Activation of ATF6 and an ATF6 DNA Binding Site by the Endoplasmic Reticulum Stress Response

Abstract

ATF6 is a member of the basic-leucine zipper family of transcription factors. It contains a transmembrane domain and is located in membranes of the endoplasmic reticulum. ATF6 has been implicated in the endoplasmic reticulum (ER) stress response pathway since it can activate expression of GRP78 and other genes induced by the ER stress response. ER stress appears to activate ATF6 by cleavage from the ER membrane and translocation to the nucleus. However, direct DNA binding by ATF6 had not been demonstrated. In this report, we have identified a consensus DNA binding sequence for ATF6. This site is related to but distinct from ATF1/CREB binding sites. The site was placed in a reporter gene and was specifically activated by ATF6 overexpression and was strongly induced by the ER stress response. A dominant negative form of ATF6 blocked ER stress induction of both ATF6 site and GRP78 reporter genes. We further found that GAL4-ATF6 could be activated by ER stress. These results demonstrate that ATF6 is a direct target of the ER stress response. A proximal sensor of the ER stress response, human IRE1 (hIRE1), was sufficient to activate the ATF6 reporter gene, while a dominant negative form of hIRE1 blocked ER stress activation, suggesting that hIRE1 is upstream of ATF6 in the ER stress signaling pathway. ATF6 is a member of the basic-leucine zipper family of transcription factors. It contains a transmembrane domain and is located in membranes of the endoplasmic reticulum. ATF6 has been implicated in the endoplasmic reticulum (ER) stress response pathway since it can activate expression of GRP78 and other genes induced by the ER stress response. ER stress appears to activate ATF6 by cleavage from the ER membrane and translocation to the nucleus. However, direct DNA binding by ATF6 had not been demonstrated. In this report, we have identified a consensus DNA binding sequence for ATF6. This site is related to but distinct from ATF1/CREB binding sites. The site was placed in a reporter gene and was specifically activated by ATF6 overexpression and was strongly induced by the ER stress response. A dominant negative form of ATF6 blocked ER stress induction of both ATF6 site and GRP78 reporter genes. We further found that GAL4-ATF6 could be activated by ER stress. These results demonstrate that ATF6 is a direct target of the ER stress response. A proximal sensor of the ER stress response, human IRE1 (hIRE1), was sufficient to activate the ATF6 reporter gene, while a dominant negative form of hIRE1 blocked ER stress activation, suggesting that hIRE1 is upstream of ATF6 in the ER stress signaling pathway. endoplasmic reticulum ER stress response element basic-leucine zipper amino acids polymerase chain reaction cAMP-response element cAMP-response element-binding protein hemagglutinin human IRE1 murine IRE1β c-Jun N-terminal kinase presenilin-1 tunicamycin The endoplasmic reticulum (ER)1 stress response is a mechanism by which cells protect themselves from many noxious insults that cause protein unfolding in the ER (reviewed in Ref. 1Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1930) Google Scholar). Inducers of this response include inhibitors of glycosylation (tunicamycin), dithiothreitol, agents that affect calcium homeostasis such as calcium ionophores and thapsigargin (an inhibitor of an ER calcium-ATPase), and agents that perturb ER function and protein movement such as brefeldin A. The ER stress response (also known as the unfolded protein response) causes an increase in gene expression of a number of ER chaperones, such as GRP78/BiP and GRP94, and erp72, which is related to protein-disulfide isomerase (1Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1930) Google Scholar). These factors probably allow the cell to refold unfolded proteins in the ER. Analysis of the promoters of these genes has revealed a consensus sequence element, CCAATN9CCACG, that is required for ER stress induction of the promoters (2Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar, 3Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (216) Google Scholar). Mutation of this ER stress response element (ERSE) has shown that both the 5′ CCAAT and 3′ CCACG boxes are required. The variable central region was not sensitive to point mutations; however, some multiple base changes abolished activity (2Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar, 3Roy B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (216) Google Scholar). Not surprisingly, NF-Y/CBP binds to the 5′ CCAAT box of the ERSE (4Roy B. Lee