From the National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan
Received for publication, November 20, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Herp is a 54-kDa membrane protein in the
endoplasmic reticulum (ER). The mRNA expression level of Herp is
increased by the accumulation of unfolded proteins in the ER.
Transcriptional changes designed to deal with this type of ER stress is
called the unfolded protein response (UPR). Most mammalian UPR-target
genes encode ER-resident molecular chaperones: GRP78, GRP94, and
calreticulin. The promoter regions of these genes contain a
cis-acting ER stress response element, ERSE, with the
consensus sequence of CCAAT-N9-CCACG. Under conditions of
ER stress, p50ATF6 (the active form of the transcription factor, ATF6)
binds to CCACG when CCAAT is bound by the general transcription factor,
NF-Y/CBF. Here, we report the genomic structure of human Herp and the
presence of a new ER stress response element, ERSE-II, in its promoter
region. The gene for Herp consists of eight exons, localized to
chromosome 16q12.2-13. The promoter region contains a single ERSE-like
sequence. In reporter gene assays, disruption of this
cis-element resulted in a partial reduction of the
transcriptional response to ER stress, suggesting that the element is
functional for the UPR. These results also suggest the involvement of
additional elements in the UPR. Further analysis, using an optimized
plasmid containing an mRNA-destabilizing sequence, revealed ERSE-II
(ATTGG-N-CCACG) as the second ER stress response element.
Interestingly, ERSE-II was also dependent on p50ATF6, in a manner
similar to that of ERSE, despite the disparate structure. The strong
induction of Herp mRNA by ER stress would be achieved by the
cooperation of ERSE and ERSE-II.
The expression of Herp, a 54-kDa endoplasmic reticulum
(ER)1-resident protein, is
induced by the unfolded protein response (UPR) (1). The UPR is a
transcriptional response to remedy the accumulation of unfolded
proteins in the ER. Most previously identified UPR-target genes encode
ER-resident molecular chaperones and folding enzymes, such as
GRP78/BiP, GRP94, protein-disulfide isomerase, and calreticulin. In
contrast to these ER luminal proteins, Herp is an integral membrane
protein, both N and C termini of which face the cytoplasmic side of the
ER. This membrane topology makes it unlikely that Herp acts as a
molecular chaperone for proteins in the ER. Herp plays an unknown role
in the cellular survival response to the accumulation of unfolded
proteins in the ER.
The ER provides an optimal environment for the synthesis, folding, and
assembly of membrane and secreted proteins. The accumulation of
unfolded or misfolded proteins in the ER under conditions of "ER
stress" threatens the normal functioning of eukaryotic cells. Although the physiological conditions inducing ER stress are not fully
understood, the cellular response to the stress is essential for
homeostasis (comprehensively reviewed by Kaufman (Ref. 2)). The
ER-stress responses are currently categorized to three mechanisms: transcriptional induction, translational attenuation, and degradation (reviewed by Mori (Ref. 3)). In addition, ER stress activates c-Jun
N-terminal kinases (4) and induces caspase-12-mediated apoptosis
(5).
The molecular mechanism of the UPR is extensively defined in the yeast,
Saccharomyces cerevisiae. The ER luminal domain of Ire1p, an
ER-resident type I transmembrane protein, senses the accumulation of
unfolded proteins in the ER, activating its cytoplasmic endoribonuclease domain through homo-oligomerization and
trans-autophosphorylation (6, 7). Activated Ire1p triggers the
production of a transcription factor, Hac1p, through an unusual
mRNA splicing mechanism (8-10). Hac1p binds to the
UPR-dependent cis-acting element, UPRE, in the
promoter regions of UPR-target genes to activate their transcription (8, 11, 12). In mammals, two Ire1p orthologs, IRE1 ERSE, possessing a consensus sequence of CCAAT-N9-CCACG, is
necessary and sufficient for the induction of at least three major ER
chaperones (GRP78, GRP94, and calreticulin) (15). This sequence is
present in the proximal promoter regions of many ER stress-responsive proteins (15, 18). The general transcription factor, NF-Y/CBF, binds to
the CCAAT motif of ERSE (17, 19, 20). Under conditions of ER stress,
p50ATF6 binds to the CCACG motif of ERSE, resulting in the
transcriptional induction of ER chaperones (17). Multiple ER
stress-responsive genes, however, possess proximal promoter regions
without an ERSE sequence, such as FKBP13 (15), asparagine synthetase
(21), ATF3 (22), and
RTP/NDRG1.2
In the present paper, we demonstrate the existence of a new ER stress
response element, ERSE-II, found in the Herp promoter region. In a
manner similar to ERSE, ERSE-II mediates the ATF6-dependent UPR.
