Discovery of genes activated by the mitochondrial unfolded protein response (mtUPR) and cognate promoter elements

Abstract

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/ebp beta gene is similarly activated and CHOP and C/EBP beta 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 MPP beta, 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.

Figure 1. Survey of promoters responsive to mtUPR.

Reporter constructs for mitochondrial proteins YME1L1, MPPβ (mitochondrial processing peptidase β subunit), Trx2 (mitochondrial thioredoxin), Tim17A, End G (Endonuclease G), NDUFB2 (subunit of complex I), and ClpP, which have CHOP consensus, and Lon, Sam50, Afg3L2, Paraplegin without CHOP consensus, or NOX3 (NADPH oxidases 3) and Typ I (Type I iodothyronine deiodinase), which are non-mitochondrial proteins but have CHOP consensus sequence in the promoter region, were tested. COS-7 cells were co-transfected with vector or vector containing OTCΔ and promoter-luciferase constructs and were used for luciferase assay 32 h after transfection. Data represent the mean±SEM from experiments performed in triplicate.

Figure 2. Nucleotide sequence alignment of the promoter region of mtUPR responsive genes.

(A): mtUPR Genes, indicating the position in the promoter relative to the transcription start site, are shown on the left hand side of the figure. Asterisks identify identical sequences and bold letters show the highly conserved elements MURE1, CHOP, and MURE2. (B): Consensus sequence of the MURE1 and MURE2 elements, as well as the sequence for CHOP taken from .

Figure 3. Effect of the mutation on MURE1 and MURE2 elements on mtUPR responsiveness.

A. Effect of MURE1 mutations on yme1l1 promoter activity.; B. Effect of MURE2 mutations on yme1l1 promoter activity; C. Effect of MURE2 mutations on mppβ promoter activity. Luciferase activity is compared for each mutation in cells co-transfected with empty vector or vector- OTCΔ and the promoter reporter constructs and is expressed as the activity relative to that obtained for the wild type promoter (RLU-relative luciferase units). The results represent the mean±SEM for 3 independent experiments. The promoter sequence for wild type and mutants is shown.

Figure 4. Spacing of MURE1, CHOP and MURE2 motifs.

MURE1-CHOP (A), CHOP-MURE2 (B) and MURE1-MURE2 (C). Each figure shows the number of motif pairs with the given separation found in the set of predicted mitochondrial proteins. Separation is defined as the distance from the first base of the first motif to the first base of the second motif.

Figure 5. Comparison of regulatory circuits for mtUPR and erUPR.

MtUPR activates transcription of both chop and c/ebpβ genes through an AP-1 site. These 2 transcription factors then bind to a CHOP element in the mtUPR responsive genes, along with transcription factors of unknown identity which bind to the MURE1 and 2 elements. ErUPR also activates transcription of CHOP (but not C/EBP genes) through an ER response element (ERSE). The function of CHOP in erUPR appears to be restricted to the induction of apoptosis through transcriptional activation of BIM.