A.S. Mol. Cell. Biol. 1995; 15: 2263-2274Crossref PubMed Scopus (86) Google Scholar,5Roy B. Li W.W. Lee A.S. J. Biol. Chem. 1996; 271: 28995-29002Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar); however, it has been less clear which factor(s) bind the element to mediate the ER stress response. In order to identify factors binding the ERSE, Yoshida et al. (2Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar) utilized a yeast one-hybrid approach and found ATF6. While they not to that ATF6 the ERSE overexpression of ATF6 reporter genes in cells in an ERSE that ATF6 can the ERSE in ATF6 is a member of the basic-leucine zipper protein family Genes Dev. PubMed Scopus Google Scholar). It is a protein amino acids Mol. Cell. Biol. PubMed Scopus Google Scholar). ATF6 contains a transmembrane domain amino acids the the K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar). and cell Haze et al. K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar) found that ATF6 in the ER. to identify an ER cleavage of which is to be an form of the this the protein was found in In expression of the domain of ATF6 in a protein that in the and that activated expression of the GRP78 gene K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar). We identified ATF6 in a yeast the response a transcription that induced expression of the gene Mol. Cell. Biol. PubMed Scopus Google Scholar). It is that ATF6 affect other genes. In order to the function and mechanism of of we the of a consensus DNA binding site for ATF6. This site is activated by overexpression of ATF6 and by ER stress In a GAL4-ATF6 protein is by ER that ATF6 can to this expression was by of the region of ATF6 amino acids This was to of of a protein was in from the and as A. PubMed Scopus Google Scholar). of binding to was by as DNA Biol. PubMed Scopus Google Scholar). the reaction of and of protein in This was for and a in The was the central It was to and the of DNA The was and The of the was by the binding of the protein to a the site in the of of This of the was from the and the DNA was of the in The DNA was and The and for the of This was for The from the of was the of and The for binding and in is a the was the and sites. was from as A. PubMed Scopus Google Scholar). and proteins for binding to the by as in of the ATF6 as a of ATF6 and of the ATF6 and a base in reporter genes. are as T. A. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar) an site 5′ to the to of the human and the contains of the ATF6 while and of sites. The reporter was from The reporter contains upstream of the and the The reporter gene to of the GRP78 was as (2Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar). contains ATF6 an the as Mol. Cell. Biol. PubMed Scopus Google Scholar). contains amino acids of ATF6 by in the of Cell. Full Text PDF PubMed Scopus Google Scholar). was from the acids from to contains the expression of DNA binding domain to ATF6 The expression for was as R.J. Genes Dev. 1998; PubMed Scopus Google Scholar). The dominant negative form of the of the kinase and contains the region for amino acids of The protein kinase expression was from cells in by the calcium J. J. B. 1998; Google Scholar). for a of reporter gene, of as an Mol. Cell. 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K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar) found that the domain of ATF6 to the and activated GRP78 gene We a ATF6 the transmembrane and ER could activate the ATF6 site We of and ATF6 to to activate the strongly activated the ATF6 site reporter as as of as strongly as of ATF6 this ATF6 protein we cell by to the the of the ATF6 We found that of of a of protein expression as of ATF6 However, of strongly activated reporter gene while of ATF6 had This that is an activated form of ATF6. We by the ATF6 to the ER the nucleus. to the results of al. K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google ATF6 was the ER while was to the not Haze et al. K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar) a cleavage of ATF6 that was induced by tunicamycin This was to ATF6. We have not been to this Mol. Cell. Biol. 