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J. Biol. Chem., Vol. 275, Issue 52, 40839-40845, December 29, 2000
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From the Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114
Received for publication, August 15, 2000, and in revised form, September 18, 2000
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CHOP/GADD153 is both an activating and repressing
transcription factor that is markedly induced in response to a variety
of cellular stresses. The CHOP/GADD153 gene was originally cloned because of its inducibility by ultraviolet light wavelength band C
(UVC) and has since been found to be activated in response to many
different cellular stresses. Some of the recent studies have questioned
the UVC responsiveness of the CHOP gene. Contradiction in our own data
led us to reexamine the UVC effects on CHOP expression. UVC is capable
of strongly activating the mouse CHOP promoter in stably transfected
NIH 3T3 cells but has only a modest and transient effect on the level
of the CHOP messenger RNA. In addition to its positive effect on CHOP
promoter activity, we show that UVC negatively affects CHOP mRNA
and protein expression. Pretreatment of NIH 3T3 cells with UVC markedly
attenuates the subsequent induction of CHOP mRNA by the cellular
stress activators methylmethane sulfate, tunicamycin, glucose
deprivation, and methionine deprivation for as long as at least 16 h. This inhibitory effect of UVC on CHOP expression in response to
stress is independent of the presence or absence of p53 and does not
involve mRNA degradation as opposed to the UVC effect that inhibits
p21 expression seen only in the absence of p53. The target of the
inhibitory effect of UVC on CHOP expression is located in the first
exon of the gene, a 5'-untranslated region that is unusually conserved
between different species. These findings suggest that an unknown
function encoded by the 5'-untranslated region somehow modifies the
response of CHOP gene transcription to UVC.
Cells of unicellular or multicellular organisms have adapted to
live and propagate in ways determined by their genetic makeup and by
the environmental cues received. Perturbations or adverse conditions
that place cells in danger trigger the stress response: an ensemble of
changes in cellular physiology including the rapid alteration of
expression of specific genes. Although many of these changes have not
been functionally characterized, they most probably participate in
mechanisms such as adaptation, protection, damage assessment, repair,
and cellular death if the damage places the rest of the organism at risk.
CHOP (C/EBP homology protein, also
called GADD 153 (growth arrest DNA
damage 153)) is a basic region leucine
zipper transcription factor and
heterodimerizes with members of the C/EBP1 family of
transcription factors (1). The expression of CHOP is markedly induced
by a variety of cellular stresses. The dimerization of CHOP with
C/EBP CHOP (GADD 153) was first identified based on the finding that it was
expressed in response to growth arrest and DNA damage (10). The
expression of CHOP has subsequently been shown to be induced by many
other cellular stresses such as amino acid deprivation (11, 12), growth
inhibition by prostaglandin A2 (13), hypoxia (14), acute phase
response (15), endoplasmic reticulum stress (16), glucose deprivation
(17), cysteine conjugates (18), tunicamycin (2, 14), and calcium
ionophore treatment (19). These findings have also questioned the
initially observed growth arrest and DNA damage inducibility of CHOP.
Glucose or amino acid limitation could have been the inducers in the
initial growth arrest studies (10, 20) in which cells were grown at high densities so that cell culture media could have become
nutrient-deprived. Also, DNA damaging agents are known to not only
target DNA but other molecules in the cell as well. Therefore, the
signals that promote CHOP gene expression may not be DNA damage
per se (16). The response of CHOP expression to UVC
irradiation is representative of the contradictions in the literature.
CHOP was first isolated as GADD153 because of its inducibility
by UVC radiation (10). Further studies showed that the
gadd153 gene promoter mediates this response (21-23). In
contradiction to these results, Wang et al. (16) showed no
increase in CHOP mRNA or protein levels following the treatment of
human fibroblasts with UVC, confirming the data obtained in
keratinocytes (24, 25). These discrepancies could reflect differences
in cell type, mode of treatment, or culture conditions.
