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Mol Cell Biol, May 1998, p. 2455-2461, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Chromatin Remodeling Mediated by Drosophila GAGA
Factor and ISWI Activates fushi tarazu Gene
Transcription In Vitro
Masahiro
Okada and
Susumu
Hirose*
Department of Genetics, The Graduate
University for Advanced Studies, and Department of Developmental
Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411, Japan
Received 30 December 1997/Returned for modification 6 February
1998/Accepted 24 February 1998
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ABSTRACT |
GAGA factor is known to remodel the chromatin structure in concert
with nucleosome-remodeling factor NURF in a Drosophila embryonic S150 extract. The promoter region of the Drosophila fushi tarazu (ftz) gene carries several binding sites
for GAGA factor. Both the GAGA factor-binding sites and GAGA factor per se are necessary for the proper expression of ftz in vivo.
We observed transcriptional activation of the ftz gene when
a preassembled chromatin template was incubated with GAGA factor and
the S150 extract. The chromatin structure within the ftz
promoter was specifically disrupted by incubation of the preassembled
chromatin with GAGA factor and the S150 extract. Both transcriptional
activation and chromatin disruption were blocked by an antiserum raised
against ISWI or by base substitutions in the GAGA factor-binding sites in the ftz promoter region. These results demonstrate that
GAGA factor- and ISWI-mediated disruption of the chromatin structure within the promoter region of ftz activates transcription
on the chromatin template.
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INTRODUCTION |
Chromatin structure appears to play
a key role in the regulation of gene expression in eukaryotes (12,
13, 34, 44). Since chromatin structure represses transcription by
preventing the binding of transcriptional regulators and transcription
machinery to their target sequences, some mechanisms such as chromatin
remodeling likely counteract this repressive effect (7, 17,
21). Indeed, transcriptional activation usually accompanies
remodeling of chromatin. For example, the promoter region of the yeast
PHO5 gene is covered by nucleosomes, but the ordered array
of nucleosomes disappears from the promoter upon induction of the gene
by phosphate starvation (1). Transactivation by certain
regulators requires recruitment of histone acetyltransferase through
coactivators (4, 25, 26, 46).
The relationship between chromatin structure and transcription was also
studied in vitro. The affinity of TATA-binding protein for the TATA
element is significantly reduced when the TATA element is covered by a
nucleosome (17). It was shown that the binding of TFIID to
the TATA element prior to chromatin assembly alleviates the
nucleosome-mediated repression of transcription (45). These observations suggested that remodeling of the chromatin structure around the promoter is crucial for transcriptional activation on the
chromatin template. Pazin et al. reported the NF-
B-mediated chromatin remodeling and transcriptional activation of the human immunodeficiency virus type 1 enhancer on a preassembled chromatin template (28). Their study highlighted the importance of
analyses on a natural promoter to understand the regulation observed in vivo.
The Drosophila fushi tarazu (ftz) gene
is expressed in a seven-striped manner during early embryogenesis
(6, 14). The promoter region of ftz carries
several binding sites for GAGA factor, which triggers chromatin
remodeling (39, 40). Topol et al. constructed a series of
transgenic flies carrying the ftz promoter-lacZ
fusion gene and found that deletion of the GAGA factor-binding sites in
the ftz promoter markedly reduced the reporter gene
expression (37). Furthermore, the striped expression of
ftz was abolished by a mutation of the
Trithorax-like gene, which encodes GAGA factor (3,
11). These observations suggested that GAGA factor-mediated
chromatin remodeling is required for the proper expression of
ftz in vivo.
To investigate the role of chromatin remodeling in transcriptional
activation directly, we have studied GAGA factor-mediated changes in
the transcriptional and structural properties of the ftz
chromatin template in vitro. Here we report that GAGA factor-mediated disruption of the chromatin structure within the promoter region of
ftz leads to transcriptional activation from the
preassembled chromatin template. This finding helps explain how the
inhibitory effect of chromatin is overcome upon induction on a natural
promoter.
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MATERIALS AND METHODS |
Preparation of proteins.
GAGA factor was expressed in
Escherichia coli by using expression vector pAR-GAGA
(32) and purified as previously described (41).
Core histones were purified from HeLa cell nuclei by the procedure of
Simon and Felsenfeld (31). Glutathione
S-transferase (GST) or GST-ISWI peptide was bacterially
expressed and purified with a glutathione Sepharose column (Pharmacia).