Cloning and Sequencing of the Human Herp Gene--
A human whole
blood, Fluorescence in Situ Hybridization (FISH) Analysis--
The
P1-derived artificial chromosome clone containing the Herp gene was
isolated from a human PAC DNA library (GenomeSystems) using Herp
cDNA as a probe. DNA from the clone, labeled by nick translation
with digoxigenin dUTP, was hybridized to normal metaphase chromosomes
derived from phytohemagglutinin-stimulated peripheral blood
lymphocytes. Specific hybridization signals were detected using
fluorescein-conjugated anti-digoxigenin antibodies, followed by
counterstaining with 4',6-diamidino-2-phenylindole dihydrochloride n-hydrate.
Construction of Plasmids--
Progressive deletion fragments of
the Herp gene 5'-flanking region were PCR-amplified using sense primers
containing an additional 5'-BglII site and an antisense
primer containing an original NcoI site at the initial Met
of Herp (5'-TTCGGTCTCGGACTCCATGGC-3'). After digestion with
BglII and NcoI, the fragments were inserted between the BglII and NcoI sites of the firefly
luciferase reporter plasmid, pGL3-Basic (Promega). Site-directed
mutations were introduced into the inserts by using oligonucleotide
primers containing the desired mutations, according to the QuikChange
site-directed mutagenesis kit protocol (Stratagene). All inserts were
sequenced to confirm the desired sequence.
Cell Culture and Luciferase Assay--
Human umbilical vein
endothelial cells (HUVECs, Clonetics) were cultured on 24-well plates
coated with type I collagen (Sumitomo Bakelite) in MCDB131 medium (Life
Technologies, Inc.), supplemented with 10 mM glutamine
(Life Technologies, Inc.), 20 mM Hepes-NaOH (pH 7.4), 2%
fetal bovine serum (Life Technologies, Inc.), and 10 ng/ml human basic
fibroblast growth factor (R&D Systems). Using 1.05 µl/well FuGENE6
transfection reagent (Roche Molecular Biochemicals), we transfected
HUVECs with 0.5 µg/well amounts of either the pGL3-Basic-derived plasmid described above or control plasmid (pGL3-Control, firefly luciferase vector with SV40 promoter and enhancer sequences, Promega) together with 0.025 µg/well of the internal control plasmid
(pRL-SV40, Renilla reniformis luciferase vector with SV40
promoter and enhancer sequences, Promega). Following a 23-h incubation,
cells were incubated for 6 h in either 1 µM
thapsigargin (Sigma), 10 µg/ml tunicamycin (Sigma), or 10 mM 2-mercaptoethanol (Nacalai Tesque). Cells were then
washed with Dulbecco's PBS (Life Technologies, Inc.) and harvested in
100 µl of Passive Lysis Buffer (Promega). We measured the firefly and
Renilla luciferase activities of 20 µl of each lysate
using a Dual-Luciferase reporter assay system (Promega). Bioluminescence was detected using a LUMINOUS CT-9000 luminometer (Dia-Iatron). After dividing luminescence intensity of firefly luciferase by that of Renilla luciferase, we determined the
"relative luciferase activity" to be the ratio of the value
obtained from each test plasmid to that of the pGL3-Control. In each
assay, the values were averaged from four independent wells.
Insertion of AT-rich Sequence to the 3'-Untranslated Region (UTR)
of the Luciferase Gene--
To make the luciferase mRNA
unstable, we inserted a synthetic double-stranded
oligonucleotide,
5'-TAATATTTATATATTTATATTTTTAAAATATTTATTTATTTATTTATTTAA-3', into
the XbaI sites of both the pGL3-Basic and pGL3-Control
plasmids. This AT-rich sequence was derived from 3'-UTR of
granulocyte-monocyte colony-stimulating factor (GM-CSF).
Transient Expression of ATF6(366)--
The cDNA encoding
human ATF6 was the kind gift of Dr. Hiderou Yoshida and Dr. Kazutoshi
Mori (Kyoto University, Kyoto, Japan). The partial open reading frame
corresponding to Met1-Asn366 was
PCR-amplified; the product was inserted into the mammalian expression
vector, pcDNA3.1(+) (Invitrogen). To express ATF6(366) transiently,
the resultant plasmid pcDNA3ATF6(366) (0.01 µg/well) was
transfected to HUVECs together with the luciferase plasmids (0.5 µg/well). As a negative control, the mock vector pcDNA3.1(+) (0.01 µg/well) was cotransfected in place of pcDNA3ATF6(366).