We have cloned the promoter region of the mouse CHOP gene. Promoter-CAT
constructs stably integrated in the genome show strong UV
responsiveness, whereas endogenous CHOP mRNA or protein do not. A
closer analysis reveals that UVC acts in a dual and antagonist way on
CHOP expression. Here we describe studies that implicate the first exon
of the CHOP gene as a novel and critical UVC-responsive modulator of
the responsivity of the expression of CHOP to cellular stress.
Cell Lines and Their Treatment--
NIH 3T3 fibroblasts were
grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% calf serum, penicillin (100 units/ml), and
streptomycin (100 µg/ml). Cells were maintained at 37 °C and 5%
CO2. The p53 +/+ and p53 Plasmids--
The CHOP promoter-CAT reporter constructs used in
this study were generated by cloning polymerase chain reaction
amplified mouse CHOP promoter fragments in the pBLCAT3 vector (27). The template for the polymerase chain reaction was a CHOP genomic clone
isolated by screening a mouse (129 Sv) genomic library (Stratagene) made of partially Sau 3A digested DNA with a probe encompassing the
mouse exon 2, exon 3, and part of exon 4. The 5' upstream regulatory
region of the CHOP gene obtained was 2.5 kilobases.2 For
preparation of construct Generation of Stably Transfected Cells--
Pools of stably
transfected cells were prepared as follows. NIH 3T3 cells were
cotransfected with 10 µg of promoter-CAT reporter construct and 2 µg of puromycin-resistant plasmid pPGKPuro (a gift of Peter W. Laird)
with the calcium phosphate precipitation technique using a reagent kit
(5 Prime Western Blot Analysis--
Cells were lysed at 4 °C in RIPA
buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.1% SDS, 1% deoxycholic acid, 1% Nonidet P-40 containing 5 µg/ml
aprotinin, 0.5 µg/ml pepstatin, 5 µg/ml leupeptin, 1 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Following SDS-polyacrylamide gel electrophoresis extracts were blotted
on polyvinylidene difluoride membranes (DuPont). Antibody for p53 was
from Santa Cruz (Pab 240). For CHOP a polyclonal antibody was
used (1). Immunoblots were revealed by ECL detection system (Amersham
Pharmacia Biotech).
Northern Blot Analysis--
Total RNA was extracted by using
trizol reagent (Life Technologies, Inc.) following the manufacturer's
protocol. RNA was size fractionated on an 1.2% agarose gel containing
formaldehyde. After transfer to a Magna Charge nylon membrane (Micron
Separation Inc.), UV treatment, and baking, the prehybridization and
hybridization were performed at 42 °C in 50% formamide, 5× SSPE,
0.1% SDS, and 5× Denhart's solution. Probe was labeled with
[ CAT Assays--
For CAT assay cells were resuspended in
0.25 m Tris-HCl, pH 7.5, and lysed by three cycles of freezing and
thawing. After heat inactivation at 65 °C for 10 min, the extracts
were cleared by centrifugation. CAT assay was performed using a
fluorescent chloramphenicol substrate FAST CAT (Molecular Probes,
Inc.). After chromatography, plates were analyzed using a fluorimager
(Molecular Dynamics). Quantification was performed with IMAGEQUANT software.
We isolated a mouse CHOP genomic clone from which we prepared CHOP
promoter-CAT reporter constructs. The reporter constructs were stably
introduced into NIH 3T3 fibroblasts by cotransfection with a plasmid
encoding puromycin resistance. Pools of puromycin-resistant colonies
were challenged with different stresses known to induce CHOP
expression. The mouse promoter (
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
inhibits the function of the latter by preventing DNA binding
to a C/EBP site in the promoters of a subset of genes. In addition,
CHOP/C/EBP heterodimer binds to a different DNA sequence of another
subset of genes (2) and stimulates the transcription of these genes
(3). CHOP also heterodimerizes with other non-C/EBP subfamilies of
basic region leucine zipper proteins, e.g. activating transcription factor 3 (4) and Jun/Fos (5). Several functions have been
proposed for CHOP. Heterotopic overexpression of CHOP induces growth
arrest in fibroblasts (6), inhibits adipocyte differentiation (7), and
induces apoptosis in vitro (8). Studies in CHOP knockout
mice also suggest a role for CHOP in apoptosis during the endoplasmic
reticulum stress response in kidney cells (9).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/ mouse embryonic fibroblasts
(26) were cultured under the same conditions except that serum was 10%
fetal bovine. For induction experiments cells were split the day before
at a density of about 2-4 × 105 cells/5-cm dish.