GST-ISWI peptide contains the N-terminal 100 amino acids of ISWI which
lack the ATPase domain.
Template DNA.
Plasmid pE(Hu)5-N carrying the ftz
promoter region from 
617 to +124 (23) and plasmid pFb205
harboring the promoter region from 860 to +728 of the
fibroin gene of Bombyx mori (23) were used as templates. To construct the templates carrying mutations in the
GAGA factor-binding sites, one, two, three, or all of the GAGAG
sequences in the ftz promoter region (GAGAG at 360 and CTCTC at 348, 158, and 46) were replaced by site-directed
mutagenesis (20) with the sequences GCGCG, CGCGC, GGATC, and
AATTC, respectively.
Reconstitution of chromatin on a ftz plasmid.
Chromatin was assembled by a salt dialysis method (33) with
a slight modification. Plasmids pE(Hu)5-N (2.5 µg) and pFb205 (2.5 µg) and purified core histones (4.5 µg) were mixed in a 50-µl reaction mixture containing 10 mM Tris-HCl (pH 7.2), 0.1 mM EDTA, 2 M
NaCl, and 10 mM 2-mercaptoethanol. The mixture was dialyzed for 90 min
each at room temperature against buffer A (10 mM Tris-HCl [pH 7.2],
0.1 mM EDTA, 0.8 M NaCl, 10 mM 2-mercaptoethanol), buffer B (10 mM
Tris-HCl [pH 7.2], 0.1 mM EDTA, 0.15 M NaCl), and then buffer C (20 mM HEPES-KOH [pH 7.9], 0.1 mM EDTA).
In vitro transcription.
Preassembled chromatin or naked
template DNA (100 ng each of ftz and fibroin
plasmid DNA) was incubated at 26°C for 2 h with various amounts
of GAGA factor and 1.5 µl of the S150 extract in 10 µl of a
solution containing 10 mM HEPES-KOH (pH 7.6), 0.5 mM EGTA, 60 mM KCl, 4 mM MgCl2, 10% glycerol, 30 mM creatine phosphate, 3 mM
ATP, 1 mM dithiothreitol, 1-µg/ml creatine phosphokinase, and 100 ng
of 
X174 replicative-form I DNA. Half of the mixture or 100 ng of
untreated naked template DNA was then subjected to a transcription
reaction containing the B. mori posterior silk gland extract
as described previously (23). Accurately initiated transcripts from the ftz and fibroin promoters
were detected by a modified S1 nuclease assay (16).
Micrococcal nuclease assay.
Chromatin containing 20 ng of
ftz plasmid DNA and 180 ng of X174 replicative-form I DNA
was incubated with GAGA factor and the S150 extract as described for
transcription reactions. The mixture (10 µl) was then treated with
2.5 U of micrococcal nuclease in 60 µl of a solution containing 50 mM
Tris-HCl (pH 8.6), 50 mM KCl, 3 mM CaCl2, and 10%
glycerol. After 2, 5, and 20 min at 26°C, a 20-µl aliquot was
removed and the reaction was terminated by addition of 1.2 µl of
2.5% Sarkosyl-0.25 M EDTA. RNase digestion was followed by proteinase
K treatment. Digested DNA was electrophoresed on a 2% agarose gel and
stained with SYBR green I. DNA was transferred to a nylon membrane and
hybridized with an end-labeled oligonucleotide probe carrying the
ftz sequence from 362 to 328 (GAWT or GAmut), from 169
to 138 (site IV), or from 56 to 26 (TATA) or the vector sequence
(5'-cttcccttcctttctcgccacgttctccggc-3') 1.2 kb downstream of
the ftz transcription start site (distal). The substituted bases in GAmut were as described above.
Restriction enzyme assay.
Chromatin was incubated with GAGA
factor and S150 extract as described for the micrococcal nuclease
assay. The mixture (10 µl) was then treated with 0.5 U of
AvaII, 2 U of FspI, or 1 U of PstI at
26°C for 1 h. After successive treatments with RNase, proteinase
K, and phenol, DNA was recovered by ethanol precipitation and digested
to completion with KpnI or HindIII. Resulting
DNA fragments were resolved on a 2% agarose gel and detected on a Southern blot with a mixture of radioactive oligonucleotide probes GAWT
and site IV or the probe distal.
