From the Institute of Developmental and Molecular Biology and Department of Biology, Texas A & M University, College Station, Texas 77843-3155
Received for publication, August 17, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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The promoter for the phaseolin (phas)
bean seed protein gene adopts an inactive chromatin structure in leaves
of transgenic tobacco. This repressive architecture, which confers
stringent spatial regulation, is disrupted upon transcriptional
activation during embryogenesis in a process that requires the presence
of both a transcription factor (PvALF) and abscisic acid (ABA). Toward determining the need for de novo synthesis of proteins
other than PvALF in transcriptional activation we explored the effect
of several eukaryotic protein synthesis inhibitors. Surprisingly, cycloheximide (CHX), emetine, and verrucarin A were able to induce transcription from the phas promoter in tobacco and bean
leaf tissue in the absence of either PvALF or ABA. This induction was decreased by the replication inhibitors hydroxyurea and aphidicolin but
not by genistein or mimosine. Since protein phosphatases and kinases are essential components of the ABA signal transduction pathway, it is conceivable that CHX is also capable of inducing phosphorylation of proteins usually involved in ABA-mediated
activation. Interestingly, okadaic acid, an inhibitor of
serine/threonine phosphatase, also strongly activated transcription
from the phas promoter. In contrast, the protein synthesis
inhibitors anisomycin and puromycin did not activate transcription from
the phas promoter, nor did the tyrosine phosphatase
inhibitors phenylarsine oxide and sodium orthovanadate. These discrete
but different results on transcriptional activation may reflect
specific modes of action of the inhibitors, or they may reflect
differential interactions of the inhibitors or of downstream events
resulting from inhibitor activity with presently unknown components of
the transcriptional activation system.
The necessity of de novo protein synthesis for gene
activation can be investigated through the use of inhibitors. For
example, the fact that the induction of most genes involved with indole acetic acid metabolism is insensitive to the protein synthesis inhibitor cycloheximide
(CHX)1 suggests that newly
synthesized protein is not necessary for activation of these genes (1).
CHX has also been reported to enhance and prolong the accumulation of
mRNA of expressing genes in a process termed superinduction (2) and
to induce de novo transcription from nonexpressing genes as
documented for PS-IAA4/5 (3), GAmyb (4),
HVA22 (5), CPRF1 (6), ATL2 (7), and
mlip15 (8). Superinduction or de novo induction
of genes by protein synthesis inhibitors is thought to occur through
several distinct processes. CHX can enhance mRNA stability by
preventing the synthesis of labile mRNA-degrading enzymes (9);
alternatively, some protein synthesis inhibitors cause RNAs to be
trapped on polysomes, thus shielding them from cytoplasmic
ribonucleases (10, 11). For autorepressive genes, the inhibited
translation of their protein product can lead to superinduction through
an inability to shut off transcription (12). CHX may lead to
transcriptional activation via the loss of labile negative regulators.
CHX can induce the uncoupling of DNA replication and chromatin assembly (because of the continued DNA replication and the absence of histone synthesis during CHX treatment), preventing the formation of a repressive chromatin structure (13); alternatively, CHX may induce
uncoupling of DNA replication and gene specific repressors, thereby
releasing chromatin constraint on transcription. CHX can lead to direct
transcription activation by eliciting chromatin-associated signals such
as H3 phosphorylation (14) or by biochemical modifications that lead to
the activation of positive (or the deactivation of negative)
transcription factors.
Phaseolin, the major seed protein of bean, Phaseolus
vulgaris, is encoded by a small gene family whose expression is
tightly regulated both temporally and spatially (15). Expression of the
Toward evaluating the need for the synthesis of protein factors
additional to PvALF to permit expression from the phas
promoter, leaves of line 58.1A plants (tobacco transformed with
ABA Induction--
Seeds collected from either 58.1A plants or
from line PvAlf-14 were surface-sterilized with 20% (v/v) household
bleach and then rinsed three times in sterile water. Seedlings (10 days) or leaves from seedlings (30 days), selected on MS medium
containing either 400 µg/ml kanamycin or 50 µg/ml hygromycin, were
incubated in liquid basal MS medium with or without 100 µM ABA (± cis/trans isomer; Sigma)
for 24 h (6 h for the RNase protection experiment) in the dark
with gentle shaking at room temperature.
Fluorometric and Histochemical GUS Assays--
Histochemical
analysis of GUS activity was according to Jefferson et al.