Cells were always subconfluent during the course of the experiments.
Tunicamycin (Sigma) was resuspended in 90% ethanol, 100 mM
Tris-HCl, pH 9, at 2 mg/ml and used at a final concentration of 10 µg/ml. Methylmethane sulfate (MMS; Aldrich) treatment was performed
at a final concentration of 50 µg/ml. Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) devoid of glucose or methionine was
used for the depletion experiments, serum was 10% normal calf serum.
The cell monolayer was washed several times with the depleted medium
before incubation. NIH 3T3 cells were irradiated with UVC for 5 s
after removal of medium and one wash with phosphate-buffered saline
with a UVC (254 nm) germicidal lamp, 6.3 J/m2/s. For
mRNA stability studies transcription was blocked with actinomycin D
(Sigma) at a final concentration of 5 µg/ml.
980/+19 and 318/+19 the forward primers
were 5'-CGAGTCGACTGTGTTTCCTCTGATGACCCAGT-3' and
5'-CGAGTCGACGGTTGCCAAACATTGCATCAT-3', respectively. For both
constructs the reverse primer was
5'-CGAGGATCCTGTTAGGCTCAAGATAACTGACCT-3'. For the exon 1 containing
construct 318/+95 the reverse primer was
5'-CGCGGATCCTCTCTCCTCAGGTTCCGGCTGTTAT-3'.
3 Prime, Inc., Boulder, CO). Selection of
puromycin-resistant colonies was performed with 1.5 µg/ml puromycin
(Sigma). Pools of at least 40 colonies were used for induction and CAT assay.
-32P]dATP by random priming reaction. For
quantification, blots were scanned with a PhosphorImager (Molecular
Dynamics), and data were analyzed with IMAGEQUANT software.
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DISCUSSION
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980/+19) behaves very similarly to
what is known from studies of the hamster promoter (778/+21) (21,
22). Strong activation of the promoter in response to MMS, actinomycin
D (data not shown), or UVC was observed. Tunicamycin, an inhibitor of
protein glycosylation and endoplasmic reticulum function, is also a
strong inducer of CHOP expression (2, 14, 16). We show that the
induction of CHOP by tunicamycin is mediated by the CHOP 980/+19
promoter (Fig. 1A). As a
control the thymidine kinase promoter activity does not change upon
treatment with UVC or tunicamycin (Fig. 1A). Under the same
stress conditions that stimulate CHOP promoter activity, we also
examined endogenous levels of CHOP mRNA and proteins (Fig. 1,
B and C). MMS and tunicamycin show the expected
induction of both mRNA and protein levels, in accordance with the
promoter data. Following UVC treatment, however, no changes in CHOP
mRNA and protein levels are seen at 4 or 24 h. A substantial
increase in p53 protein level is seen, demonstrating the effectiveness
of the UVC treatment (Fig. 1B).
View larger version (28K):
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Fig. 1.