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RESULTS |
GAGA factor activates transcription on a ftz chromatin
template.
To test whether GAGA factor can activate transcription
from the preassembled ftz chromatin template, we performed
an in vitro transcription assay (Fig.
1A). We used a plasmid template carrying the ftz promoter sequence from 617 to +124 (nucleotides
are numbered relative to the transcription initiation site). This
promoter region is known as the zebra element that is sufficient for
the seven-striped expression of ftz (15). As a
control, a plasmid harboring the promoter region from 860 to +728 of
the fibroin gene of B. mori that does not contain
the GAGAG sequence was included in the reaction mixtures. Chromatin was
reconstituted from core histones, ftz, and
fibroin plasmid DNA by a salt dialysis method (33). When the reconstituted chromatin was subjected to
transcription reactions without preincubation with GAGA factor and the
S150 extract, transcription of both ftz and
fibroin was repressed completely (Fig. 1B, S/D).
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FIG. 1.
Transcription on the preassembled ftz
chromatin template is activated by preincubation with GAGA factor. (A)
Schematic diagram of a transcription experiment. The preassembled
chromatin template containing 200 ng of DNA was preincubated with or
without GAGA factor in the presence of S150 extract and ATP.
Subsequently, transcription was carried out by addition of the
posterior silk gland (psg) extract and the remaining three nucleotides
(XTP) to one-half of each preincubation mixture. (B) Transcription on
the ftz chromatin template. Chromatin was preincubated with
the indicated amounts of GAGA factor and then transcribed as described
above. The signal intensity of each transcript relative to that
obtained without GAGA factor is indicated at the bottom. Transcription
on the same amount of naked template DNA without preincubation with
GAGA factor is shown as a control. S/D shows transcription on the
chromatin template (100 ng of DNA) without preincubation with GAGA
factor and the S150 extract.
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To test the effect of GAGA factor on transcription of the chromatin
template, the reconstituted chromatin was first incubated
with GAGA
factor and a small amount of S150 extract from
Drosophila early embryos (
2) to allow remodeling and then tested for
transcription
activity. The S150 extract contains a
nucleosome-remodeling factor
(NURF) that acts with GAGA factor to
disrupt the ordered array
of nucleosomes near the GAGA factor-binding
sites (
41). Since
the S150 extract was highly inhibitory to
transcription in vitro
(data not shown), the amount of the extract was
adjusted to 1/10
of that required for reconstitution of the chromatin
structure
on the template DNA to minimize the carryover of the extract
to
transcription reactions. The transcripts from the
ftz and
fibroin promoters were analyzed by a modified S1 nuclease
assay (
16).
Transcription on the
ftz chromatin
template was activated up to
14-fold by preincubation with GAGA factor
and the S150 extract,
but it was not activated in the absence of GAGA
factor (Fig.
1B).
The transcriptional activation reached its maximum
level when
the chromatin template was preincubated with 25 ng of GAGA
factor.
Under these conditions, GAGA factor was present in 13-fold
molar
excess over the chromatin template that carries four GAGAG
sequences
within the
ftz promoter region. No activation
occurred in the
absence of the S150 extract or ATP in the preincubation
mixture
(data not shown). By contrast, transcription on the
fibroin chromatin
template was not activated by the
preincubation with GAGA factor
and the S150 extract (Fig.
1B).
GAGA factor does not activate transcription on a naked DNA
template.
GAGA factor has been reported to activate transcription
on naked DNA templates in crude extracts but not in transcription systems reconstituted from purified components (8, 19). GAGA factor-mediated transcriptional activation on a naked DNA template required the presence of a nonspecific DNA-binding protein, suggesting that GAGA factor functions as an antirepressor by preventing
nonspecific inhibitory proteins such as histone H1 from binding to DNA
(8, 19, 29). It is therefore possible that GAGA factor may
activate transcription by a similar mechanism in our transcription
assay. To test this possibility, we carried out experiments similar to those in Fig. 1, starting from the naked DNA template. Since the amount
of S150 extract in the preincubation reactions was 1 order of magnitude
lower than that required for full assembly of the chromatin structure
on the template, nucleosomes were barely detectable by supercoiling
assay after preincubation (data not shown). In contrast to the
preassembled chromatin, we observed little activation (up to 1.5-fold)
of ftz transcription after preincubation of the naked
template DNA with GAGA factor and the S150 extract (Fig. 2). This indicates that only trace levels
of activation may be caused by elimination of nonspecific DNA-binding
proteins in the presence of GAGA factor. These results also suggest
that the GAGA factor-mediated transcriptional activation occurs
specifically on the chromatin template.