(20). For fluorometric (MUG) assay, leaves or callus were homogenized
in the GUS extraction buffer (50 mM
NaH2PO4, pH 7.0, 10 mM EDTA, 0.1%
Sarkosyl, 0.1% Triton X-100, 10 mM RNase Protection Assay (RPA)--
Antisense constructs for
generating riboprobes were prepared by subcloning fragments containing
the 3' end of the uidA and the region encoding 18 S
RNA into the vector pPCR-script amp sk(+) (Stratagene, La Jolla, CA).
Antisense uidA and 18 S rRNA riboprobes of 310 and 200 nucleotides, respectively, were synthesized by transcription in vitro, using T3 or T7 polymerase on a
HindIII- or EcoRI-linearized plasmid.
RPAs were performed in reactions containing 5-10 µg of total RNA
using an RPAII kit (Ambion, Austin, TX). The protected fragments were
analyzed by electrophoresis on a 5% polyacrylamide, 8 M
urea gel.
Isolation of Histones--
Approximately 5 g of leaf tissue
was powdered in a mortar and pestle using liquid nitrogen, then treated
with nuclei isolation buffer NIB1 (0.5 M hexylene glycol,
20 mM KCl, 20 mM PIPES pH 6.5, 0.5 mM EDTA, 0.4% Triton X-100, 0.05 mM spermine,
0.125 mM spermidine, 7 mM 2-mercaptoethanol,
0.5 mM phenylmethylsulfonyl fluoride, and 0.5% (v/v)
aprotonin) in the presence of protein phosphatase inhibitors (10 mM NaF, 1 mM sodium orthovanadate). Nuclei were
recovered by centrifugation at 1400 × g for 10 min and washed
twice in NIB2 (NIB1 without Triton X-100). All centrifugations were
carried out at 4 °C. Nuclei were resuspended in 3 ml RSB (22) buffer
(10 mM Tris-HCl, pH 7.6, 3 mM
MgCl2, 10 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 0.5% (v/v) aprotonin, 10 mM NaF, 1 mM sodium orthovanadate) and
extracted with 0.4 N H2SO4 to
isolate total histones. The samples were precipitated with trichloroacetic acid and then resuspended in TE buffer containing protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 0.5% v/v aprotonin, 10 mM NaF).
Electrophoresis and Western Blotting--
Proteins were analyzed
by SDS-15% polyacrylamide gels. The proteins were visualized by
Coomassie Brilliant Blue staining or transferred to nitrocellulose
membranes (23). The anti-phosphorylated H3 (anti-pH3) was purchased
from Upstate Biotechnology, Lake Placid, NY. The membrane containing
the histones was immunochemically stained with anti-pH3 and
peroxidase-conjugated goat anti-rabbit antibody (Sigma) using
SuperSignal West Pico detection (Pierce).
Phas Expression Can Be Induced in Leaf Tissue by CHX--
To
examine whether de novo protein synthesis is required for
PvALF-dependent induction of expression from the
phas promoter in the presence of ABA, leaves from either
58.1A (tobacco transformed with
Although the above experiments show that the phas promoter
can be activated in the absence of de novo protein
synthesis, it is likely that this expression is not through ABA- or
PvALF-mediated processes. As shown in Fig. 1B, treatment of
PvAlf-14 leaves with CHX in the absence of ABA or treatment of 58.1A
plants with CHX in the presence or absence of ABA can also induce
phas mRNA accumulation (Fig. 1B, lanes
2 and 4). This reveals that the CHX treatment can
bypass the absolute requirement for PvALF and ABA to activate phas in transgenic tobacco (19). The samples were cultured
in CHX containing MS liquid medium for 6 h in these and
subsequent CHX experiments reported in this paper because this period
gave maximal accumulation of mRNA from 58.1A leaves (Fig.
1C).
A similar induction of expression from the phas promoter by
CHX to that seen in transgenic tobacco was obtained using leaf tissue
of its native species, P. vulgaris (Fig. 1D).
This shows conclusively that the induction of expression seen in
tobacco is not due to the use of a heterologous system.
Phas Activation by CHX Is
Concentration-dependent--
To investigate whether
phas activation by CHX results from the inhibition of
protein synthesis, we tested phas induction under different
concentrations of CHX. As shown in Fig.