UVC irradiation strongly activates the CHOP
gene promoter but has no effect on endogenous CHOP mRNA and protein
levels. A, pools of NIH 3T3 cells stably transfected
with the mouse CHOP promoter ( 980/+19) or the thymidine kinase
promoter linked to the CAT reporter gene were treated with UVC (30 J/m2) or tunicamycin (TUN, 10 µg/ml) and
harvested at the indicated times (hours). Extracts were prepared, and
CAT activity was estimated by using a fluorescent chloramphenicol
substrate. Fold induction in CAT activity compared with the untreated
cells are shown. B, NIH 3T3 cells were treated with UVC (30 J/m2), tunicamycin (10 µg/ml), or MMS (50 µg/ml) and
harvested at the indicated times (hours). Protein extracts were subject
to SDS-polyacrylamide gel electrophoresis and blotted on polyvinylidene
difluoride membranes. CHOP and p53 were detected with specific
antisera and revealed with ECL. C, NIH 3T3 cells were
treated as in B. At the indicted times total RNA was
prepared. After electrophoresis and blotting, the CHOP RNA was detected
by hybridization with a 32P-labeled probe.
The absence of CHOP induction by UVC is in contradiction with our
promoter data obtained using the same cell line and same treatment
conditions. One possible explanation is that UVC induces CHOP gene
transcription in a transient manner, such that at the time points we
examined (4 and 24 h) the mRNA and protein levels are either
not yet induced or have already returned to the non-induced level. The
high stability of the CAT protein on the other hand may have allowed
its accumulation and detection even long after the promoter is in the
induction state. To explore this hypothesis, we examined CHOP mRNA
levels at more frequent time intervals (Fig. 2). A rapid but modest elevation of CHOP
mRNA is observed as early as 5 min after UVC exposure (2.5-fold
above control). The induction of CHOP mRNA peaks at 1 h
(4.5-fold above control) and decreases after 1 h, reaching control
levels at 3 h. This modest increase in CHOP mRNA levels,
however, does not correspond to the large increase in the activity of
the CHOP promoter-CAT reporter activity observed in response to UVC.
This discrepancy could be explained by a dual action of UVC on CHOP
expression: a positive action on the CHOP promoter activity (as
revealed by our construct) and a negative action (acting on
transcription initiation, elongation, or RNA stability) that
counteracts the promoter activity and consequently attenuates the
induction of expression. If so, this hypothesis would predict that UVC
may inhibit the induction of CHOP expression by other inducers of
cellular stress. This hypothesis was explored. UVC treatment performed
1 h or 30, 15, or only 5 min (lanes 6, 5,
4, and 3, respectively) prior to MMS treatment
significantly inhibits the strong induction of CHOP expression observed
with MMS treatment alone (lane 2 and control level of CHOP
mRNA, lane 1) (Fig. 3).
This effect of UVC is long lasting, because 16 h after UVC
treatment cells that were challenged with MMS show no or very little
induction of CHOP mRNA or protein levels (Fig. 3B). This
inhibition by UVC of CHOP induction is not restricted to MMS induction,
because the induction of CHOP in response to tunicamycin, glucose
deprivation, or methionine deprivation is also inhibited by
pretreatment of the cells with UVC (Fig.
4).
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Recently, Gorospe et al. (28) have analyzed in detail
the p53-dependent elevation of p21 expression in response
to UV light. The study showed that in the absence of p53 not only did
induction of p21 by UVC not occur, but that the induction of p21 by
mimosine (which occurs independently of p53) is suppressed by
UVC. The study showed that p21 mRNA degradation was the mechanism
by which p21 expression was suppressed. The effect of UVC that we
observed in our studies here is reminiscent of what was described for
p21. The NIH 3T3 fibroblasts used in our studies have been reported to
be mutated in the p53 gene (29). Therefore, we tested whether the
inhibitory effect of UVC on CHOP expression is dependent on the
presence of a functional p53 protein and whether CHOP mRNA degradation is involved. We find that UVC inhibition of CHOP expression occurs independently of p53 because it is observed in both p53 +/+ and
p53 / mouse embryonic fibroblasts (Fig.
5). We then analyzed the effect of UVC on
the stability of the CHOP mRNA. Cells treated with tunicamycin
during 4 h with or without receiving a UVC pretreatment were
subjected to a block in transcription by actinomycin D. The fate of the
CHOP mRNA was analyzed at different times (Fig.