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Full Text PDF PubMed Scopus Google Scholar, K. J. Cell. Full Text PDF PubMed Scopus Google Scholar). contains a protein kinase domain a domain to Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). can be activated by ER stress which to of This activity causes the cleavage of an for the transcription an The is by a This results in of which ER genes in yeast Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. PubMed Scopus Google Scholar, Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of yeast have been that have and ER membrane R.J. Genes Dev. 1998; PubMed Scopus Google J. 1998; PubMed Scopus Google Scholar). of IRE1 and gene and dominant negative IRE1 was to tunicamycin induction of GRP78 expression R.J. Genes Dev. 1998; PubMed Scopus Google J. 1998; PubMed Scopus Google Scholar). These results that IRE1 is a of the ER stress response in GRP78 is by ATF6 (2Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1014) Google we to IRE1 is sufficient for ATF6 as of in of the ATF6 site which could be by tunicamycin We a dominant negative which has a of the kinase and for an ATF6 We found that strongly tunicamycin induction of the ATF6 site reporter gene We point and that the kinase and J. for for of the ATF6 reporter of these to activate the suggesting that both are required for IRE1 function of these point tunicamycin however, they both by not The was shown to tunicamycin induction of the GRP78 gene in cells R.J. Genes Dev. 1998; PubMed Scopus Google Scholar). This be to the expression of genes in cells In the was found to be a inhibitor of in and J. results that overexpression is sufficient to ATF6 and that IRE1 is required for ATF6 This that IRE1 is upstream of ATF6 in the ER stress response pathway. both the kinase and of IRE1 to be required for ATF6 We have found that ATF6 binds to a consensus DNA binding site in This site could be activated by overexpression of ATF6 in placed upstream of a reporter gene, the consensus ATF6 site was activated by agents that the ER stress response. ATF6 appears to mediate this response, since dominant negative of ATF6 blocked the A of ATF6 to DNA binding domain that it could mediate ER stress induction of a reporter These results that ATF6 is a target of the ER stress response. (2Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar) identified ATF6 in a yeast as the consensus ERSE We found that dominant negative ATF6 could ER stress induction of the GRP78 that contains ERSE In activated expression of the but not of in the not These results further that ATF6 is in ER stress induction of the GRP78 can bind to the CCAAT box of the ERSE site (4Roy B. Lee A.S. Mol. Cell. Biol. 1995; 15: 2263-2274Crossref PubMed Scopus (86) Google Scholar, B. Li W.W. Lee A.S. J. Biol. Chem. 1996; 271: 28995-29002Abstract Full Text Full Text PDF PubMed Scopus (58) Google and it has been that ATF6 and binds to the CCACG of the site K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar). this site is the of the of the ATF6 consensus site we have a member of the ATF6 probably binds to the ATF6 site as a In of we have found that ATF6 can while it not in and In the ERSE, it is that ATF6 binds to the DNA a the binding by This ATF6 not bind to this site (2Yoshida H. Haze K. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar). J. and A mechanism is by binding response to the response element of the gene Dev. PubMed Scopus Google Scholar). We have the domain of ATF6 to identify DNA binding It is that other in the domain affect the DNA binding We have not been to the domain in a form in order to this However, the DNA binding site was activated strongly and specifically by suggesting that the domain the of the the however, affect other factors such as and affect binding to other sites. We have been to ATF6 binding activity in of tunicamycin induced cells not the ATF6 binding site in a we binding of and but not ATF6. This probably the of and and the of the ATF6 cleavage the cell can the ATF6 site from ATF1/CREB binding since we found that the site was activated by and tunicamycin We that some ER genes are activated by direct ATF6 binding the consensus ERSE sites. However, of the of ER genes that have been identified has not revealed an ATF6 consensus site that is required for ER stress A of was found in the and GRP78 promoters and of the but was not in the human GRP78 This region of the was not required for tunicamycin induction of a reporter gene, since it contains from to However, of to in the strongly the of expression from to the tunicamycin induction was from to B. Lee A.S. Nucleic Acids Res. 1999; 27: 1437-1443Crossref PubMed Scopus (216) Google Scholar). further be required to the ATF6 site to ER stress induction of the GRP78 reporter ATF6 site be the in other ER genes the promoters have not been We it that ER stress induction of the ATF6 site in since the induction was the reporter a point in the site abolished and the site was not induced by A of the the for revealed promoters that the ATF6 consensus site the base is and to of the this is In the of a is not the that contains the sequence was the of the human is a to the that Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) such