2A, lanes 2 and
3, the phas promoter is not activated at low
concentrations (0.1 and 1 µM) of CHX that do not greatly
reduce protein synthesis (Fig. 2B). However, a low level of
transcription (detected as uidA mRNA; Fig.
2A, lane 4) occurred after 5 h of 10 µM CHX treatment, when protein synthesis was reduced to
25% compared with the control. At 100 µM, CHX was 98%
effective in inhibiting protein (GUS) synthesis (Fig. 2B),
and transcription from the phas promoter was strongly induced (Fig. 1A).
Differential Effects of Replication Inhibitors on CHX-induced phas
Expression--
It has been hypothesized that treatment of cells with
an efficient inhibitor of protein synthesis, such as CHX, would be
expected to uncouple DNA synthesis and chromatin assembly and that this may have widespread consequences in relieving chromatin-mediated general transcriptional repression (13). To determine whether uncoupling of DNA replication and chromatin assembly is responsible for
protein inhibitor-inducible phas expression, we tested the effect of DNA replication inhibitors on CHX-induced phas
expression. As shown in Fig.
3A, pretreatment with 10 mM hydroxyurea (HU), a replication inhibitor that inhibits
ribonucleotide reductase, for 30 min before adding CHX substantially
reduced the degree of induction by CHX. Pretreatment of leaves with 10 mM HU for 24 h before adding CHX further reduced the
degree of induction of uidA expression by CHX (Fig.
3A, lanes 4 and 5 compared with lanes 1 and 3). This reduction was not due to HU
toxicity, since HU concentrations of up to 100 mM did not
reduce transcription from the CaMV35S promoter (Fig.
3B). We also found that CHX-mediated activation of
phas is higher in young leaves where there is more DNA
replication than that in older leaves (Fig. 3C, compare
lanes 2 and 4). However, aphidicolin, another DNA
replication inhibitor that inhibits DNA polymerase phas Can Be Strongly Induced by Some, but Not All, Protein
Synthesis Inhibitors--
To further evaluate the mechanism of
CHX-induced phas activation, we examined the effect of
alternative protein synthesis inhibitors (verrucarin A, emetine,
anisomycin, and puromycin) on activation in leaf tissues of transgenic
tobacco plants. Like CHX, verrucarin A and emetine strongly induced
phas expression (Fig. 4,
A and B). However, puromycin and anisomycin did
not induce phas activation when used at concentrations that
are effective for inhibition of protein synthesis and activation of
gene expression in animal cell lines (Fig. 4, B and
C). Overexposure of the gel revealed uidA
mRNA expression at levels barely above background for two different
concentrations of puromycin and anisomycin (Fig. 4C). The
trace levels of uidA mRNA in the presence of 100 µg/ml puromycin and 10 µg/ml of anisomycin could be attributed to poor inhibition of protein synthesis at these concentrations, as indicated by MUG assays (Fig. 4D). However, 10-fold higher
concentrations of these inhibitors (1 mg/ml puromycin or 100 µg/ml
anisomycin) are very effective in inhibiting protein synthesis (Fig.
4D), yet no uidA mRNA was detected in
58.1A leaves. This suggests that inhibition of protein synthesis
alone will not lead to transcriptional activation from the
phas promoter.
Induction of phas Expression in Leaf Tissues in Response to
Phosphatase Inhibitors--
In addition to inhibiting protein
synthesis, CHX has been shown to induce histone H3 phosphorylation
(14). Because of the role of chromatin in silencing expression from the
phas promoter, we decided to explore the use of okadaic
acid, a serine/threonine phosphatase inhibitor that has been shown to
induce H3 phosphorylation and early-response gene expression in
mammalian cells (14). Okadaic acid was able to induce transcriptional
expression from the phas promoter in leaf tissue of both
58.1A and PvAlf-14 plants (Fig.
5A, lanes 2 and
4). The induction of phas expression from leaves
of PvAlf-14 was about twice as strong as that from leaves of 58.1A. A
similar serine/threonine phosphatase inhibitor, cantharidin, can also
induce phas expression (data not shown).
We then tested whether CHX- or okadaic acid-induced phas
activation is related to bulk H3 phosphorylation, as has been shown for
early-response genes (14). Western blotting with antibodies to pH3
showed that there is no discernible difference between the amount
of pH3 in transgenic tobacco leaves in the presence or absence of CHX
treatment (Fig. 5B, compare lanes 3 and
4), suggesting that bulk H3 phosphorylation is not
responsible for phas activation.