Fluorescent Immunocytochemistry--
We constructed an
expression plasmid coding for a FLAG-tagged version of ATF6
Met1-Asn366 and transfected this plasmid into
HUVECs. After a 21-h incubation, cells were rinsed with Dulbecco's
PBS, fixed in 2% paraformaldehyde for 15 min, and permeabilized with
0.05% Triton X-100 for 2 min. Following an incubation in 5% normal
goat serum and 5% fish gelatin for 30 min, we detected endogenous Herp
and transiently expressed FLAG-ATF6(366) simultaneously in 1-h
incubation of 20 µg/ml anti-Herp rabbit polyclonal antibody (1) and
10 µg/ml anti-FLAG M2 mouse monoclonal antibody (Eastman Kodak Co.).
Cells were then incubated with Oregon Green 514-conjugated goat
anti-rabbit IgG (Molecular Probes) and Rhodamine Red-X-conjugated goat
anti-mouse IgG (Molecular Probes) for 1 h. After washing with PBS,
fluorescence was visualized using a confocal laser-scanning microscope
with FLUOVIEW (Olympus).
Genomic Structure of Human Herp--
We obtained a complete
sequence of the human Herp gene (GenBankTM accession no. AB034990) by
comparison of two partially overlapping clones, isolated from the human
genomic DNA library, to the Herp cDNA sequence (GenBankTM
accession no. AB034989). The gene was officially designated
HERPUD1 by the HUGO Gene Nomenclature Committee. The
schematic structure, sequences across the exon-intron junctions, and
the sizes of exons and introns are shown in Fig. 1 (A and B). The
Herp gene contains eight exons and spans 11,738 bp in length. The
identified exon-intron junctions agreed with the intron 5'-GT and 3'-AG
consensus sequences. The 5'-terminal transcription start site had been
previously determined by cap-site hunting (1). Exon 1 encoded the
5'-UTR and the first 49 N-terminal residues including the initial Met
codon. The stop codon and the 3'-UTR were encoded by exon 8.
To localize the Herp gene on human chromosomes, we performed FISH
analysis utilizing DNA from the P1-derived artificial chromosome clone
containing the Herp gene. Labeled Herp DNA was hybridized to
chromosomes derived from peripheral blood lymphocytes. Eighty metaphase
cells were analyzed; 73 exhibited specific labeling of the 16q12.2-13
region (Fig. 1C).
Sequence of the 5'-Flanking Promoter Region--
We sequenced the
~6-kilobase pair 5'-flanking region of the Herp gene. Computer
analysis by TFSEARCH using the TRANSFAC data base (24) revealed many
potential transcription factor-binding sites within the sequence. The
proximal 200-bp sequence upstream of the transcriptional start site,
including several putative cis-acting regulatory elements,
is shown in Fig. 1D. The canonical TATA box, specifying the
transcriptional start site, is found in close proximity to exon 1. Two
CAAT boxes were also identified. The 5'-flanking region contained
several GC boxes (GGCG), suggesting multiple Sp1-binding sites.
The Herp promoter region contains one ERSE-like sequence,
Functional Mapping of the Herp Promoter--
A series of reporter
plasmids containing sense fragments of the Herp 5'-flanking region
(from nucleotide
The basal luciferase activity of plasmid containing the longest
5'-flanking sequence ( Disruption of ERSE in the Herp Promoter--
One ERSE-like
sequence, Optimization of the Reporter Plasmid to Monitor the Induction
Effectively--
Observation of the effects of stimulants on
transcriptional induction in reporter gene assays is contingent on a
faster turnover of mRNA produced from the test plasmid DNA. We,
therefore, introduced an AT-rich sequence into the 3'-UTR of the
firefly luciferase plasmids. The 51-nucleotide stretch
(TAATATTTATATATTTATATTTTTAAAATATTTATTTATTTATTTATTTAA), known to
selectively destabilize mRNA, was identified from the 3'-UTR of
GM-CSF cDNA (25). Insertion of this sequence into the luciferase
3'-UTR of the plasmid containing the Herp Identification of ERSE-II--
To identify additional
cis-elements involved in thapsigargin-induction, we made a
series of mutant plasmids that also contained the disrupted ERSE.