6). As expected, the level of CHOP
mRNA is reduced in cells pretreated with UVC. However, the
stability of the CHOP mRNA is not affected by UVC. On the contrary,
the decay of the CHOP mRNA is slightly more rapid in the cells that
were not exposed to UVC. Based on the results of our studies,
suppression of CHOP and p21 expression by UVC appear to involve
different mechanisms.
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Because of the remarkably high evolutionary conservation of the
5'-untranslated first exon of the human, hamster, rat, and mouse CHOP
genes (Fig. 7), we examined the first
exon (exon 1) of the mouse CHOP gene as a potential cause of the UVC
inhibitory effects on the stress-mediated inducibility of CHOP gene
expression. Remarkably, we found that the inclusion of the exon 1 sequence in the CHOP promoter in the context of the CHOP promoter-CAT
reporter constructs (see Fig. 7 for map of the constructs used)
markedly impaired its activation in response to UVC (3.5-fold
induction) compared with reporter constructs lacking exon 1 (53-fold
induction) (Fig. 8A).
Moreover, this marked inhibitory effect of the exon 1 sequence on the
UVC induction of CHOP promoter activity appears to be unique to UVC
because induction by tunicamycin of the promoter activity in the
presence (228-fold induction) or the absence (145-fold induction) of
exon 1 is essentially unaltered.
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Thus, the behavior of the activations of the CHOP promoter constructs
with exon 1 by UVC compared with tunicamycin and MMS mimics the effects
of these stimuli on the activation of the endogenous CHOP gene (Figs.
1, B and C, and 2). Based on these findings we next investigated the effects of the pretreatment of NIH cells, expressing CHOP promoter reporters with and without exon 1, with UVC on
subsequent responses to the stress-inducing agent tunicamycin. Surprisingly, an exposure of the cells to UVC inhibits the induction of
CHOP gene expression by tunicamycin for at least 16-20 h (Fig. 8B). That the inhibition of CAT activity is not due to UVC
toxicity is shown by the robust induction of the CHOP promoter without exon 1 16 h after UVC treatment (Fig. 8B).
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DISCUSSION |
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The UV stress response is an ensemble of changes in gene and
protein expression occurring in cells irradiated with UV light (30,
31). DNA damage has been the initial main focus of studies on UV
action. The short wavelength component of UV light, UVC, has been used
extensively as a source of genotoxic stress. In fact DNA damage was
shown to be one of the initial signals in two important pathways
induced by UVC: the p53 (32) and the NF
B (33). Other DNA
damage-dependent pathways leading to the production of
secreted factors have been described (34, 35). But UVC is not only a
genotoxic stress (reviewed in Ref. 31). It is absorbed by other
biomolecules in the cell, like proteins and lipids. Suspicions about
extra-nuclear targets capable of initiating UVC stress signaling (36)
were confirmed by the demonstration that plasma membrane receptors are
activated very early after UVC exposure (37-39). Ligand-independent
receptor dimerization upon UV irradiation was suggested as a mechanism
of activation, but several reports have now shown that it is the
inactivation of tyrosine phosphatases by UVC that cause phosphorylation
and activation of the receptors (40, 41).
Downstream events in the UV stress response have been characterized in more detail and originate at least in part from the activation of membrane receptors. These include the activation of several kinases cascades ultimately leading to transactivation of several MAP kinases: c-Jun N-terminal kinase MAP kinase (42), p38 MAP kinase (43-45), and extracellular regulated kinase MAP kinase (45). Consequently, multiple transcription factors in the nucleus, e.g. c-Jun, ternary complex factor, activating transcription factor 2, and the cAMP response element-binding protein, become phosphorylated by these kinases and regulate transcriptional programs of the cell. Among the target genes of these transcription factors are those encoding for transcription factors. Rapidly developing transcriptional cascades are thus initiated. The AP-1 complex (Jun/Fos heterodimers) is the prototype of these immediate early transcriptional responses and is induced at the transcriptional level in a few minutes after UVC irradiation.
