Identification of ERSE-II, a New cis-Acting Element Responsible for the ATF6-dependent Mammalian Unfolded Protein Response*

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 acis-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 thiscis-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.

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 stressresponsive 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.

EXPERIMENTAL PROCEDURES
Cloning and Sequencing of the Human Herp Gene-A human whole blood, 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 5Ј-TGG-TTTCTCCGGTTACAC-3Ј and 5Ј-AGAGACCACAGGTATCTC-3Ј as primers with the plate lysates as templates. Two positive phages were cloned by limiting serial dilution. The insert DNAs isolated from these clones (ϳ17 kilobases each) were sequenced using a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (PerkinElmer Life Sciences).
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 antidigoxigenin antibodies, followed by counterstaining with 4Ј,6-diamidino-2-phenylindole dihydrochloride n-hydrate.
Construction of Plasmids-Progressive deletion fragments of the Herp gene 5Ј-flanking region were PCR-amplified using sense primers containing an additional 5Ј-BglII site and an antisense primer containing an original NcoI site at the initial Met of Herp (5Ј-TTCGGTCTCG-GACTCCATGGC-3Ј). 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 l/well FuGENE6 transfection reagent (Roche Molecular Biochemicals), we transfected HUVECs with 0.5 g/ 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 g/ 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 M thapsigargin (Sigma), 10 g/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 l of Passive Lysis Buffer (Promega). We measured the firefly and Renilla luciferase activities of 20 l 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 3Ј-Untranslated Region (UTR) of the Luciferase Gene-To make the luciferase mRNA unstable, we inserted a synthetic double-stranded oligonucleotide, 5Ј-TAATATTTA-TATATTTATATTTTTAAAATATTTATTTATTTATTTATTTAA-3Ј, into the XbaI sites of both the pGL3-Basic and pGL3-Control plasmids. This AT-rich sequence was derived from 3Ј-UTR of granulocyte-monocyte colony-stimulating factor (GM-CSF).
Fluorescent Immunocytochemistry-We constructed an expression plasmid coding for a FLAG-tagged version of ATF6 Met 1 -Asn 366 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 g/ml anti-Herp rabbit polyclonal antibody (1) and 10 g/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).

RESULTS
Genomic Structure of Human Herp-We obtained a complete sequence of the human Herp gene (GenBank accession no. AB034990) by comparison of two partially overlapping clones, isolated from the human genomic DNA library, to the Herp cDNA sequence (GenBank 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 5Ј-GT and 3Ј-AG consensus sequences. The 5Ј-terminal transcription start site had been previously determined by cap-site hunting (1). Exon 1 encoded the 5Ј-UTR and the first 49 N-terminal residues including the initial Met codon. The stop codon and the 3Ј-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 5Ј-Flanking Promoter Region-We sequenced the ϳ6-kilobase pair 5Ј-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 cisacting 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 5Ј-flanking region contained several GC boxes (GGCG), suggesting multiple Sp1-binding sites.
The Herp promoter region contains one ERSE-like sequence, Ϫ88 CCAATGGGCGGCAGCCACA Ϫ70 , 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-N 9 -CCACG, is necessary and sufficient for the induction of the ER-resident molecular chaperones, GRP78, GRP94, and calreticulin. Although the G nucleotide at the 3Ј 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.
Functional Mapping of the Herp Promoter-A series of reporter plasmids containing sense fragments of the Herp 5Јflanking region (from nucleotide Ϫ5000 to Ϫ200) 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.
The basal luciferase activity of plasmid containing the longest 5Ј-flanking sequence (Ϫ5000/ϩ98) exhibited approximately half the activity of an SV40 promoter control (Fig. 2). Thapsigargin, an inhibitor of ER-resident Ca 2ϩ -ATPase, is used experimentally to activate the UPR. Following a 6-h treatment with 1 M thapsigargin, luciferase activity increased significantly (ϳ4.3-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 Ϫ5000 to Ϫ1800 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 Ϫ1000/ϩ98, Ϫ800/ϩ98, Ϫ600/ϩ98, and Ϫ400/ϩ98 were similar in magnitude to Ϫ1800/ϩ98. Al-though removal of the Ϫ400 to Ϫ200 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 Ϫ200/ϩ98 region to analyze this hypothesis in detail.
Disruption of ERSE in the Herp Promoter-One ERSE-like sequence, Ϫ88 CCAATgggcggcagCCACA Ϫ70 , is contained within the Herp 5Ј-flanking region. As the A nucleotide at the 3Ј end was different from a G in the ERSE consensus, CCAAT-N 9 -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 induc- tion. 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.
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 3Ј-UTR of the firefly luciferase plasmids. The 51-nucleotide stretch (TAATATTTATATATTTATATTTTTAAAATATTTATT-TATTTATTTATTTAA), known to selectively destabilize mRNA, was identified from the 3Ј-UTR of GM-CSF cDNA (25). Insertion of this sequence into the luciferase 3Ј-UTR of the plasmid containing the Herp Ϫ200/ϩ98 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.
Identification of ERSE-II-To identify additional cis-ele-ments involved in thapsigargin-induction, we made a series of mutant plasmids that also contained the disrupted ERSE. First, we searched the region from nucleotide Ϫ196 to Ϫ89, 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, Ϫ186 GCGGGT-TGCA Ϫ177 and Ϫ176 TCAGCCCGTG Ϫ167 were mutated, although the induction by thapsigargin treatment remained intact (lines 4 and 5). Mutation of Ϫ126 GCCGATTGGG Ϫ117 or Ϫ116 CCACGTTGGG Ϫ107 , however, resulted in a significant de- crease 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 Ϫ122, Ϫ121, Ϫ120, Ϫ119, Ϫ118, Ϫ116, Ϫ115, Ϫ114, Ϫ113, and Ϫ112 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, Ϫ122 ATTGGgC-CACG Ϫ112 , 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 ciselement, ERSE-II.
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 5Ј-flanking region (Ϫ200/ϩ98) 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 cisacting elements, contributing equally to the UPR-dependent induction of Herp mRNA.
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 FLAGtagged 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 Ϫ200/ϩ98 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 ATF6dependent UPR.
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), sug- gesting 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. DISCUSSION 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-N 9 -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-N 9 -CCACA. Therefore, the ERSE consensus should be described as containing the sequence: CCAAT-N 9 -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 ATF6mediated 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.
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 ( Ϫ93 CCAATgagcgc-ccgCCgCG Ϫ75 ) in the promoter region (27). We found two ERSE-II-like sequences, Ϫ267 ATTGGaCCACG Ϫ277 and Ϫ160 AT-TGGaCCACG Ϫ170 , 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 (Ϫ257 to Ϫ136). 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. 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.