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FIG. 2.
Transcription on the DNA template is not activated by
preincubation with GAGA factor. Naked template DNA (100 ng each of the
ftz and fibroin plasmids) was preincubated with
the indicated amounts of GAGA factor in the presence of S150 extract
and ATP. Half of each mixture was then used for transcription.
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GAGA factor disrupts the chromatin structure in the ftz
promoter region.
Next, we analyzed changes in the chromatin
structure of the ftz template triggered by GAGA factor.
After incubation of the chromatin template with GAGA factor in the
presence of a small amount of S150 extract, chromatin was subjected to
a micrococcal nuclease assay. After digestion with micrococcal
nuclease, DNA was purified and electrophoresed on an agarose gel and
then stained with SYBR green I dye. No change in the pattern of
nucleosome ladders was observed upon incubation with GAGA factor,
indicating that GAGA factor did not affect the bulk chromatin structure
(Fig. 3A, SYBR). The DNA in the gel was
then transferred to a nylon membrane, and successive Southern
hybridizations were carried out. We observed GAGA factor-dependent
smearing of nucleosome ladders when the Southern blot was probed with
an oligonucleotide carrying the sequence in the ftz promoter
(Fig. 3A, GAWT and TATA). The smearing of nucleosome ladders was not
detectable when we omitted the S150 extract (data not shown). It also
appeared that GAGA factor had little effect on the chromatin structure
1.2 kb downstream of the transcription start site (Fig. 3A, distal). These data indicate that the chromatin structure in the ftz
promoter was specifically disrupted by incubation with GAGA factor and the S150 extract.
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FIG. 3.
GAGA factor-mediated disruption of the chromatin
structure in the ftz promoter. Preassembled chromatin (200 ng of DNA) was incubated with 0 or 50 ng of GAGA factor in the presence
of S150 extract and ATP, and the chromatin structure was subsequently
analyzed. (A) Micrococcal nuclease (MNase) assay. After incubation with
GAGA factor, chromatin was digested with micrococcal nuclease for 2, 5, and 20 min. Deproteinized digestion products were resolved on a 2%
agarose gel and stained with SYBR green I fluorescent dye. Salt
dialysis showed the digestion pattern without incubation with GAGA
factor and S150 extract. DNA was transferred to a nylon membrane and
detected by successive Southern hybridizations using the
oligonucleotide probes indicated by the thick bars. Probe distal
corresponds to the vector sequence 1.2 kb downstream of the
transcription start site (position not shown). The filled ovals and the
rectangle represent the GAGAG sequences and TATA element, respectively.
The arrow shows the start site of ftz transcription. (B)
Restriction enzyme assay. After incubation with GAGA factor, chromatin
was treated with AvaII, FspI, or PstI.
The DNA was purified and digested to completion with KpnI or
HindIII. The probes used for Southern hybridizations are
indicated. Uncut represents DNA that was not digested with
AvaII, FspI, or PstI. The middle
regions of the gel lacking relevant hybridization signals are not
shown. Thick arrows represent the restriction sites. The other symbols
are the same as in panel A.
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To get independent evidence for GAGA factor-mediated disruption of
chromatin, we analyzed the accessibility of the
ftz promoter
region to restriction enzymes. After incubation of the chromatin
with
GAGA factor in the presence of a small amount of S150 extract,
AvaII,
FspI, or
PstI was added to the
reaction mixture and incubation
was continued. DNA was purified and
then cut to completion with
KpnI or
HindIII.
The digested products were detected on a Southern
blot. The recognition
sites for
AvaII (
9),
FspI (
90 and
317),
and
PstI (
267) in the
ftz promoter region were more
susceptible
to these enzymes when the chromatin template was incubated
in
the presence of GAGA factor than in its absence (Fig.
3B). However,
GAGA factor did not affect digestion at the
FspI site 2.5 kb
downstream
from the
ftz promoter region (Fig.
3B, bottom).
These results
confirm that specific disruption of the chromatin
structure in
the
ftz promoter region occurs in a GAGA
factor-dependent manner.
Transcription is not activated by alteration of nucleosome
spacing.