that it is a for a gene induced to for ER promoters the consensus the human and human genes. be required to these ATF6 The for ER stress of ATF6 by Haze et al. K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar) is that ATF6 is the ER membrane and that ER stress it is the membrane such that the N-terminal domain can to the and activate gene we have not been to the cleavage of results are this We by of ATF6 that it to the ER not the domain of ATF6 was it to the nucleus. In this of ATF6 was a of to the cleavage of ATF6 be to of and the of this that are We of ATF6 to for while Haze et al. to ATF6. It is that of ATF6 results in cell and that of ATF6 are in Haze et al. the ATF6 in the cells and of the ATF6 cleavage that we have not We for this to ATF6 Mol. Cell. Biol. PubMed Scopus Google Scholar) had a in such that we not to a clear and be required for to the and of the ATF6 cleavage we and K. Yoshida H. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar) have found that ATF6 is an of gene the of cleavage of ATF6 and movement to the is the which is that ATF6 transcription as a transmembrane protein the of the membrane that the protein from the membrane and to the nucleus. A for of a transcription by cleavage from the ER membrane is (reviewed in Ref. A. 1999; PubMed Scopus Google Scholar). is an ER transmembrane protein that is activated in response to This results in cleavage of sites. it is in the domain by site by cleavage by site in the transmembrane domain the This results in of the DNA binding domain of which to the nucleus. The site site in was found to have the sequence cleavage the and J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and while the other could be such that the for the cleavage site is J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a is in the domain of ATF6. We are this site and site are required for ATF6 The cleavage of is further by which and to the A. 1999; PubMed Scopus Google Scholar). The of the of ATF6 is by factors and cleavage in the domain as as the of the domain the ER The proximal sensor of the ER stress response that has been identified is In this protein is required for of the ER stress response Cell. Full Text PDF PubMed Scopus Google Scholar, K. J. Cell. Full Text PDF PubMed Scopus Google Scholar). It is an ER transmembrane protein in the a protein kinase domain and a domain activity that is to Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). has the to the of a transcription by of in a that is Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. PubMed Scopus Google Scholar, Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of have been and which have ER and domain as yeast R.J. Genes Dev. 1998; PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google Scholar). IRE1 appears to be required for the ER stress response, since it can GRP78/BiP and target genes of the ER stress response. In dominant negative of IRE1 in the kinase and blocked ER stress induction of target genes R.J. Genes Dev. 1998; PubMed Scopus Google J. 1998; PubMed Scopus Google We found that overexpression activated the ATF6 reporter and that point in the kinase abolished this IRE1 a of the kinase and in as a dominant negative to ER stress of the ATF6 reporter These results that IRE1 is upstream of ATF6 in the ER stress signaling pathway. direct IRE1 of ATF6 is to be we have had the ATF6 cleavage we have not been to IRE1 ATF6 results have not of ATF6 such that direct of ATF6 by IRE1 not be mechanism for of ATF6 by IRE1 could be and by the and It was found that tunicamycin and IRE1 can activate kinase activity A. PubMed Scopus Google Scholar). It has been shown that can ATF6 in and activate transcription activity in J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, we found that and and not activate the ATF6 reporter gene not These results that and are not sufficient for ER stress of ATF6. In dominant negative blocked IRE1 of A. PubMed Scopus Google Scholar) but had IRE1 of the ATF6 reporter by suggesting that is not required not IRE1β overexpression induced J. 1998; PubMed Scopus Google Scholar). ER stress can cause as as induction of ER that protect cells from it is to that IRE1 can mediate both of these It was found that is the ER membrane and is required for induction of by ER stress T. H. Li J. J. PubMed Scopus Google Scholar). ER stress induced the cleavage of to a that was the ER T. H. Li J. J. PubMed Scopus Google Scholar). IRE1 can it be to it ATF6 is required for A of the ER stress signaling pathway is the of presenilin-1 is a ER membrane protein that is in some of H. K. T. 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