Tyrosine phosphatase inhibitors, phenylarsine oxide and sodium
orthovanadate, a specific inhibitor for dual specificity phosphatase and tyrosine phosphatase, were not effective in inducing
uidA mRNA expression from the phas promoter.
Slightly higher than background levels of uidA mRNA were
detected only after overexposure of the gel for more than 2 days using
a PhosphorImager (Fig. 5C).
CHX and Okadaic Acid Can Induce the Expression of Various
Seed-specific Genes--
Given the remarkable stimulation of
expression from the phas promoter, we were interested in
exploring the ability of CHX and okadaic acid to induce expression of
other seed storage protein genes. As shown in Fig.
6, RPA analysis clearly demonstrated that expression of uidA from the normally seed specific
DC3 (26), HaG3 (27), and Arcelin (28)
promoters was induced in leaves of tobacco transformed with these
constructs by treatment with CHX or okadaic acid, albeit at different
efficiencies: DC3 was strongly induced but HaG3
and Arcelin were induced only weakly.
We have shown previously that the phas promoter adopts
an inactive chromatin structure in leaf tissue of transgenic tobacco and that the repressive chromatin structure is disrupted upon transcriptional activation during embryogenesis, processes that require
both PvALF and ABA (17, 18). The presence of PvALF results in
remodeling of the chromatin architecture over the phas promoter, allowing ABA-stimulated transcriptional activation (19). Here
we show that several eukaryotic protein synthesis inhibitors can induce
transcription from the phas promoter in tobacco and bean
leaf tissue in the absence of either PvALF or ABA. The expression seen
in leaves from bean, the plant from which phas was isolated (29), confirms that the induction observed in the case of tobacco does
not result from the use of a heterologous system. We also demonstrated
that CHX-induced phas activation is associated with inhibition of protein synthesis, although this is not the only requirement. Since phosphatase inhibitors can also induce
phas activation, it is possible that protein synthesis
inhibitor-induced expression from the phas promoter is
mediated through a general signal transduction pathway involving
phosphorylation/dephosphorylation events. The stimulation of
transcription from the Arcelin, Dc3, and
HaG3 seed-specific promoters by CHX and okadaic acid (Fig. 6) indicates the generality of transcriptional activation by these compounds.
CHX-induced phas Expression and Uncoupling of DNA Replication and
Chromatin Assembly--
CHX-induced mRNA stability or
CHX-inhibited autorepression can only contribute to superinduction of
genes after their transcription has been activated. Since
phas is not expressed in leaf tissues of transgenic tobacco
plants, as shown both by nuclei run-on and by RPA experiments (17, 19),
accumulation of its mRNA in the presence of CHX cannot result from
mRNA stabilization or release from autorepression. Further support
that mRNA stability is not involved is provided by the decreased
level of uidA mRNA seen in leaves after 6 h of
exposure to CHX (Fig. 1C). Additional evidence against the
involvement of mRNA stability is provided by the fact that
verrucarin A also induces phas expression (Fig.
4A), even though its mode of action is to dissociate
mRNAs from ribosomes, thereby exposing them to cytoplasmic ribonucleases.
An attractive explanation for protein synthesis inhibitor-inducible
phas expression is the uncoupling of DNA replication and chromatin assembly. In the presence of protein synthesis inhibitors, the synthesis of both histones and/or non-nucleosomal repressors will
be affected. If DNA replication occurs during CHX treatment, histone-
or gene-specific repressors may not be present in sufficient quantity
to cover the newly replicated phas promoter. In such cases,
phas will be expressed, since the transcription complex is
still functional (13). This scenario suggests that CHX-induced transcription should be reduced upon addition of DNA replication inhibitors. Pretreatment of leaf tissue with the DNA replication inhibitor HU did indeed reduce induction by CHX and greater reduction was observed if more HU was added or if the pretreatment was longer (Fig. 3A). This reduction is not due to HU toxicity, since
higher levels of HU did not affect transcription from the constitutive CaMV35S promoter (Fig. 3B). Furthermore,
CHX-mediated induction was much greater for young leaves (in which
replication is active) than for mature leaves (Fig. 3C),
where little replication occurs. A slight reduction in CHX-mediated
activation was also observed in the presence of aphidicolin (Fig.
3A).