First, we searched the region from nucleotide Functional Contribution of ERSE and ERSE-II to the UPR--
To
compare the activity of ERSE and ERSE-II, we measured the luciferase
activity of plasmids containing combination of mutations in these two
cis-elements. We utilized not only thapsigargin but also
tunicamycin (N-glycosylation inhibitor) and mercaptoethanol (reducing agent) as ER-stress inducers to see specific induction by the
UPR. Plasmid DNA containing the 5'-flanking region ( Effect of ATF6 Overexpression on the Herp Promoter
Activity--
The general transcription factor, NF-Y,
constitutively binds the CCAAT motif of ERSE (17, 18). The
transcription factor, ATF6 (p90ATF6), on the ER membrane is activated
by proteolysis in response to ER stress; the resultant N-terminal
soluble form (p50ATF6) moves into nuclei to bind directly to the CCACG
motif (16, 17). We, therefore, examined the effect of p50ATF6
overexpression on the induction of Herp expression. As the cleavage
site involved in conversion from p90ATF6 to p50ATF6 is unknown, we
utilized ATF6(366), an N-terminal soluble fragment containing the
entire basic region and majority of the leucine zipper region of ATF6. ATF6(366) translocates to the nucleus to enhance the levels of GRP78
mRNA (16). Upon transfection of the expression plasmid encoding
FLAG-tagged ATF6(366) into HUVECs, a fraction of transfected cells
possessed nuclei recognized by an anti-FLAG-tag antibody, indicating
that the expressed ATF6(366) was present in nuclei (Fig.
6A, red signal).
Cells with immunonegative nuclei were also observed, likely due to a
failure of transfection. Following staining of cells with an anti-Herp
antibody, immunopositive signals of the ER in ATF6(366)-expressing
cells were stronger than those in cells without ATF6(366) (Fig.
6A, green signal). This suggests that
overexpressed ATF6(366) functions in vivo to induce the
expression of Herp in the ER.
To demonstrate that p50ATF6 induces the transcriptional activity of the
Herp promoter, the plasmid containing the
p50ATF6 binds directly to the CCACG portion of ERSE to exert its
ability as a trans-factor (17). Mutation of CCACA/G motifs of both ERSE
and ERSE-II in the Herp promoter abrogated the inducible effect of
ATF6(366) (Fig. 6B, line 5), suggesting that the
enhancer activity of p50ATF6 requires the CCACG sequences of both
ERSE-II and ERSE. p50ATF6 binds to CCACG only when CCAAT is bound by
NF-Y, exactly 9 bp upstream of CCACG (17). Mutation of the CCAAT motifs of both ERSE and ERSE-II abrogated the ATF6 effect as well (line 6). The indispensability of NF-Y binding is also applicable to ERSE-II as well as ERSE, despite the differences in both the direction and interval of CCAAT and CCACG in ERSE-II from those in ERSE.
We identified two cis-acting elements responsible for
the UPR-dependent transcriptional induction in the proximal
promoter region of the Herp gene. CCAATgggcggcagCCACA is almost
identical to the 19-nucleotide consensus sequence of ERSE,
CCAAT-N9-CCACG (15). The other, ATTGG-N-CCACG, is a new
element, termed ERSE-II. ERSE-II also contains two motifs, CCAAT
(complementary to ATTGG) and CCACG, although the orientation and the
interval between them are different from ERSE. Moreover, ERSE-II
functions as an ER stress response element in an
ATF6-dependent fashion, in the same manner as the original
ERSE.
The A nucleotide at position 19 in ERSE of the Herp promoter differs
from a G of the ERSE consensus. Our data, however, could not
demonstrate a significant functional difference in response to
thapsigargin treatment between A and G at this position (Fig. 3A). Yoshida et al. (15) demonstrated that
substitution of the nucleotide G to T was a crucial mutation, impairing
the UPR; they did not, however, examine the effect of substitution to
A. Furthermore, ERSE-like sequences also appear in the human ER
stress-responsive genes, GRP58 (15) and SERCA2
(26), with a sequence of CCAAT-N9-CCACA. Therefore, the
ERSE consensus should be described as containing the sequence:
CCAAT-N9-CCACG/A.
