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. 2000 Sep;20(18):6755-67.
doi: 10.1128/MCB.20.18.6755-6767.2000.

ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response

H Yoshida  1 , T Okada, K Haze, H Yanagi, T Yura, M Negishi, K Mori
Affiliations

ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response

H Yoshida et al. Mol Cell Biol. 2000 Sep.

Abstract

Transcription of genes encoding molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) is induced by accumulation of unfolded proteins in the ER. This intracellular signaling, known as the unfolded protein response (UPR), is mediated by the cis-acting ER stress response element (ERSE) in mammals. In addition to ER chaperones, the mammalian transcription factor CHOP (also called GADD153) is induced by ER stress. We report here that the transcription factor XBP-1 (also called TREB5) is also induced by ER stress and that induction of CHOP and XBP-1 is mediated by ERSE. The ERSE consensus sequence is CCAAT-N(9)-CCACG. As the general transcription factor NF-Y (also known as CBF) binds to CCAAT, CCACG is considered to provide specificity in the mammalian UPR. We recently found that the basic leucine zipper protein ATF6 isolated as a CCACG-binding protein is synthesized as a transmembrane protein in the ER, and ER stress-induced proteolysis produces a soluble form of ATF6 that translocates into the nucleus. We report here that overexpression of soluble ATF6 activates transcription of the CHOP and XBP-1 genes as well as of ER chaperone genes constitutively, whereas overexpression of a dominant negative mutant of ATF6 blocks the induction by ER stress. Furthermore, we demonstrated that soluble ATF6 binds directly to CCACG only when CCAAT exactly 9 bp upstream of CCACG is bound to NF-Y. Based on these and other findings, we concluded that specific and direct interactions between ATF6 and ERSE are critical for transcriptional induction not only of ER chaperones but also of CHOP and XBP-1.