The CHOP/GADD153 gene was initially isolated in a screen for genes
whose expression is induced by UVC (10). The strongest data supporting
the UVC inducibility of the CHOP/GADD153 gene come from studies
performed with the isolated promoter of the gene. Strong inducibility
has been described with the hamster gene promoter spanning from
position 778 to +21 relative to the start of transcriptional
initiation (21-23). We have cloned 2.5 kilobases of the upstream
regulatory region of the mouse CHOP gene.2 Promoter-CAT
reporter constructs were derived from this mouse sequence and stably
transfected in NIH cells. Strong UV responsiveness (up to 100-fold)
were obtained and are in agreement with the data on the isolated
hamster promoter in the context of transfected transcriptional reporter constructs.
Data on the endogenous CHOP mRNA and protein are more conflicting in the literature. An increase of the GADD 153 mRNA of about 5-fold was reported in the initial cloning publication (10). A similar significant increase was observed in HeLa cells following UVC irradiation (46). Other reports demonstrated a small or even no response (16, 24, 25, 28, 47, 48). In all of these studies the UVC dose was quite similar: 15-30 J/m2. One study used a more natural range of UV light source and reported a significant increase in CHOP mRNA (24).
Our findings appear to reconcile the conflicting findings in earlier
reports. We observe a 4-5-fold increase in CHOP mRNA levels.
Levels of CHOP mRNA increase within 5 min after irradiation, peak
at about 1 h and return to the uninduced level at about 3 h.
This transient increase in CHOP mRNA levels in response to UVC may
explain the lack of an increase observed by others at times later than
2 h after irradiation. This relatively modest and transient
increase in CHOP mRNA contrasts with the strong effect that UVC
exerts on activity of the CHOP promoter under identical conditions of
exposure to UVC and in the same cell line. This discrepancy led us to
hypothesize that the CHOP gene responds to two opposing signals from UV
light: an induction of CHOP gene transcription mediated by the promoter
of the gene between positions 318 to +19 relative to the start of
transcription and an inhibitory action mediated by exon 1 of the gene
that prevents the induction of CHOP by the subsequent application of
stress-inducing agents for as along as 20 h after UVC irradiation.
The mechanism(s) by which the presence of the first exon confers a UVC-induced inhibition of subsequent induction of the expression of the CHOP gene for as long as 20 h is unknown. The first exon flanks the promoter and is transcribed and present in the mature CHOP mRNA. Therefore, the UVC inhibitory effect observed in the presence of exon 1 could arise at at least three levels of gene expression: (i) transcriptional initiation by influencing the activity of the upstream promoter, (ii) transcriptional elongation by blocking the passage of RNA polymerase, or (iii) RNA stability because exon 1 is present in the mature mRNA.
No such destabilization of CHOP mRNA was observed. Rather, UVC appeared to increase the stability of the CHOP mRNA. These findings are in agreement with those of Jackman et al. (47), who showed stabilization of the GADD153 mRNA in several situations of cellular stress, one of them being UVC irradiation. Stabilization of mRNA was shown recently to be the mechanism by which UV induces the expression of several important growth regulating genes: the p21 cyclin-dependent kinase inhibitor (28, 49) and the later induction of c-Jun transcripts that follows its early transcriptional induction (50). Interestingly, the CHOP gene contains a 3' AU-rich region known to be involved in mRNA stability (28, 51). The AU-rich region sequence is also found in the p21 and the c-Jun transcripts. This sequence is able to bind several proteins; one of these, HuR, is capable of stabilizing the mRNA to which it is bound (52). HuR translocates from nucleus to cytoplasm upon UV irradiation and stabilize the p21 message (49).
Gorospe et al. (28) describe that in cells lacking the p53 tumor suppressor protein UV was not only not able to induce p21 but also prevented the induction of p21 by mimosine. However, in our studies UVC inhibition is not dependent on the presence or absence of p53, therefore revealing a new mechanism and pathway by which UV is acting.