It has been reported that the transcription factors, NF-Y and ATF6,
simultaneously bind to the CCAAT and CCACG portions of ERSE,
respectively (17). The former is considered to bind in a constitutive
manner, independent of the UPR. The latter binds only when it is
converted from the ER membrane-embedded p90ATF6 to the soluble p50ATF6
by processing induced by ER stress (16, 17). Binding of ATF6 to CCACG
requires the binding of NF-Y to the CCAAT sequence at a position
exactly 9 bp upstream of CCACG (17). This 9-bp distance is critical;
neither 8 nor 10 bp is acceptable (17). The unidirectional necessity,
however, of CCAAT and CCACG was not investigated. Our data indicate
that the role of ERSE-II as a cis-acting element was exerted
by ATF6 as was the case with that of ERSE (Fig. 6). Both the CCACG and
the ATTGG (complementary to CCAAT) sequences of ERSE-II were critical
for ATF6-mediated transcription. Although direct evidence is not
available, it is likely that both NF-Y and p50ATF6 bind to ERSE-II to
enhance transcription (Fig. 7). We
observed specific binding of NF-Y to the CCACG sequence of ERSE-II
in vitro (data not shown). If our model is correct, the
inverse direction of two motifs may be necessary when the distance
between them is 1 bp, not 9. A study of the steric structure of
protein-DNA interaction will help determine the validity of this
argument. By analogy to ERSE, other transcription factors, such as
CREB-RP (15) and XBP-1 (17), may bind to the CCACG sequence of
ERSE-II.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
and IRE1
, have
been identified (13, 14), although the substrates of their
endoribonuclease activities are unknown. ATF6 has been identified as
the transcription factor responsible for the UPR (15). The quiescent
form of ATF6 (p90ATF6), a type II-transmembrane protein, is embedded in
the ER membrane and proteolyzed in an ER stress-dependent
manner (16). The liberated N-terminal fragment (p50ATF6) translocates
to the nucleus, binding to the mammalian UPR-dependent
cis-acting element, designated the ER stress response element (ERSE) (15, 17).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
genomic library (Stratagene) was screened to obtain genomic
clones encoding Herp using the PCR-based screening method described
previously (23). Positive phages were cloned utilizing the
Escherichia coli XL1-Blue MRA strain as host. PCR was
performed using 5'-TGGTTTCTCCGGTTACAC-3' and 5'-AGAGACCACAGGTATCTC-3'
as primers with the plate lysates as templates. Two positive
phages were cloned by limiting serial dilution. The insert DNAs
isolated from these clones (~17 kilobases each) were sequenced using
a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (PerkinElmer
Life Sciences).
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ABSTRACT
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Fig. 1.
Human Herp gene. A, genomic
organization. Exons are indicated by black boxes and
numbered. The initial Met and stop codons appear in exons 1 and 8, respectively. B, exon-intron boundaries. The exon
sequences, deduced from comparison to the Herp cDNA, are given in
uppercase letters, whereas the intron sequences are in
lowercase letters. All introns start with GT and end with
AG. The potential polyadenylation signal is underlined, and
the polyadenylation site at the 3' end of exon 8 is indicated by an
arrowhead. The complete sequence data have been submitted to
the GenBankTM/EMBL/DDBJ data bases under the accession number
AB034990. The gene has been officially designated HERPUD1 by
the HUGO Gene Nomenclature Committee. C, FISH analysis.
Labeled DNA containing the Herp gene was hybridized to metaphase
chromosomes. The hybridization signals on chromosome 16 are indicated
by arrows. D, sequence of 5'-flanking region. Exon 1 is
shaded. Negative numbers indicate the
distance of the nucleotide from the transcription start site. Putative
regulatory motifs are labeled with lines. The motif
corresponding to ERSE is shown in black-boxed white
letters.
88CCAATGGGCGGCAGCCACA
70, located upstream
of the TATA box (Fig. 1D). ERSE is a cis-acting regulatory element identified in the promoters of mammalian UPR target
genes (15). ERSE, with a consensus of CCAAT-N9-CCACG, is
necessary and sufficient for the induction of the ER-resident molecular
chaperones, GRP78, GRP94, and calreticulin. Although the G nucleotide
at the 3' end of the consensus sequence is replaced by an A in the Herp
ERSE, we predict this sequence functions in the
UPR-dependent induction of Herp expression at the
transcriptional level.
5000 to
200) upstream of the firefly luciferase
gene were transfected into HUVECs. The firefly luciferase activity in
each assay was normalized to a cotransfected Renilla
luciferase plasmid, pRL-SV40, to compensate for a varied efficiency of transfection.
5000/+98) exhibited approximately half the
activity of an SV40 promoter control (Fig.