In contrast to the above results, incubation of PvAlf-14 leaves with
mimosine and genistein, two other inhibitors of replication, led to
enhancement of CHX-mediated induction of expression from the
phas promoter (Fig. 3D). Although the different
outcomes of experiments using replication inhibitors are puzzling, it
is feasible that mimosine and genistein have other functions in
addition to the inhibition of replication.
CHX-inducible phas Expression Is Not Entirely Due to the Loss of
Labile Negative Regulators--
It has been well documented that many
protein synthesis inhibitors, such as CHX, superinduce mRNA
synthesis by de novo transcriptional activation (2, 30-33).
A widely accepted interpretation of this effect is that a labile
transcription repressor is degraded following protein synthesis
inhibition, resulting in transcriptional activation. The strong
correlation seen here between protein synthesis inhibition and
phas promoter activation supports this interpretation:
cycloheximide (100 µM), verrucarin A, and emetine are
effective protein synthesis inhibitors and all induce expression from
the phas promoter. In contrast, puromycin (100 µg/ml) and
anisomycin (10 µg/ml) are not effective inducers of phas
activation and are relatively weak inhibitors of protein synthesis in
plants (puromycin at 100 µg/ml was only 45% effective in inhibiting
GUS protein synthesis, and anisomycin at 10 µg/ml was 64%
effective). However, at higher concentrations, puromycin (1 mg/ml) and
anisomycin (100 µg/ml) are very effective in inhibiting protein
synthesis (Fig. 4D), but transcription was not activated in
their presence (Fig. 4C). While these results appear to
detract from the possibility that a labile repressor is involved in
regulating expression from the phas promoter, or that
uncoupling of DNA replication and chromatin assembly is the cause of
phas induction, they may reflect the inhibition of specific
components of the activation processes. For example, the individual
protein synthesis inhibitors may be differentially effective with
regard to specific components or those inhibitors that do induce
expression may have side effects that lead to transcriptional activation.
CHX-inducible phas Expression Is Not Due to Global H3
Phosphorylation--
Another possible explanation for CHX-inducible
expression from the phas promoter is that CHX can actively
elicit chromatin-associated signals that lead to transcription
activation. CHX has been shown to superinduce the c-jun gene
in human cells (14), which was later shown to be correlated with bulk
histone H3 phosphorylation (34). In this case, activation of gene
expression was not related to the inhibition of protein synthesis, and
a subinhibitory concentration of protein synthesis inhibitor was able
to superinduce c-jun transcription (35). Although the
phosphatase inhibitors okadaic acid and cantharidin can induce
phas activation (Fig. 5), immunoblotting of bulk histone with anti-pH3 antibodies showed that CHX treatment does not increase the level of bulk histone pH3 level. This suggests that CHX-induced phas expression is not through global H3 phosphorylation,
but certainly does not eliminate the possibility that localized H3 phosphorylation of phas chromatin is involved.
A Possible Explanation for Inhibitor-induced phas Transcriptional
Activation--
It is likely that the inhibition of protein
phosphatase by protein phosphatase inhibitors or of its synthesis by
protein synthesis inhibitors has several biochemical consequences on
signal transduction mechanisms. These could include phosphorylation or
dephosphorylation events that lead to the activation of positive, or
the deactivation of negative, transcription regulators (36). Indeed,
our experiments suggest that the inhibitors can act through a passive
activation pathway (Fig. 7, steps
A or B) or an active pathway (Fig. 7, steps C-F).
We favor the active pathway as being the predominant explanation for
our results, because the passive events require significant depletion
of histones; this should be especially apparent for cells that are
actively replicating. However, while our experiments with the
replication inhibitor hydroxyurea (Fig. 3A) and to a lesser
extent, aphidicolin, do show a decrease in protein synthesis inhibitor-induced activation, the effect is not striking and does not
appear to be sufficient to account for the rapid induction of
expression from the phas promoter upon exposure of leaves to protein synthesis inhibitors (Fig. 1C). In contrast,
post-translational modifications, such as phosphorylation of
pre-existing substrates, appear to provide a feasible basis for the
rapid induction of phas expression.