S150 extract contains an activity that adjusts the
nucleosome spacing from the tightly packed state to the regularly
spaced one observed in biological samples (2). By incubation
with S150 extract, the distance between the nucleosomes of the
chromatin template assembled by the salt dialysis method was changed
from 160 bp (Fig. 3A, salt dialysis) to 180 bp (Fig. 3A, SYBR) in the presence or absence of GAGA factor. However, the transcription of
ftz was not activated by incubation with S150 extract when GAGA factor was absent (Fig. 1B, lanes 2 and 3). These results showed
that the alteration of the nucleosome spacing was not sufficient for
transcriptional activation of the ftz chromatin template. The increase in nucleosome repeat length did not occur in the absence
of ATP (data not shown).
Anti-ISWI serum prevents both transcriptional activation and
chromatin remodeling.
Three chromatin-remodeling complexes termed
NURF, ACF, and CHRAC, all of which contain ISWI as a component, were
isolated from Drosophila embryonic extracts (18, 40,
41, 43). ISWI shares similarity with yeast SWI2/SNF2 and
Drosophila Brahma in its DNA-dependent ATPase domain but not
in the N-terminal region (10, 22, 36, 40). To test the
correlation between GAGA factor-mediated transcriptional activation and
chromatin remodeling, we added a polyclonal antiserum raised against
the N-terminal region of ISWI to the reaction mixtures. The antiserum,
but not the preimmune serum, abolished the transcriptional activation when added at the beginning of preincubation (Fig.
4, lanes 4 and 5). Prebinding of a
GST-ISWI peptide to the antibody restored the transcriptional
activation (Fig. 4, lane 9). The antiserum had no effect on the GAGA
factor-mediated transcriptional activation when it was added just
before transcription (Fig. 4, lanes 6 and 7). Transcription on the
naked template DNA was not influenced by addition of the antiserum
(data not shown). This finding indicates that ISWI is involved in the
GAGA factor-mediated transcriptional activation on the chromatin
template. The results also showed that the chromatin template becomes
competent for transcription during preincubation with GAGA factor and
the S150 extract and not after transcription has been initiated.
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FIG. 4.
Anti-ISWI serum prevents GAGA factor-mediated
transcriptional activation on the ftz chromatin template. A
polyclonal antiserum raised against the N-terminal region of ISWI or
preimmune serum (0.5 µl) was added at the beginning of (lanes 4 and
5) or after (lanes 6 and 7) preincubation, and then transcription was
carried out. The antiserum (0.5 µl) was mixed with 1.5 µg of
bacterially expressed GST-ISWI (lane 9) or GST (lane 8) and incubated
for 2 h on ice prior to addition to the preincubation mixture.
Samples in lanes 1 to 7 were electrophoresed on the same gel, while
those in lanes 8 and 9 were run on a different gel.
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Both restriction enzyme susceptibility and micrococcal nuclease assay
results showed that addition of ISWI antiserum (Fig.
5A, lanes 5, 6, 13, and 14, and B), but
not preimmune serum (Fig.
5A, lanes 3, 4, 11, and 12), to the
preincubation mixture abolished
the GAGA factor-mediated chromatin
remodeling. Addition of the
antiserum that had been incubated with the
GST-ISWI peptide had
no effect on the chromatin remodeling (Fig.
5A,
lanes 8 and 16).
These data indicate that the chromatin remodeling
mediated by
GAGA factor and ISWI is necessary for transcriptional
activation
on the
ftz chromatin template. Interestingly,
regularly spaced
nucleosomes were formed in the presence of ISWI
antiserum (Fig.
5B). This suggests that the ISWI antiserum blocked
transcriptional
activation on the chromatin template by inhibiting the
GAGA factor-mediated
chromatin remodeling but not by affecting the
nucleosome-spacing
activity.
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FIG. 5.
Anti-ISWI serum blocks GAGA factor-mediated chromatin
remodeling. (A) Restriction enzyme assay. Preassembled chromatin (200 ng of DNA) was incubated with 0 or 50 ng of GAGA factor, the S150
extract, and ATP in the presence (lanes 5 to 8 and 13 to 16) or absence
(lanes 1, 2, 9, and 10) of antiserum against ISWI (0.5 µl) or in the
presence of preimmune serum (lanes 3, 4, 11, and 12), and the chromatin
structure was subsequently analyzed by restriction enzyme assay as
described in the legend to Fig. 3B. The anti-ISWI serum was prebound
with GST-ISWI (lanes 8 and 16) or GST (lanes 7 and 15) as described in
the legend to Fig. 4. (B) Micrococcal nuclease (MNase) assay.
