Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt)

Abstract

Mitochondrial diseases and aging are associated with defects in the oxidative phosphorylation machinery (OXPHOS), which are the only complexes composed of proteins encoded by separate genomes. To better understand genome coordination and OXPHOS recovery during mitochondrial dysfunction, we examined ATFS-1, a transcription factor that regulates mitochondria-to-nuclear communication during the mitochondrial UPR, via ChIP-sequencing. Surprisingly, in addition to regulating mitochondrial chaperone, OXPHOS complex assembly factor, and glycolysis genes, ATFS-1 bound directly to OXPHOS gene promoters in both the nuclear and mitochondrial genomes. Interestingly, atfs-1 was required to limit the accumulation of OXPHOS transcripts during mitochondrial stress, which required accumulation of ATFS-1 in the nucleus and mitochondria. Because balanced ATFS-1 accumulation promoted OXPHOS complex assembly and function, our data suggest that ATFS-1 stimulates respiratory recovery by fine-tuning OXPHOS expression to match the capacity of the suboptimal protein-folding environment in stressed mitochondria, while simultaneously increasing proteostasis capacity.

Fig. 1. ATFS-1 interacts with the promoters of mitochondrial protective genes induced during mitochondrial stress

A. Venn diagram illustrating the genes induced in an atfs-1-dependent manner during mitochondrial stress (Nargund et al., 2012), and the gene promoters that ATFS-1 bound during mitochondrial stress identified by ChIP-seq, and the overlap. B-E. ChIP-seq profiles of the mthsp-70, ymel-1, gpd-2 and hsp-4 promoters in wild-type worms raised on spg-7(RNAi), using ATFS-1 antibody (upper panel) or no antibody (lower panel). The y-axis is the number of sequence reads and the x-axis is approximately 2.5 kilobases with the start codon marked with an arrowhead. F-I. ChIP of the promoters in Figures 1B-E in the presence or absence of ATFS-1 antibody from wild-type worms raised on control or spg-7(RNAi) as measured by qPCR (N = 3, ± SD, p* (student t-test) < 0.05). J-M. Expression levels of mthsp-70, ymel-1, gpd-2 and hsp-4 mRNA in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05) (right panels). See also Figure S1 and Tables S1 and S2.

Fig. 2. ATFS-1 binds to the UPR^mtE and induces mitochondrial chaperone transcription during stress

A. The UPR^mtE consensus sequence. B. ChIP-seq profile of the hsp-60 promoter in wild-type worms raised on spg-7(RNAi) using ATFS-1 antibody or no antibody. The three UPR^mtEs within the promoter are marked with red boxes, and the element mutated in Figure 2E is marked with a black dot. The y-axis is the number of sequence reads and the x-axis is approximately 2.5 kilobases with the start codon marked with an arrowhead. Of note, the majority of the reads come from the multi-copy hsp-60_pr∷gfp transgene likely accounting for the high number of reads and the sharp profile borders. C. ChIP of the hsp-60 promoter in the presence or absence of ATFS-1 antibody from wild-type worms raised on control or spg-7(RNAi) as measured by qPCR (N = 3, ± SD, p* (student t-test) < 0.05). D. Expression levels of hsp-60 mRNA in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05). E. Photomicrographs of hsp-60_pr∷gfp (top panels) or hsp-60_pr∷gfp lacking a UPR^mtE (*) (bottom panels) transgenic worms raised on control or spg-7(RNAi). Scale bar, 0.5 mm. See also Figure S2.

Fig. 3. ATFS-1 interacts with multiple metabolic gene promoters

A. Gene ontology-KEGG Pathway analysis of the genes obtained by ATFS-1 ChIP-seq indicating the five most enriched pathways. B-C. ChIP-seq profiles of representative genes in the glycolysis (B) and TCA cycle (C) pathways of wild-type worms raised on spg-7(RNAi), using ATFS-1 antibody or no antibody. The y-axis is the number of sequence reads and the x-axis is approximately 2.5 kilobases with the start codon marked with an arrowhead. D-E. Expression levels of the glycolysis transcripts (D) enol-1 and ldh-1, and the TCA cycle transcripts (E) aco-2 and cts-1 in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05). See also Figure S3 and Table S3.

Fig. 4. ATFS-1 limits the accumulation of nuclear-encoded OXPHOS transcripts

A-F. ChIP-seq profiles of representative OXPHOS genes nuo-4, sdha-1, ucr-2.1, cyc-2.1, cco-1, and atp-3 of wild-type worms raised on spg-7(RNAi), using ATFS-1 antibody or no antibody. The y-axis is the number of sequence reads and the x-axis is approximately 2.5 kilobases with the start codon marked with an arrowhead. G-L. Expression levels of nuo-4, sdha-1, ucr-2.1, cyc-2.1, cco-1, and atp-3 mRNA in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05) (right panels). See also Figure S4 and Table S4.