In the putative active pathway, we postulate that a key protein
phosphatase exists. The depletion of this phosphatase by protein synthesis inhibitors (Fig. 7, step F) and the inhibition of
its activity by the inhibitors okadaic acid and cantharidin (Fig. 7,
step C1) result in the induction of expression from the
phas promoter. Either or both depletion and inhibition of
the phosphatase (Fig. 7, steps C1-C3 or step F)
could lead to the modification of the activity of transcription
factors, directly or indirectly through the activation of additional
phosphatase(s) or kinase(s). An alternative or additional set of events
resulting from the depletion or inactivation of the postulated protein
phosphatase could lead to chromatin potentiation as shown in Fig. 7,
steps D and E. Potentiation can be achieved by
targeted alterations in the chromatin environment encompassing specific
genes, both directly, by phosphorylation of nucleosomal and chromatin
proteins, or indirectly by allowing the recruitment of histone
acetyltransferases to specific phosphoepitopes on transcription
factors (Fig. 7, step E).
Our experiments provide clues regarding the nature of the putative
protein phosphatase. Because okadaic acid and cantharidin can induce
phas expression while sodium orthovanadate cannot suggests that this protein phosphatase is a serine/threonine protein phosphatase (PP1, PP2A, or PP2B) that normally negatively regulates expression from
the phas promoter. Interestingly, okadaic acid has been
shown to inhibit most gibberellic acid-inducible events and partly
inhibits the induction of a HVA gene by ABA (37). Kinase and
phosphatase inhibitors have been shown to affect gene expression in
response to ABA (38-40). For example, the DNA binding activity of the
G-box-binding bZIP factor GBF1 can be stimulated through
phosphorylation by a kinase from nuclear extracts (41), and it is known
that phosphorylation of bZIP factors in vivo initiates their
translocation from the cytoplasm to the nucleus (42). Since CHX is a
protein synthesis inhibitor, its inhibition of the putative phosphatase
synthesis may mimic the loss of phosphatase activity and hence lead to
a gain of function through the phosphorylation of kinases or
transcription factors. The factor phosphorylated as a result of the
presence of CHX should therefore be constitutively present in
vegetative tissues, e.g. an EmBP-like bZIP factor or an
helix-loop-helix factor such as PG1 (43).
Phosphatase inhibition resulting from exposure to the protein
inhibitors CHX, emetine, or verrucarin A could lead to both chromatin
modification and transcriptional activation. Since we have shown that
there is no bulk H3 phosphorylation as a result of CHX treatment,
chromatin modification is unlikely to operate through direct H3
phosphorylation. However, it is possible CHX-triggered phosphorylation
or dephosphorylation can activate a factor that can attract a
nucleosome remodeling complex, or a histone acetyltransferase, or even a histone kinase that leads to local H3 phosphorylation. The
same transcription factor or another factor activated by CHX must be a
component of the ABA signal transduction pathway that leads to
transcriptional activation of the phas gene (19).
The results obtained here are dramatic with regard to our understanding
of the regulation of expression from the phas promoter. First, they show that the normally tight repression of expression from
the phas promoter in vegetative tissue can be overcome
rapidly by exposure to the potent inhibitor of protein synthesis, CHX. Second, while tyrosine phosphatase inhibitors had no discernible effect, inhibition of by okadaic acid activated transcription from the
phas promoter, strongly implicating the presence of a serine/threonine phosphatase as a key regulator. Third, our findings extend previous studies that have demonstrated the ability of CHX to
induce or superinduce transcription (2) by showing that other protein
synthesis inhibitors can act similarly. Fourth, the findings provide
the insight that phosphorylation of a kinase or a transcription factor
are likely components of the ABA-mediated activation pathway.
Additional insight may be gained through future experiments to
determine why the protein inhibitors anisomycin and puromycin do not
effectively activate phas expression as do CHX, emetine, and
verrucarin A. Similarly, the basis for the differential effects of the
replication inhibitor HU compared with the inhibitors genistein
and mimosine on CHX-mediated activation remains to be resolved. The
differences may reflect specific modes of action of the inhibitors, or
they may reflect differential interactions of the inhibitors or of
downstream products resulting from inhibitor activity with components
of the signal transduction and transcriptional activation systems that
remain to be identified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phaseolin gene (phas) is totally inactive during
vegetative phases of plant development (16). This is achieved by a
repressive chromatin architecture (17) that is remodeled concomitant
with gene activation in the developing seed, resulting in disruption of
histone-mediated DNA wrapping and permitting abundant factor binding to
the phas promoter (18). Activation of the phas
promoter is a two-step process: chromatin modification mediated by the transcription factor PvALF, followed by abscisic acid (ABA)-mediated transcriptional activation (19).