2). Thapsigargin, an inhibitor of
ER-resident Ca2+-ATPase, is used experimentally to activate
the UPR. Following a 6-h treatment with 1 µM
thapsigargin, luciferase activity increased significantly (~4.3-fold)
over basal activity, consistent with previous results demonstrating the
induction of Herp mRNA by thapsigargin (1). The SV40 promoter
encoded by the pGL3-Control vector did not respond to thapsigargin
treatment. Removal of the
5000 to
1800 region of Herp resulted in
an increase of basal activity, suggesting the existence of silencing
element in the region; little effect, however, was observed in the
response to thapsigargin. Both the basal and thapsigargin-treated
activities of plasmids containing
1000/+98,
800/+98,
600/+98, and
400/+98 were similar in magnitude to
1800/+98. Although removal of
the
400 to
200 region resulted in a reduction of basal activity,
the strong induction of luciferase activity in response to thapsigargin
remained intact. We, therefore, concluded that the
cis-elements responsible for the response to thapsigargin
treatment would lie within the region 200 bp upstream of the
transcription start site. In the following experiments, we used a
plasmid containing the
200/+98 region to analyze this hypothesis in
detail.
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Fig. 2.
Map of the functional elements in the Herp
promoter. HUVECs were transfected with luciferase reporter
plasmids containing progressive 5' end deletions from nucleotide
5000, made from pGL3-Basic and the Herp gene. After 23 h, the
cells were incubated with or without 1 µM thapsigargin
for 6 h and assayed for luciferase activity. Basal and
thapsigargin-treated activities are shown by open and
closed bars, respectively. Values averaged from four
independent experiments are shown with standard deviations relative to
the basal activity of pGL3-Control containing the SV40 promoter.
Tg, thapsigargin.
88CCAATgggcggcagCCACA
70, is
contained within the Herp 5'-flanking region. As the A nucleotide at
the 3' end was different from a G in the ERSE consensus,
CCAAT-N9-CCACG, we examined the transcriptional
effect of this nucleotide difference. Both basal and
thapsigargin-treated activities of the plasmid containing
CCACg (Fig. 3A,
line 2) were similar to those of the original plasmid
(line 1), suggesting that the A nucleotide functions similarly to a G nucleotide in the response to thapsigargin. We performed site-directed mutagenesis on two motifs of ERSE and examined
the effects on the transcriptional induction following thapsigargin
treatment. Throughout this paper, the term "mutation" is defined as
the substitution of A, C, G, and T for C, A, T, and G, respectively.
Disruptive mutation of either of the two motifs, CCAAT or CCACA,
resulted in a partial reduction of the thapsigargin-dependent induction of luciferase activity
(Fig. 3A, lines 3 and 4), indicating
their involvement in the induction. Mutation of both motifs, however,
did not completely abrogate the response to thapsigargin (line
5). These results suggest that other cis-elements are
involved in thapsigargin-dependent transcriptional induction. Under these experimental conditions, however, the observed inducibilities were not high enough to define the elements. We, therefore, modified the plasmid DNAs to effectively monitor the difference in activity with or without thapsigargin treatment.
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Fig. 3.
Disruption of ERSE and optimization of the
reporter plasmid. A, mutational effects of ERSE on Herp
promoter activity. The ERSE-like sequence in the Herp 5' region,
CCAATgggcggcagCCACA, was mutated to CCAATgggcggcagCCACg
(line 2), aaccggggcggcagCCACA (line
3), CCAATgggcggcagaacac (line 4),
aaccggggcggcagaacac (line 5); the
luciferase activities of each were then measured. Basal and
thapsigargin-treated activities are shown in open and
closed bars, respectively. B, effect of an
mRNA-destabilizing sequence on induction. The AT-rich sequence in
the 3'-UTR of GM-CSF was inserted downstream of the luciferase gene of
both the plasmid containing the 200/+98 region of Herp (line
2) and the pGL3-Control plasmid (line 4). We compared
the luciferase activities of these plasmids to the parent plasmids
(lines 1 and 3) in the absence (open
bars) or presence (closed bars) of thapsigargin.
Tg, thapsigargin.
200/+98 region resulted in
dramatic reduction of the basal activity; the activity in the presence
of thapsigargin was relatively unchanged (compare lines 1 and 2 in Fig. 3B). As a result, the induction rate of thapsigargin treatment increased from 2.8 to 7.7 in this assay.
Insertion of the AT-rich sequence into the control pGL3-Control plasmid, containing the SV40 promoter, had little effect on the ratio
of basal to thapsigargin-treated activities (1.1 to 1.4, lines
3 and 4), although the luciferase activities were
reduced in both cases. We utilized this optimized plasmid to identify additional transcriptional control elements in the Herp promoter region.
196 to
89, making 11 sets of consecutive 10-bp mutations. After measuring the resulting
luciferase activities (Fig.
