When unfolded proteins are accumulated in the ER, eukaryotic cells from yeast to humans induce transcription of ER chaperones and ERAD components to maintain the homeostasis of the ER. In yeast, ER chaperones and ERAD components are induced by the same mechanism via the Ire1p-Hac1p pathway. In contrast, mechanisms underlying their induction have diverged in mammals, as transcriptional induction of ERAD components but not ER chaperones depends on the IRE1-XBP1 pathway, the mammalian counterpart of the yeast Ire1p-Hac1p pathway (). However, the molecular basis of this divergence remains unclear, mainly because the cis
-acting elements responsible for induction of mammalian ERAD components have not been identified to date. This is in marked contrast to the situation of mammalian ER chaperones, whose induction is known to be mediated by cis
-acting ERSE (7
). UPRE has the properties of those expected of a cis
-acting element responsible for the transcriptional induction of mammalian ERAD components, as UPRE-mediated transactivation depends on the IRE1-XBP1 pathway (17
). Nonetheless, UPRE is an artificially selected sequence and its presence has yet to be demonstrated in any promoters of mammalian ERAD components.
In this report, we analysed the promoter of human HRD1, an important component of mammalian ERAD, and found that induction of HRD1 upon ER stress is mediated by two cis
-acting elements. One is a canonical ERSE and the other is a novel element we designate as UPRE II, on the basis that it has properties closely similar to those of UPRE but differs from UPRE in sequence. The presence of UPRE II, to which XBP1 but not ATF6 binds directly (), explains at least in part the dependency of HRD1 induction on the IRE1-XBP1 pathway. We emphasize that the identification of XBP1 binding sites is not straightforward. A recent report (29
) identified several XBP1 binding sites using chromatin immunoprecipitation on chip analysis, namely GCCACG
(underlined sequence is identical to CCACG of ERSE), GACGTG (part of UPRE consensus), ACGT core and CGGAAG. However, the UPRE II we identified in HRD1 promoter (CCGCGT) differed from all of the above sequences. Careful work is necessary to unravel XBP1 binding sites. Perhaps the most unexpected finding of this study is that a sequence perfectly matching the consensus of UPRE at -3262 to -3255 plays almost no role in the induction of HRD1 in response to ER stress (). There must be inhibitory activity or an inhibitory chromatin structure around the UPRE from -3262 to -3255 that blocks UPRE-mediated transactivation, because XBP1 is able to bind to the UPRE when tested in EMSA (). Solving this discrepancy will require analysis at the chromatin level. Nonetheless, given that it is not present in mouse and rat promoters (), the UPRE sequence may have been created in human HRD1 promoter by chance, without functional implications.
We showed here that the induction of HRD1 is mediated by ERSE and UPRE II (). While this article was under revision, Kaneko et al
) reported the characterization of human HRD1 promoter. They found that human HRD1 promoter carries a complete ERSE (designated ERSE2) and an incomplete ERSE (designated ERSE1) downstream of ERSE2; their ERSE2 is identical to our ERSE at -1476 to -1458 (). Using reporter luciferase assays, they showed that ERSE2 but not ERSE1 is involved in the induction of HRD1 (30
), consistent with our results (). Nevertheless, notwithstanding their conclusion that ER stress-induced HRD1 expression is mediated by the IRE1-XBP1 pathway, the molecular basis of their conclusion remains obscure, because ATF6 but not XBP1 bound to ERSE2 in EMSA (30
). Because XBP1 but not ATF6 bound in EMSA to the UPRE II we identified in this report (), our results explain at least in part the dependency of HRD1 induction on the IRE1-XBP1 pathway.
Given that the induction of HRD1 is mediated by ERSE and UPRE II (), induction should be considerable even in the absence of IRE1α (to an extent approximately half that observed in IRE1α+/+ MEFs, based on the results of , lanes 1 and 2), as ATF6 binds to ERSE and activates transcription. However, induction of HRD1 mRNA on ER stress was markedly reduced in IRE1α-/- or XBP1-/- MEFs (). The promoter of Herp, a component of ERAD (31
), is well characterized; induction of Herp on ER stress is mediated by ERSE and ERSE II (27
). Given this, induction of Herp should occur normally even in the absence of IRE1, as ATF6 binds to both ERSE and ERSE II and activates transcription. However, induction of Herp is greatly diminished in IRE1α-/- or XBP1-/- MEFs as compared with that in IRE1α+/+ MEFs [ and (26
)]. It was recently reported that Herp ERSE II-mediated transactivation is not affected by the absence of IRE1α (33
), contrary to our previous results (26
). However, we consistently and reproducibly observe diminished transactivation through Herp ERSE II in IRE1α-/- MEFs, and the results of Liang et al
) are not consistent with the greatly mitigated induction of Herp mRNA in IRE1α-/- in MEFs as compared with that IRE1α+/+ MEFs [ and (26
)]. Further, the analysis of Liang et al
) did not include necessary controls such as measurement of the UPRE reporter, the transcriptional activity of which is absolutely dependent on IRE1α.
These results require consideration of why the induction of Herp or HRD1 is affected by the absence of IRE1 more severely than the extent expected from promoter analysis. One common feature is that the two cis-acting elements regulating the induction of Herp or HRD1 are close to each other: ERSE II is only 23 bp upstream of ERSE in the Herp promoter, while UPRE II is only 13 bp upstream of ERSE in the HRD1 promoter. Chromatin structures around closely spaced regions may affect the recruitment of certain transcription factors. Further analysis combined with chromatin-level analysis will improve our understanding of the molecular mechanisms underpinning the specific regulation of promoters of mammalian ERAD components during the UPR.