binding
site. Chromatin was isolated from strains FY24 (WT) and
FY716 (
TATA) grown under derepressing
conditions (low glucose,
) or repressing conditions (high glucose,
). Data presented in B and C were normalized
against input DNA (% Input).
TATA). Indeed, the levels of H3 acetylation observed
for this allele in both high and low glucose are nearly identical to
those observed for wild-type SUC2. Thus, the increased
acetylation of H3 observed at the SUC2 promoter in low
glucose is not dependent upon transcription per se.
2/Mcm1 or Mig1p. Others have shown
that these recruiting proteins can be bypassed by the fusion of Ssn6p
or Tup1p to the lexA DNA binding domain. Ssn6p-lexA and Tup1p-lexA
fusion proteins can repress artificial promoters containing multiple
lexA operators. Here we examined whether direct recruitment of such
fusion proteins is sufficient to direct repression of a more natural
a cell-specific promoter containing a single lexA
operator, and, if so, whether decreased acetylation of H3 and H4
accompanies this repression.
2/Mcm1 operator in the promoter region of the
endogenous MFA2 gene (see "Experimental Procedures").
This derivative (MFA2.1) is still susceptible to Tup1p-Ssn6p
repression in
cells but is not repressed in a cells, as
expected. Insertion of the lexA operator sequences therefore did
not disrupt the natural function or regulation of this promoter.
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Fig. 3.
Transcriptional repression mediated by
artificial recruitment of Tup1p or Ssn6p. A, schematic
of the MFA2.1 reporter gene located at the endogenous
MFA2 locus. The promoter region ( ), the MFA2
gene (
), and the lacZ gene (
) are shown. The positions
of the lexA operator, the
2/MCM1 operator, the TATA box, and the
start of translation (+1) are indicated. The region corresponding to
the PCR product amplified by specific primers for quantitation of
MFA2.1 sequences in chromatin immunoprecipitation experiments is also
depicted. Positions (in base pairs) are relative to the start of
translation. B, summary of results obtained from
-galactosidase assays examining the effect of targeting either Tup1p
(Tup1p-lexA) or Ssn6p (Ssn6p-lexA) to the
MFA2.1 reporter gene. Results are expressed as a percentage
of the
-galactosidase activity observed in MFA2.1
tup1 expressing
only the lexA DNA binding domain (lexA DBD). The genotype of
TUP1 and SSN6 is indicated as either wild-type
(+) or deleted (
). Assays were carried out in triplicate and
repeated. Data shown are representative of all assays, and assay
variability was
10%.
) relative to
recruitment of the lexA DNA binding domain alone (
), and this
decreased acetylation did not require endogenous Tup1p. Direct
recruitment of Ssn6p, however, was not sufficient to confer a stable
decrease in H3 or H4 acetylation at the MFA2.1 promoter,
consistent with the inefficient repression conferred by this fusion
protein (data not shown). Taken together, these data demonstrate that
direct recruitment of Tup1 is sufficient to induce decreased histone acetylation associated with repression of target promoters, furthering the link between repression and chromatin remodeling in
vivo.
View larger version (15K):
[in a new window]
Fig. 4.
Decreased acetylation of the
MFA2.1 gene upon targeting of Tup1p. Chart
summarizing results of chromatin immunoprecipitation assays examining
the acetylation of histone H3 (lysines 9 and 18) and histone H4 in a
strain in which the endogenous TUP1 gene has been deleted.
Results of PCR quantitation for a strain expressing only the lexA DNA
binding domain ( ) were compared with those from a strain expressing
a Tup1p-lexA fusion protein (
). Experiments were repeated, and the
results were averaged. All values are normalized to the amount of PCR
product generated using DNA isolated from a fraction of the chromatin
used in the immunoprecipitation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Diane Edmondson for providing the MFA2.1 reporter strain and the Tup1p-lexA expression vector, Yukio Mukai for the Ssn6p-lexA expression vector, Fred Winston for providing the FY716 strain containing the suc2-104 allele, Steve Elledge for donating additional strains, Bryan Turner for providing the anti-unacetylated histone H4 antibodies, and Dave Allis for contributing the anti-H3 Ac9,14 antibodies. We also thank members of the Roth laboratory for many lively discussions regarding these experiments.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Research Grant GM51189 (to S. Y. R.) and American Cancer Society Postdoctoral Fellowship PF4398 (to J. R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 117, Houston, TX 77030. Tel.: 713-794-4908; Fax: 713-790-0329; E-mail: syr@mdacc.tmc.edu.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008668200
1 D. Stillman, personal communication.
3 J. R. Bone, unpublished observations.
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ABBREVIATIONS |
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The abbreviation used is: PCR, polymerase chain reaction.
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