4A), we found that basal
activities were reduced when two regions,
186GCGGGTTGCA
177 and
176TCAGCCCGTG
167 were mutated, although the
induction by thapsigargin treatment remained intact (lines 4 and 5). Mutation of
126GCCGATTGGG
117 or
116CCACGTTGGG
107, however, resulted in a
significant decrease of luciferase activity upon thapsigargin
treatment, despite little effect on basal activity (lines 10 and 11). To identify the nucleotides involved in the thapsigargin response, we assessed the effects of 14 nucleotide mutations crossing these two regions on luciferase activity (Fig. 4B). Mutations at
122,
121,
120,
119,
118,
116,
115,
114,
113, and
112 demonstrated inhibitory effects on the
thapsigargin-induced response of luciferase activity (lines
3-13 except line 8). These results indicate that the
11-bp stretch,
122ATTGGgCCACG
112, in the
Herp promoter region is responsible for the transcriptional response to
thapsigargin. This 11-bp sequence contains two motifs forming the ERSE
consensus, CCAAT (complementary to ATTGG) and CCACG, although the
orientation of the first sequence is inverted. We termed this
cis-element, ERSE-II.
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Fig. 4.
Identification of ERSE-II. A,
effect of 10-bp mutations on Herp promoter activity. Each of 10-bp
units upstream of the ERSE was disrupted; the effect on luciferase
activities was analyzed. Luciferase induction by thapsigargin treatment
was reduced when nucleotides 126/
117 (line 10) and
116/
107 (line 11) were mutated. B, effect of
1-bp mutations on Herp promoter activity. Nucleotides from
123 to
110, upstream of the disrupted ERSE, were separately mutated; the
effect on luciferase activity was then analyzed. Luciferase induction
by thapsigargin treatment was reduced when nucleotides
122
(line 3),
121 (line 4),
120 (line
5),
119 (line 6),
118 (line 7),
116
(line 9),
115 (line 10),
114 (line
11),
113 (line 12), and
112 (line 13)
were mutated. Tg, thapsigargin.
200/+98) of the
Herp gene demonstrated enhanced activity in the presence of all the
reagents used (Fig. 5, line
1), in contrast to the control plasmid containing the SV40
promoter (line 5). Disruption of the original ERSE resulted
in decrease of the response to the ER-stress inducers but not in a
complete loss (line 2). In a similar way, disruption of the
novel ERSE-II also exhibited a weakened response (line 3).
When both elements were disrupted, the transcriptional induction by ER
stress was abrogated (line 4). These results suggest that
ERSE and ERSE-II would function independently as cis-acting elements, contributing equally to the UPR-dependent
induction of Herp mRNA.
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Fig. 5.
Comparative analysis of ERSE and ERSE-II as
cis-elements for the UPR. The luciferase activity
of plasmids under the control of the Herp 200/+98 region containing
either the disrupted ERSE (line 2), the disrupted ERSE-II
(line 3), or both (line 4) were compared with the
parent plasmid (line 1). Activity was measured in the
absence (open bars) or presence of either 1 µM
thapsigargin (closed bars), 10 µg/ml tunicamycin
(light gray bars), or 10 mM mercaptoethanol
(dark gray bars). Tg, thapsigargin;
Tm, tunicamycin; Me, mercaptoethanol.
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Fig. 6.
Effect of ATF6 overexpression on Herp
promoter activity. A, fluorescent immunocytochemical
analysis. FLAG-tagged ATF6(366) was transiently expressed in
HUVECs and double-stained with both anti-FLAG-tag and anti-Herp
antibodies. Bar, 20 µm. B, reporter gene
assays. Plasmid DNA encoding ATF6(366) was cotransfected into HUVECs
together with plasmids containing luciferase under the control of the
Herp 200/+98 region with various mutations. The activity of each was
measured (closed bars). The mock vector, pcDNA3.1(+),
was used as a control (open bars).
200/+98 region of Herp was
cotransfected into HUVECs in conjunction with the ATF6(366)-expression
plasmid. As expected, coexpression of ATF6(366) resulted in an
enhancement of luciferase activity (Fig. 6B, line
1). The induction was partially reduced when the two motifs, CCAAT
and CCACA, of ERSE were disrupted (line 2), indicating both
that the effect of ATF6(366) is dependent on the cis-element and that other elements are involved in this induction. Disruption of
both motifs, ATTGG (complementary to CCAAT) and CCACA, of ERSE-II also
demonstrated a partial reduction in induction (line 3).