Preassembled chromatin (200 ng of DNA) incubated with 0 or 50 ng of
GAGA factor, the S150 extract, and ATP in the presence of anti-ISWI
serum (0.5 µl) was subjected to a micrococcal nuclease assay as
described in the legend to Fig. 3A.
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Both GAGA factor-mediated transcriptional activation and chromatin
remodeling require GAGA factor-binding sites.
To test whether
transcriptional activation and chromatin remodeling are dependent on
GAGA factor-binding sites, we designed a template in which all four of
the GAGAG sequences in the ftz promoter were replaced with
other sequences. Preassembled chromatin on the mutant template was
preincubated with GAGA factor and the S150 extract, and then
transcription was carried out. The mutations abolished the GAGA
factor-mediated transcriptional activation on the chromatin template.
In the same reactions, naked fibroin template DNA was
transcribed efficiently (Fig. 6, lanes 3 to 5). The mutations had no effect on transcription from the naked
template DNA (Fig. 6, lanes 1 and 2).
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FIG. 6.
Mutations in the GAGA factor-binding sites abolish
transcriptional activation on the chromatin template. The preassembled
chromatin template (200 ng of DNA) carrying base substitutions in four
GAGAG sequences was preincubated with the indicated amounts of GAGA
factor in the presence of S150 extract and ATP; subsequently, half of
each preincubation mixture was subjected to a transcription reaction.
As a control, naked fibroin plasmid DNA (50 ng) was added to
the transcription reaction mixtures (lanes 3 to 5). The wild-type (WT;
lane 1) or mutant (lane 2) naked template DNAs were also tested for
transcriptional activity without preincubation with GAGA factor.
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GAGA factor-mediated disruption of the chromatin structure in the
ftz promoter region was not observed on the mutant template
when either a micrococcal nuclease assay (Fig.
7A) or a restriction
enzyme assay (Fig.
7B) was used. When we compared transcription
and restriction enzyme
sensitivity among chromatin templates carrying
a mutation(s) in one to
four GAGAG sequences, the levels of both
transcriptional activation and
nuclease sensitivity decreased
in proportion to the number of altered
GAGAG sequences (data not
shown). These results showed that the GAGA
factor-binding sites
in the
ftz promoter are necessary for
both chromatin remodeling
and subsequent transcriptional activation.
The alteration of nucleosome
spacing occurred on the mutant template
lacking GAGAG sequences
(Fig.
7A). However, it did not lead to
transcriptional activation,
supporting our conclusion that the
formation of regularly spaced
nucleosomes is not sufficient for
transcriptional activation.
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FIG. 7.
Mutations in GAGA factor-binding sites prevent chromatin
remodeling. (A) Micrococcal nuclease (MNase) assay. Preassembled
chromatin (200 ng of DNA) carrying mutations in four GAGAG sequences
was incubated with 0 or 50 ng of GAGA factor in the presence of S150
extract and ATP and then subjected to a micrococcal nuclease assay as
described in the legend to Fig. 3A. (B) Restriction enzyme assay.
Preassembled chromatin carrying mutations was incubated with GAGA
factor and the S150 extract under conditions identical to those used
for panel A and subjected to a restriction enzyme assay.
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DISCUSSION |
In this study, we showed chromatin remodeling and transcriptional
activation on the preassembled ftz chromatin template. Both of these processes require GAGA factor, S150 extract, and intact GAGA
factor-binding sites on the template, and both can be inhibited by an
antiserum against ISWI. From these results, we conclude that the GAGA
factor- and ISWI-mediated chromatin remodeling activates transcription
on the preassembled ftz chromatin template.
Recent studies have suggested the existence of distinct multiprotein
complexes that can act by chromatin remodeling (5, 18, 41,
43). Four such complexes have been identified in Drosophila. The first contains brm, a Drosophila
homolog of yeast SWI2/SNF2 (9, 36). The second is NURF,
which consists of four proteins that include ISWI (40, 41).