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Figures

FIG. 1
FIG. 1
Induction of XBP-1 and CHOP transcripts by ER stress. (A) Effects of various ER stress-inducing reagents on the levels of XBP-1 and CHOP mRNA. Total RNA was extracted from HeLa cells in 60-mm dishes that had been treated for 8 h with either 2 μg of tunicamycin (TM)/ml, 3 μM A23187, or 100 nM thapsigargin (Tg) and analyzed by Northern blot hybridization. The filter was hybridized with one of the 32P-labeled DNA probes specific for GRP78, XBP-1, CHOP, or GAPDH mRNA and then stripped for hybridization with a different probe. (B) Time course of induction. HeLa cells were treated with 2 μg of tunicamycin/ml for the indicated periods before total RNA was extracted and analyzed by Northern blot hybridization as for panel A.
FIG. 2
FIG. 2
Deletion analysis of the human XBP-1 (A) and CHOP (B) promoters. Various fragments derived from the human XBP-1 and CHOP promoter regions are shown schematically on the left. Numbers indicate nucleotide positions from the transcription start site. Solid and hatched boxes indicate the locations of ERSE motifs with perfect and considerable matches to the consensus, respectively (see Fig. 3A). Each of these fragments cloned immediately upstream of the firefly luciferase gene in the pGL3-Basic vector was transiently introduced into HeLa cells together with the pRL-SV40 reference plasmid as described in Materials and Methods. Relative luciferase activity in transfected cells incubated for 16 h with (solid boxes) or without (open boxes) 2 μg of tunicamycin (TM)/ml was determined, and averages from four independent experiments are presented with standard deviations (error bars). Fold induction was calculated by dividing the relative luciferase activity in TM-treated cells by that in untreated cells.
FIG. 3
FIG. 3
Presence of ERSE-like sequences in the human XBP-1 and CHOP promoters. (A) Comparison of ERSE-like sequences in the XBP-1 and CHOP promoters with the consensus. Nucleotides identical to the ERSE consensus are shaded. Numbers indicate the locations relative to the transcription start site. (B) Transcriptional activities of ERSE-like sequences in the XBP-1 and CHOP promoters. Oligonucleotides encoding each of the ERSE-like sequences (shaded) with indicated flanking nucleotides were inserted into the pGL3-Promoter vector, and the resulting plasmids were transiently introduced into HeLa cells with the pRL-SV40 reference plasmid. The relative luciferase activity in transfected cells incubated for 16 h with (solid boxes) or without (open boxes) 2 μg of tunicamycin (TM)/ml was determined, and averages from four independent experiments are presented with standard deviations (error bars).
FIG. 4
FIG. 4
Effects of disrupting ERSE on the activities of the human XBP-1 (A) and CHOP (B) promoters. Structures of the XBP-1 and CHOP promoters are shown schematically as described for Fig. 2. XBP-1–ERSE1, CHOP–ERSE1, and CHOP–ERSE2 were disrupted by mutating their sequences to agAtcN9CCACG, gatcTN9tacat, and gatccN9tgcga, respectively (mutated nucleotides are indicated by lowercase letters); disrupted ERSEs are marked by crosses. Each of the intact and mutant promoters was cloned immediately upstream of the firefly luciferase gene in the pGL3-Basic vector; their activities then were determined and are presented as described for Fig. 3.
FIG. 5
FIG. 5
Identification of the nucleotides in CHOP–ERSE1 required to mediate transcriptional induction. (A) Effects of various nucleotides on the activities of GRP78–ERSE1 and CHOP–ERSE1. Nucleotide C at position −47 of GRP78–ERSE1 and nucleotide G at position −89 of CHOP–ERSE1 (marked by the arrow) were changed to other nucleotides as indicated by lowercase letters. The activities of wild-type and mutant ERSEs were determined and are presented as described for Fig. 3. (B) Effects of point mutations on the activity of CHOP–ERSE1. Each of the nucleotides (−74 to −94) in the human CHOP promoter was mutated by transversion as indicated by lowercase letters. The activities of wild-type and mutant ERSEs were determined and are presented as described for Fig. 3.
FIG. 6
FIG. 6
Involvement of ER stress-induced proteolysis of ATF6 in transcriptional induction of the XBP-1 and CHOP genes. (A) Schematic structures of full-length ATF6, ATF6 (670), and its C-terminal deletion mutants ATF6 (373) and ATF6 (366). The locations of the basic region, leucine zipper (ZIP), and transmembrane domain (TMD) are indicated. Numbers indicate amino acid positions from the N terminus. (B) 293 cells cultured in 60-mm dishes were transiently transfected with 10 μg of pCGN alone (vector) or various pCGN-based ATF6 expression plasmids as indicated. At 48 h after transfection, total RNA was extracted and analyzed by Northern blot hybridization as for Fig. 1.
FIG. 7
FIG. 7
Mapping of the transactivation domain of ATF6. (Left) Schematic structures of ATF6 as well as fusion proteins between GAL4DB and various ATF6 subregions. The dotted lines delineate the region deleted from the construct. The positions of the bZIP region and transmembrane domain (TMD) are indicated. (Right) Transcriptional activities of various fusion proteins. HeLa cells in 24-well plates were transiently transfected with each of the fusion plasmids together with the reporter plasmid pG5luc containing five Gal4p binding sites upstream of the firefly luciferase gene. Constitutively expressed luciferase activities were determined and normalized as described in Materials and Methods. Relative activities are presented as averages with standard deviations (error bars) from triplicate determinations of four independent transfections. The positive control supplied by the manufacturer (pBIND-Id and pACT-MyoD control vectors; Promega) showed a relative activity of 4.6 ± 0.3 in this assay.
FIG. 8
FIG. 8