Inhibition of transcription initiation could be mediated by a cis-acting element located in exon 1 in the DNA. Many examples of regulatory elements downstream of the initiation site have been reported. We have used exon 1 DNA sequences as a probe in a bandshift assay and have not detected binding of proteins in nuclear extracts from cells treated or not treated with UV (data not shown). Transcription initiation could also be modulated by regulatory elements present in the RNA. Such regulation has been demonstrated for example for the HIV trans-activator protein protein that binds the Tat-responsive sequence element in the newly synthesized mRNA and enhances transcription initiation by interacting with the basal transcription machinery.
Finally, transcript elongation could be the target of UVC action. This type of regulation is widely observed in nature and can utilize various mechanism (see Ref. 53 for review). DNA-binding proteins can interfere with the elongation of the nascent RNA. In addition, RNA binding-proteins like trans-activator protein or Trp RNA-binding attenuation protein bind to the newly transcribed RNA and arrest progression of RNA polymerase (54).
DNA damage per se is capable of inhibiting transcript elongation (see Ref. 55 for review). Cyclobutane pyrimidine dimers, the most prevalent lesion formed by short wavelength UV radiation, are known to block DNA replication and gene transcription. At the dose used in this study (30 J/m2), the frequency of cyclobutane pyrimidine dimers in the irradiated genome should be of about 1 in 3,000 base pairs (56). Because lesion in the untranscribed strand of DNA is not subject to transcription arrest (57), this calculates a frequency of about 1 in 6,000 base pairs in the transcribed strand. The CHOP gene is a short gene, and cyclobutane pyrimidine dimers are a good substrate for transcription-coupled repair and should therefore be rapidly removed. Furthermore, inhibition of CHOP inducibility could be seen at lower UVC dose of 12 J/m2 (data not shown). It is therefore unlikely that DNA lesions in the CHOP gene are responsible for the lack of CHOP induction by UVC. Also the CAT reporter gene under the CHOP promoter is strongly activated by UVC, which could not occur if transcription elongation was blocked by DNA lesions. It is, however, possible that the exon 1 of the CHOP gene represents a hot spot for UVC-induced lesions. These hot spots have been described (58). Also changes in the kinetic of repair of DNA have been described, for example, in the p53 gene leading to mutational hot spots where repair is slow (59).
In summary, we report here a new finding about the regulation of CHOP
gene expression by UVC, namely an inhibitory effect on expression Most
studies on UVC have concentrated on the activation of gene and protein
expression. The inhibitory effect we describe here is interesting in at
least two respects: it reconciles the contradictions observed in the
literature and also in our own studies, and it reveals a new mechanism
by which UVC acts, namely on an untranslated 5' exon. This mechanism of
inhibition of gene expression by UVC may represent a quick and
economical way (in a period of high stress) of turning off genes whose
expression is not favorable for the cell at a given moment but might be
needed later on.
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ACKNOWLEDGEMENTS |
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We thank Edward V. Maytin for help in
preparing the UVC-irradiation protocol. We thank Peter W. Laird for the
pPGKPuro vector and Tyler Jacks and member of his lab for the p53 +/+
and p53 / mouse embryonic fibroblasts. We thank Townley Budde for
help in preparation of the manuscript and Richard Larraga for
electronic formatting of the manuscript and figures.
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FOOTNOTES |
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* 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.
Investigator with the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Lab. of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit St., WEL320, Boston, MA 02114. Tel.: 617-726-5190; Fax: 617-726-6954; E-mail: jhabener@partners.org.
Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.M007440200
2 M. Schmitt-Ney, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; CAT, chloramphenicol acetyltransferase; MAP, mitogen-activated protein; MMS, methylmethane sulfonate; UVC, ultraviolet light wavelength band C; HIV, human immunodeficiency virus.
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REFERENCES
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