Disruption of both elements, ERSE and ERSE-II, resulted in a complete
loss of the ATF6 effect (line 4). These data suggest that
both ERSE and ERSE-II are involved in the ATF6-dependent
UPR.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Model for UPR-dependent
transcriptional induction of Herp. The up-regulation of Herp
mRNA expression resulting from ER stress is regulated by two
cis-acting elements, ERSE and ERSE-II. NF-Y binds constantly
to the CCAAT sequence in these elements. Upon ER stress, p90ATF6 in the
ER membrane is activated to form p50ATF6, a soluble molecule that
translocates to the nucleus and binds to the CCACG/A sequence of ERSE
and ERSE-II, activating transcription. TBP, TATA-binding
protein.
Most UPR-target genes, such as GRP78, GRP94, and
the gene for calreticulin, are fully activated by multiple copies of
ERSE (15, 18). Despite a single ERSE, however, Herp mRNA induction by the UPR is very strong as compared with other ER chaperones (1).
ERSE-II may cooperate with ERSE to facilitate the strong induction of
Herp in response to ER stress. We searched for ERSE-II in other ER
stress-responsive genes to demonstrate the function of this sequence in
the response to cellular stress. ORP150 is an ER-resident protein whose
expression is induced by hypoxia; three distinct mRNA species are
produced by alternative promoters (27). One of them was preferentially
induced by hypoxia and ER stress, in a manner dependent on a single
ERSE-like sequence (93CCAATgagcgcccgCCgCG
75) in the promoter
region (27). We found two ERSE-II-like sequences,
267ATTGGaCCACG
277 and
160ATTGGaCCACG
170, upstream of the ERSE.
They also might be involved in the UPR.
Recently, van Laar et al. (28) identified a human methyl
methanesulfonate (MMS)-inducible gene, Mif1, identical to
Herp. The mRNA is also induced by tunicamycin, osmotic shock, and
UV irradiation. Although they demonstrated that one
cis-element, ERSE, was involved in the response to
tunicamycin, ERSE-II was not mentioned. The induction of
Mif1 by MMS was mediated by neither ERSE nor ERSE-II but by
a 122-bp fragment (257 to
136). As MMS also induces the mRNA
expression of GRP78 (28, 29), GRP94 (28), and
CHOP (29), known UPR-target genes, these genes and Herp may
share an additional cis-acting MMS response element.
The function of Herp is still unknown. It was believed that all proteins encoded by UPR-target genes functioned as molecular chaperones and folding enzymes to relieve the disturbance of the ER. As the majority of the molecule is exposed to the cytoplasm, Herp may play a role independent of molecular chaperones (1). ER stress also up-regulates the mRNA expression of the transcription factors, CHOP (29), ATF3 (22), and XBP-1 (17). In addition, a large number of UPR-target genes have been identified in yeast using microarray techniques that are not limited to proteins involved in ER folding (30). Further research from a wide viewpoint will be required to determine the physiological function of Herp.
To facilitate our study, we modified the plasmid DNA for reporter gene
assays. We reduced basal luciferase activity in cells by preventing the
accumulation of superfluous mRNA and enzyme prior to stimulation by
destabilizing the luciferase mRNA. This technique allowed us to
identify a new cis-element responsible for stimulation of
gene expression. Although we used this technique to detect response to
ER stress, the destabilization of reporter gene plasmids will be widely
applicable to the search for cis-elements responsible for
other conditions.
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ACKNOWLEDGEMENTS |
---|
We thank Akemi Fukumoto and Chikako Yasuda for technical assistance, and Dr. Hiderou Yoshida and Dr. Kazutoshi Mori for their kind donation of the ATF6 cDNA.
![]() |
FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Health and Welfare of Japan; from the Ministry of Education, Science, Sports, and Culture of Japan; from the Japan Society for the Promotion of Science; and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan.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.
The nucleotide sequence reported in this paper has been submitted to the GenBankTM/EMBL/DDBJ Data Bank with accession number AB034990.
To whom correspondence should be addressed: National
Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Tel.: 81-6-6833-5012 (ext. 2589); Fax: 81-6-6872-8091; E-mail: kame@ri.ncvc.go.jp.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M010486200
2 K. Kokame, H. Kato, and T. Miyata, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; UPR, unfolded protein response; ERSE, ER stress response element; FISH, fluorescence in situ hybridization; HUVEC, human umbilical vein endothelial cell; UTR, untranslated region; GM-CSF, granulocyte-monocyte colony-stimulating factor; MMS, methyl methanesulfonate; bp, base pair(s); PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
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