Fig. 5. ATFS-1 limits mtDNA-encoded transcript accumulation and directly binds to the non-coding region of mtDNA

A. ChIP-seq profiles of the entire mitochondrial genome (mtDNA) of wild-type worms raised on spg-7(RNAi), using ATFS-1 antibody or no antibody. The y-axis is the number of sequence reads and the x-axis is the 13,794 nucleotide mitochondrial genome. The protein coding genes are marked with blue arrows, the rRNA genes with purple arrows, the non-coding region in green, and the putative UPR^mtE is marked with a red box. B. ChIP of the mtDNA non-coding region in the presence or absence of ATFS-1 antibody from wild-type worms raised on control or spg-7(RNAi) as measured by qPCR (N = 3, ± SD, p* (student t-test) < 0.05). C. mtDNA non-coding region sequence with the putative UPR^mtE in red. D. EMSA using recombinant ATFS-1 and wild-type D-loop oligonucleotides or D-loop oligonucleotides with the 9 base pair UPR^mtE scrambled. Unbound oligos (black square), ATFS-1-DNA complex (black arrow) and the loading well (gray arrow) are indicated. E. Immunoblots of lysates from worms raised on control or spg-7(RNAi) following fractionation into total lysate (T), postmitochondrial supernatant (S) and mitochondrial pellet (M) (Nargund et al., 2012). Endogenous NDUFS3 serves as a mitochondrial marker and hsp-60_pr∷gfp as a stress-induced cytosolic marker. Full-length (black arrow) and the mitochondrial forms (gray arrows) of ATFS-1 are indicated. F. Relative mtDNA copy number in wild-type and atfs-1(tm4525) worms raised on control or spg-7(RNAi) as determined by qPCR (N = 3, ± SD, p* (student t-test) < 0.05). G-I. Expression levels of the mtDNA-encoded mRNAs nd6, cox2 and atp6 in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05). J. Expression level of the mtDNA-encoded ctb-1 mRNA in wild-type or atfs-1(tm4525) worms expressing no transgenes (-) or transgenic wild-type ATFS-1, ATFS-1^Δ1-32.myc or ATFS-1^ΔNLS raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05). K. Schematics of wild-type ATFS-1, ATFS-1 with an impaired mitochondrial targeting sequence (MTS) (ATFS-1^Δ1-32.myc), and ATFS-1 with a mutated nuclear localization sequence (NLS) (ATFS-1^ΔNLS). L. Fluorescent photomicrographs of atfs-1(tm4525);hsp-60_pr∷gfp worms expressing transgenic ATFS-1, ATFS-1^Δ1-32.myc, or ATFS-1^ΔNLS raised at 20°C. Scale bar, 0.5 mm. See also Figure S5.

Fig. 6. ATFS-1 promotes OXPHOS complex assembly during mitochondrial dysfunction

A-C. ChIP-seq profiles of nuaf-1, lpd-8, and Y17G9B.5 of wild-type worms raised on spg-7(RNAi), using ATFS-1 antibody or no antibody. The y-axis is the number of sequence reads and the x-axis is approximately 2.5 kilobases with the start codon marked with an arrowhead. D-F. Expression levels of nuaf-1, lpd-8, and Y17G9B.5 mRNA in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05) (right panels). G. SDS-PAGE immunoblots of extracts from wild-type and atfs-1(tm4525) worms raised on control or spg-7(RNAi). COX1 is a component of the cytochrome c oxidase complex (IV) and tubulin was used as a loading control. H. SDS-PAGE immunoblots of extracts from wild-type and atfs-1(tm4525) worms raised on control or spg-7(RNAi) (upper three panels). The lower two panels are blue-native PAGE immunoblots of the same extracts. NDUFS3 is a component of the NADH ubiquinone oxidoreductase complex, ATP5A is an ATP synthase component and tubulin was used as a loading control. I. Oxygen consumption in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) (N = 3, ± SD, p* (student t-test) < 0.05).

Fig. 7. Mitochondrial and nuclear accumulation of ATFS-1 promotes respiration and development during mitochondrial dysfunction

A. Photomicrographs of wild-type hsp-60_pr∷gfp and afts-1(tm4525);hsp-60_pr∷gfp worms expressing transgenic ATFS-1, or ATFS-1^Δ1-32myc raised on spg-7(RNAi). Scale bar, 0.5 mm. B. SDS-PAGE immunoblots of extracts from atfs-1(tm4525) worms expressing transgenic ATFS-1, or ATFS-1^Δ1-32myc raised on spg-7(RNAi) (upper three panels). The lower two panels are blue-native PAGE immunoblots of the same extracts. C. Oxygen consumption in atfs-1(tm4525) worms expressing transgenic ATFS-1 or ATFS-1^Δ1-32myc raised on spg-7(RNAi) (N = 3, ± SD, p* (student t-test) < 0.05). D. SDS-PAGE immunoblots of extracts from atfs-1 (tm4525) worms expressing transgenic ATFS-1 or ATFS-1^ΔNLS raised on spg-7(RNAi) (upper three panels). The lower two panels are blue-native PAGE immunoblots of the same extracts. E. Oxygen consumption in atfs-1(tm4525) worms expressing transgenic ATFS-1 or ATFS-1^ΔNLS raised on spg-7(RNAi) (N = 3, ± SD, p* (student t-test) < 0.05). F. Representative photomicrographs of synchronized afts-1(tm4525);hsp-60_pr∷gfp worms expressing transgenic ATFS-1, ATFS-1^Δ1-32myc or ATFS-1^ΔNLS allowed to develop for 72 hours on spg-7(RNAi). Note the increased progeny in the ATFS-1^WT panel relative to the ATFS-1^Δ1-32myc and ATFS-1^ΔNLS panels. Scale bar, 0.5 mm. G. Representative photomicrographs of synchronized wild-type or afts-1(tm4525) worms expressing transgenic ATFS-1 ^Δ1-32myc. Scale bar, 0.5 mm. H. Signaling model depicting ATFS-1's function during mitochondrial stress. See also Figure S6.