1470phas/uidA, see Ref. 19) and line PvAlf-14 plants (a
58.1A plant retransformed with CaMV35S/PvAlf, see Ref. 19)
and bean leaves were treated with protein synthesis inhibitors. To our
surprise, we found that three potent eukaryotic protein synthesis
inhibitors (CHX, verrucarin A, and emetine) were able to induce
transcriptional expression from the phas promoter in these
vegetative tissues without the presence of either PvALF or ABA. We
demonstrated that phas activation can also be induced by the
protein phosphatase inhibitors okadaic acid and cantharidin. The
stimulation of phas expression by protein synthesis
inhibitors and by phosphatase inhibitors suggests that transcription
from the phas promoter is regulated through a complex network of interactive signaling components and pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol) and centrifuged for 5 min in a microcentrifuge. Then, 200 µl
of extract were mixed with 200 µl of substrate solution (GUS
extraction buffer + 4-methylumbelliferyl
-D-glucuronide:
4-MUG, Fluka) and incubated at 37 °C; 100-µl aliquots were removed
at 0, 60, or 120 min and the reaction terminated by addition of 900 µl of Na2NO3. Fluorescence was measured on a
fluorometer (VersaFluorTM fluorometer, Bio-Rad). Protein
concentrations were determined using the colorimetric assay of Bradford
(21). Specific GUS activity was calculated as pmol 4-MU
h
1 µg
1 protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1470phas/uidA, see Ref.
19) or PvAlf-14 plants (line 58.1A additionally transformed with
CaMV35S/PvAlf) were subjected to CHX treatment in the
presence or absence of ABA. Half of the CHX-treated leaves was used for
MUG assays, the other half was used for RPA experiments. MUG analysis
showed that CHX was very effective in preventing de novo
protein synthesis, since no GUS accumulation was detected in PvAlf-14
seedling leaves treated with both ABA and CHX (Fig.
1A). Despite the complete
inhibition of de novo protein synthesis, phas
mRNA was still synthesized (Fig. 1B, compare lanes
5 and 6), suggesting that de novo protein
synthesis is not required for phas activation. The synthesis
of uidA mRNA appeared to be slightly reduced in the
presence of CHX. However, this difference may be due to the variation
of PvALF levels among plants (19). The similar levels of 18 S rRNA seen
in each lane of the RPA assay show that comparable amounts of RNA were
loaded.
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Fig. 1.
CHX induces ectopic phaseolin gene
expression. A, CHX is very effective in preventing
protein synthesis. CHX treatment for 5 h blocks the production of
GUS activity in PvAlf14 plants in the presence of ABA. B,
RPA of uidA and 18 S rRNA in transgenic 58.1A and
PvAlf 14 plants in the presence or absence of ABA or CHX. The
32P-labeled probes are specific for uidA and 18 S rRNA. C, time course of phas/uidA induction by
CHX. 58.1A leaves were treated with CHX for various periods of time,
total RNA was extracted, and RPA was conducted with both
uidA and 18 S probes. D, RPA of bean leaves in
the presence of ABA and/or CHX. Expression of phas (not
uidA, as indicated) was obtained using a 244-nucleotide
riboprobe corresponding to a 3' region of the phas coding
region.
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Fig. 2.
CHX-inducible phas
expression is positively correlated with protein synthesis
inhibition. A, the effect of different concentrations
of CHX on phas induction in leaves of 58.1A. B,
the effect of different concentrations of CHX on protein (GUS)
synthesis in leaves of PvAlf-14.
, had only a
small inhibitory effect on CHX-induced phas expression (Fig.
3A, compare lanes 7 and 8 with
lane 1). Contrary to the histone depletion hypothesis,
mimosine and genistein, compounds shown to inhibit DNA
replication in animal (24, 25) and plant (rice, data not shown) cells,
were found to superinduce phas expression in the presence of
CHX (Fig. 3D).
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Fig. 3.
The influence of DNA replication and its
inhibitors on the induction of phas expression by
CHX. A, the induction of phas/uidA
expression in 58.1A leaves is inhibited by hydroxyurea but only
slightly by aphidicolin. B, hydroxyurea does not inhibit the
expression of uidA in tobacco transformed with
CaMV35S/uidA. C, cycloheximide induces
phas/uidA expression differentially in young and mature
leaves of 58.1A. D, mimosine and genistein can
increase the activation of phas by CHX in mature 58.1A leaf
tissues.
