In an accompanying paper, we show that the mitochondrial Unfolded Protein Response or mtUPR is initiated by the activation of transcription of chop through an AP-1 element in the chop promoter. Further, we show that the c/ebpβ gene is similarly activated and CHOP and C/EBPβ subsequently hetero-dimerise to activate transcription of mtUPR responsive genes. Here, we report the discovery of six additional mtUPR responsive genes. We found that these genes encoding mitochondrial proteases YME1L1 and MPPβ, import component Tim17A and enzymes NDUFB2, endonuclease G and thioredoxin 2, all contain a CHOP element in their promoters. In contrast, genes encoding mitochondrial proteins Afg3L2, Paraplegin, Lon and SAM 50, which do not have a CHOP element, were not up-regulated. Conversely, genes with CHOP elements encoding cytosolic proteins were not induced by the accumulation of unfolded proteins in mitochondria. These results indicate that mtUPR responsive genes appear to share a requirement for a CHOP element, but that this is not sufficient for the regulation of the mtUPR. A more detailed analysis of promoters of mtUPR responsive genes revealed at least two additional highly conserved, putative regulatory sites either side of the CHOP element, one a motif of 12 bp which lies 14 bp upstream of the CHOP site and another 9 bp element, 2 bp downstream of the CHOP site. Both of these additional elements are conserved in the promoters of 9 of the ten mtUPR responsive genes we have identified so far, the exception being the Cpn60/10 bidirectional promoter. Mutation of each of these elements substantially reduced the mtUPR responsiveness of the promoters suggesting that these elements coordinately regulate mtUPR.
References
[1]
Lindquist S (1986) The heat-shock response. Annu Rev Biochem 55: 1151–1191.
[2]
Gething MJ, Sambrook J (1992) Protein folding in the cell. Nature 355: 33–45.
[3]
Schroder M (2006) The unfolded protein response. Mol Biotechnol 34: 279–90.
[4]
Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107: 881–91.
[5]
Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D (2000) Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell 5: 897–904.
[6]
Ye J, Rawson RB, Komuro R, Chen X, Dave UP, et al. (2000) ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell 6: 1355–1364.
[7]
Schroder M, Kaufman RJ (2002) The mammalian unfolded protein response. Annu Rev Biochem 74: 739–789.
[8]
Ubeda M, Wang XZ, Zinszner H, Wu I, Habener JF, et al. (1996) Stress-induced binding of the transcription factor CHOP to a novel DNA control element. Mol Cell Biol 16: 1479–1489.
[9]
Puthalakath H, O'Reilly LA, Gunn P, Lee L, Kelly PN, et al. (2007) ER stress triggers apoptosis by activating BH3-only protein Bim. Cell. 129: 1337–49.
[10]
Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39: 359–407.
[11]
Martinus RD, Garth GP, Webster TL, Cartwright P, Naylor DJ, et al. (1996) Selective induction of mitochondrial chaperones in response to loss of the mitochondrial genome. Eur J Biochem 240: 98–103.
[12]
Zhao Q, Wang J, Levichkin IV, Stasinopoulos S, Ryan MT, et al. (2002) A mitochondrial specific stress response in mammalian cells. Embo J 21: 4411–4419.
[13]
Horibe T, Hoogenraad NJ (2007) The CHOP gene contains an element for the positive regulation of the mitochondrial unfolded protein response. PLoSONE (In Press).
[14]
Calvo S, Jain M, Xie X, Sheth SA, Chang B, et al. (2006) Systematic identification of human mitochondrial disease genes through integrative genomics. Nat Genet 38: 576–582.
[15]
Yoshida H, Okada T, Haze K, Yanagi H, Yura T, et al. (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. Mol Cell Biol 20: 6755–6767.
[16]
Xie X, Lu J, Kulbokas EJ, Golub TR, Mootha V, et al. (2005) Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals. Nature 434: 338–345.
[17]
Matys V, Fricke E, Geffers R, Gossling E, Haubrock M, et al. (2003) TRANSFAC: transcriptional regulation, from patterns to profiles. Nucleic Acids Res 31: 374–8.
[18]
Kelly SE, Bachurski CJ, Burhans MS, Glasser SW (1996) Transcription of the lung-specific surfactant protein C gene is mediated by thyroid transcription factor 1. J Biol Chem 271: 6881–6888.
[19]
Kimura S, Hara Y, Pineau T, Fernandez-Salguero P, Fox CH, et al. (1996) The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev 10: 60–69.
[20]
Travers K J, Patil CK, Wodicka L, Lockhart DJ, Weisman JS, et al. (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101(3): 249–258.
[21]
Scheurer D, Song B, McEwen E, Lui C, Laybutt R, et al. (2001) Translational control is required for the unfolded protein response and in vivo glucose homeostasis Molec Cell 7(6): 1165–1176.
[22]
Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, et al. (2003) An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Molec Cell 11(3): 619–633.
[23]
Langer T, Neupert W (1996) Regulated protein degradation in mitochondria. Experientia 52: 1069–1076.
[24]
Ohsato T, Ishihara N, Muta T, Umeda S, Ikeda S, et al. (2002) Mammalian mitochondrial endonuclease G. Digestion of R-loops and localization in intermembrane space. Eur J Biochem 269: 5765–5770.
[25]
Li LY, Lio X, Wang X (2001) Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 412: 95–99.
[26]
Parrish J, Li L, Klotz K, Ledwich D, Wang X, et al. (2001) Mitochondrial endonuclease G is important for apoptosis in C.elegans. Nature 412: 90–94.