The third and fourth are ACF and CHRAC, which also contain ISWI
(18, 43). While NURF perturbs the regularly spaced
nucleosome, ACF and CHRAC serve as an ATP-dependent nucleosome assembly
and spacing factor, respectively (18, 43). The present study
shows that formation of regularly spaced nucleosomes is not sufficient
but GAGA factor-mediated chromatin remodeling is required for
transcriptional activation of the ftz chromatin template.
Moreover, CHRAC is less effective in GAGA factor-dependent chromatin
remodeling than NURF (43). These findings suggest that NURF
plays a key role in the GAGA factor-dependent transcriptional
activation in our system. The transcription extract may also contain
some chromatin-remodeling activities, possibly containing ISWI
homologs. But these activities, if any, do not seem to contribute so
much to transcriptional activation in our system, because addition of
the ISWI antiserum just before transcription had no effect on
transcription. However, we cannot exclude the possibility that these
activities might reduce the extent of the activation observed.
Although GAGA factor activated transcription on the chromatin template
in the presence of S150 extract, it barely activated transcription on
the naked template DNA. This supports the notion that GAGA factor is
not an ordinary transcriptional regulator which controls the efficiency
of transcription by interacting with general transcription factors
(13). Instead, the factor may serve as a landmark to recruit
a remodeling factor close to its binding site. However, there is no
evidence for direct interaction between GAGA factor and
chromatin-remodeling factors, and mechanisms of the recruitment of
these factors remain unknown. Genetic analyses have suggested that
products of the trithorax group genes, including Trithorax-like, form a multimeric protein complex to execute their function (27). This hypothetical complex may be responsible for the recruitment of remodeling factor. The in vitro system employed
in this study enabled us to remodel a chromatin assembled from purified
histones and DNA. The system will be useful for functional
characterization of the putative protein complex.
Micrococcal nuclease assays showed that the nucleosome structure around
350 and the TATA element of ftz was disrupted by incubation with GAGA factor and the S150 extract. A restriction enzyme
assay demonstrated that the AvaII site at 9, the
FspI sites at 90 and 317, and the PstI site
at 267 on the ftz chromatin template were more susceptible
to digestion after incubation with GAGA factor and the S150 extract.
Furthermore, base substitutions of all four GAGAG sequences in the
ftz promoter at 360, 348, 158, and 46 were required
to completely suppress the GAGA factor-mediated chromatin remodeling.
These results indicate that chromatin is remodeled throughout the
proximal region of the ftz promoter.
Analyses using transgenic fly lines have identified two major groups of
cis elements within the ftz promoter that are
required for the striped expression of ftz (37, 38,
42). One group comprises the GAGA factor-binding sites described
above. The other is a binding site for the nuclear receptor FTZ-F1
located 280 bp upstream of the transcription initiation site. Our in
vitro transcription studies revealed that activation of ftz
by FTZ-F1 requires two coactivators, termed MBF1 and MBF2
(23). MBF1 is a bridging molecule that interconnects FTZ-F1
and TATA-binding protein and recruits positive cofactor MBF2 to a
promoter carrying the FTZ-F1-binding site. MBF2 activates transcription
through its contact with TFIIA. This allows the selective activation of ftz in a FTZ-F1 binding site-dependent manner (24,
35). It is most likely that the GAGA factor-mediated chromatin
remodeling in the proximal region of the ftz promoter is a
prerequisite for the formation of active complexes containing FTZ-F1,
MBF1, MBF2, TFIIA, and TBP. Shopland et al. have reported that a
mutation in the GAGA site on the hsp70 promoter affects the
accessibility of the heat shock transcription factor (30).
This suggests that a mechanism similar to that described here may
operate in the activation of the hsp70 promoter.
| |
ACKNOWLEDGMENTS |
We thank T. Tsukiyama and C. Wu for pAR-GAGA and important
suggestions on preparation of the S150 extract, T. Ohta for ISWI antiserum, H. Ueda for helpful discussions during the course of this
work, and J. Tomizawa, M. Muramatsu, and M. Jindra for critical reading
of the manuscript.
This work was supported by Grants-in-Aid for Scientific Research from
the Ministry of Education, Science, Sports and Culture of Japan (S.H.)
and the Research Fellowship of the Japan Society for the Promotion of
Science for Young Scientists (M.O.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Developmental Genetics, National Institute of Genetics, Mishima,
Shizuoka-ken 411, Japan. Phone: 81-559-81-6771. Fax: 81-559-81-6776. E-mail: shirose{at}lab.nig.ac.jp.
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Abstract
Introduction