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Fig. 4.
The effect of various protein synthesis
inhibitors on phas/uidA expression in transgenic
tobacco leaves. A, the effect of verrucarin A on
phas expression in 58.1A and PvAlf-14. B,
expression from phas is induced by emetine but not by
puromycin. C, the effect of different concentrations of
puromycin and anisomycin on phas expression in 58.1A.
D, the effect of different protein synthesis inhibitors on
MUG activity in PvAlf-14.
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Fig. 5.
The effect of protein phosphatase inhibitors
on phas expression. A,
activation of phas by okadaic acid. B, histone H3
phosphorylation status is not changed by PvALF or CHX treatment. Leaves
of 58.1A were cultured in the presence or absence of CHX. Histones (10 µg) isolated from treated leaves were electrophoretically resolved on
a 15% SDS-polyacrylamide gel electrophoresis gel, transferred to a
polyvinylidene difluoride membrane, and immunochemically
stained with anti-pH3. Lanes 1 and 2 show
Coomassie Blue-stained proteins. Lanes 3 and 4 show the immunochemically stained membrane. C, the influence
of tyrosine phosphatase inhibitors on phas activation in
58.1A.
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Fig. 6.
CHX and okadaic acid can induce
transcriptional expression from several seed storage protein
genes. A, the effect of CHX on the expression of
DC3, HaG3, and Arcelin genes.
B, okadaic acid can activate the expression of
uidA from DC3, HaG3, and
Arcelin promoters in transgenic tobacco.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Possible modes of action of protein
synthesis, DNA replication, and protein phosphatase inhibitors in
phas promoter activation. Expression from
the phas promoter typically involves potentiation (chromatin
remodeling instigated by PvALF, dark green arrow) and
ABA-mediated activation (light green arrow) steps. Two
pathways for inhibitor-induced activation are portrayed. In the
Passive Pathway, inhibition of translation (A) by
protein synthesis inhibitors (cycloheximide, emetine, and verrucarin A)
leads to a decrease in histone synthesis and failure to establish an
inactive nucleosomal architecture over the phas promoter
after DNA replication, yielding a potentiated state that is accessible
to the activators (black arrow). B, inhibition of
DNA replication (by hydroxyurea) eliminates the need for new histones;
nucleosome architecture over the promoter remains unperturbed and the
inactive state is maintained (magenta arrows). In the
Active Pathway, protein phosphatase inhibitors (okadaic acid
and cantharidin) inhibit the activity of a postulated protein
phosphatase (C1). The absence of dephosphorylation
(C2) causes an accumulation of the phosphorylated (active)
state of a putative transcription factor (orange ball) that
activates transcription (C3; red arrows).
Alternatively, protein phosphatase inhibitors may prevent
dephosphorylation of histones, leading to hyperphosphorylated
nucleosomes (D) that can potentiate the promoter by
recruiting nucleosome remodeling activity, such as histone
acetyltransferase (blue arrows). E, if the
unknown factor (orange ball) is a protein kinase, rather
than a transcription factor, its activated state may hyperphosphorylate
histones and hence result in nucleosome remodeling (purple
arrows), as in D. F, it is also possible
that the protein synthesis inhibitors cycloheximide, emetine and
verrucarin A function by preventing the synthesis of a labile protein
phosphatase (black arrow), leading to hyperphosphorylation
and activation by pathways C, D, or
E.
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ACKNOWLEDGEMENT |
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We thank Mahesh Chandrasekharan for his detailed suggestions and Tom McKnight, Mike Manson, Dorothy Shippen, and Terry Thomas for valuable discussions.
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FOOTNOTES |
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* This work was supported by Grant MCB99-74706 from the National Science Foundation.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.
Present address: Sangamo Biosciences Inc., 501 Canal Blvd., Suite
A100, Richmond, CA 94804.
§ To whom correspondence should be addressed. Fax: 979-862-4098; E-mail: tim@idmb.tamu.edu.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M007504200
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ABBREVIATIONS |
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The abbreviations used are:
CHX, cycloheximide;
ABA, abscisic acid;
GUS, -glucuronidase;
MUG (or 4-MUG), 4-methyl-umbelliferyl glucuronide;
RPA, RNase protection assay;
HU, hydroxyurea;
PIPES, 1,4-piperazinediethanesulfonic acid;
pH3, phosphorylated H3.
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