Effects of overexpression of ATF6 mutants on the activities of the human GRP78, XBP-1, and CHOP promoters. (A) Schematic structures of full-length ATF6 and two mutants, ATF6 (373) and ATF6 (373) ΔAD. The locations of the activation domain (AD), bZIP region, and transmembrane domain (TMD) are marked. Numbers indicate amino acid positions relative to the N terminus. (B) Structures of intact and mutant promoters cloned immediately upstream of the firefly luciferase gene are shown schematically as in Fig. 4. A reporter plasmid and the pRL-SV40 reference plasmid were transfected into HeLa cells together with pcDNA3.1(+) (vector) or one of the mutant ATF6 expression plasmids pcDNA-ATF6 (373) and pcDNA-ATF6 (373) ΔAD as described in Materials and Methods. The relative luciferase activity in transfected cells incubated for 16 h with (solid boxes) or without (open boxes) 2 μg of tunicamycin (TM)/ml was determined, and averages from four independent experiments are presented with standard deviations (error bars).
FIG. 9
FIG. 9
Direct binding of ATF6 to ERSE in the presence of NF-Y. (A) 32P-labeled ERSE-CC containing CCAAT-N9-CCACG (lanes 1 to 4), ERSE-CM containing CCAAT-N9-gatgt (lanes 5 and 6), ERSE-MC containing gacta-N9-CCACG (lanes 7 and 8), or ERSE-MM containing gacta-N9-gatgt (lanes 9 and 10) was incubated with in vitro-translated ATF6 (373) in the presence (+) or absence (−) of recombinant NF-Y as indicated. Protein-DNA complexes formed were analyzed by EMSA as described in Materials and Methods. The positions of complexes I and II are indicated. (B) A mixture of in vitro-translated ATF6 (373) and recombinant NF-Y was treated with (+) or without (−) various antisera as indicated prior to incubation with 32P-labeled ERSE-CC. EMSA was carried out as for panel A. (C) The specific binding of in vitro-translated ATF6 (373) and recombinant NF-Y to 32P-labeled ERSE-CC was competed by unlabeled oligonucleotides in 100-fold molar excess as indicated. EMSA was carried out as for panel A.
FIG. 10
FIG. 10
Direct binding of ATF6 to XBP-1–ERSE1 (A) and CHOP-ERSE (B). A mixture of in vitro-translated ATF6 (373) and recombinant NF-Y was treated with (+) or without (−) various antisera as indicated prior to incubation with 32P-labeled XBP-1–ERSE1 (lanes 1 to 6) or 32P-labeled CHOP-ERSE (lanes 7 to 12). The protein-DNA complexes formed were analyzed by EMSA as for Fig. 9. The positions of complexes I, I*, and II are indicated.
FIG. 11
FIG. 11
ERSE-binding activities in nuclear extracts of HeLa cells. (A) 32P-labeled ERSE-CC was incubated with nuclear extracts prepared from HeLa cells that were left untreated (−TM) or treated (+TM) with 2 μg of tunicamycin/ml for 4 h. Protein-DNA complexes formed were analyzed by EMSA as described in Materials and Methods. The position of complex I is indicated. (B) Nuclear extracts of untreated HeLa cells (−TM) or those treated (+TM) with 2 μg of tunicamycin/ml for 4 h were incubated with (+) or without (−) various antisera as indicated prior to incubation with 32P-labeled ERSE-CC (lanes 3 to 8). Formation of complex I was competed by a 100-fold molar excess of unlabeled ERSE-CC (lane 9) or ERSE-MM (lane 10). EMSA was carried out as for panel A. Only specific binding is shown. The position of complex I is indicated. (C) Nuclear extracts of untreated HeLa cells (−TM) or those treated (+TM) with 2 μg of tunicamycin/ml for 4 h were mixed with in vitro-translated ATF6 (373) or control reticulocyte lysates (vector) and then incubated with (+) or without (−) various antisera as indicated prior to incubation with 32P-labeled ERSE-CC. EMSA was carried out as for panel A. Only specific binding is shown, and the positions of complexes I and II are indicated.
FIG. 12
FIG. 12
Effects of altering the spacing on the ATF6-binding and transcription-inducing activities of ERSE. (A) Nucleotide sequences of wild-type and mutant forms of XBP-1–ERSE1 analyzed. A 41-bp sequence containing XBP-1–ERSE1 and its surrounding nucleotides is shown and referred to as CCAAT-N9-CCACG. Sequences matching the consensus ERSE are shaded. One nucleotide, G, between the CCAAT and CCACG sequences was deleted to create CCAAT-N8-CCACG, while one nucleotide, A (indicated by the underlined lowercase letter), was inserted to create CCAAT-N10-CCACG. (B) Binding of ATF6 to wild-type or mutant XBP-1–ERSE1. The oligonucleotide probe CCAAT-N9-CCACG, CCAAT-N8-CCACG or CCAAT-N10-CCACG, the sequences of which are delineated in panel A, was incubated after labeling with 32P with (+) or without (−) in vitro-translated ATF6 (373) in the presence of NF-Y. Protein-DNA complexes formed were analyzed by EMSA. The positions of complexes I and II are indicated. C, Transcriptional response to ER stress of human XBP-1 promoter containing wild-type or mutant XBP-1-ERSE1. XBP-1-ERSE1 (CCAAT-N9-CCACG) was mutated to CCAAT-N8-CCACG or CCAAT-N10-CCACG as indicated in part A in the XBP-1 promoter which was then cloned immediately upstream of the firefly luciferase gene in the pGL3-Basic vector. Their transcriptional activities were determined and are presented as described in Fig. 3.
FIG. 13
FIG. 13
Model for the mammalian UPR. ATF6 is synthesized as a precursor protein (p90ATF6) that anchors in the ER membrane under normal conditions. Upon accumulation of unfolded proteins in the ER, membrane-bound p90ATF6 is processed into a soluble and active form (p50ATF6). p50ATF6 translocates into the nucleus and directly binds to the CCACG part of ERSE, the CCAAT part of which is constitutively occupied by NF-Y. Thus, ERSF composed of NF-Y and p50ATF6 activates transcription of target genes. Target proteins include ER chaperones and two transcription factors, CHOP and XBP-1. Induced ER chaperones cope with unfolded proteins accumulated in the ER. Induced CHOP is likely to help the cells prepare for apoptosis by stimulating transcription of its target genes (referred to as DOCs). The roles of induced XBP-1 and its target genes are currently unknown. XBP-1 may function as a regulator of the UPR through ERSE.
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