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ATFS-1 counteracts mitochondrial DNA damage by promoting repair over transcription

Nature Cell Biology volume 25, pages 1111–1120 (2023)Cite this article

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Abstract

The ability to balance conflicting functional demands is critical for ensuring organismal survival. The transcription and repair of the mitochondrial genome (mtDNA) requires separate enzymatic activities that can sterically compete1, suggesting a life-long trade-off between these two processes. Here in Caenorhabditis elegans, we find that the bZIP transcription factor ATFS-1/Atf5 (refs. 2,3) regulates this balance in favour of mtDNA repair by localizing to mitochondria and interfering with the assembly of the mitochondrial pre-initiation transcription complex between HMG-5/TFAM and RPOM-1/mtRNAP. ATFS-1-mediated transcriptional inhibition decreases age-dependent mtDNA molecular damage through the DNA glycosylase NTH-1/NTH1, as well as the helicase TWNK-1/TWNK, resulting in an enhancement in the functional longevity of cells and protection against decline in animal behaviour caused by targeted and severe mtDNA damage. Together, our findings reveal that ATFS-1 acts as a molecular focal point for the control of balance between genome expression and maintenance in the mitochondria.

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Fig. 1: Mitochondrial ATFS-1 counteracts cellular dysfunction caused by mtDSBs.
Fig. 2: Mitochondrial ATFS-1 reduces age-dependent mtDNA damage in a manner dependent on BER enzymes.
Fig. 3: Mitochondrial ATFS-1 requires the bZIP domain to interact with HMG-5 and reduce mtDNA transcript levels.
Fig. 4: ATFS-1 inhibits the formation of the mitochondrial pre-initiation complex.
Fig. 5: Mitochondrial function of ATFS-1 is non-tissue specific and evolutionarily conserved.

Data availability

Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank R. Tweedale, M. Hilliard and V. Anggono for the comments on the paper and members of the Zuryn laboratory for discussions and comments. This work was supported by NHMRC project grants GNT1128381 and GNT1162553 (to S.Z.), GNT2010332 (to A.F. and O.R.), ARC Discovery grant DP200101630 (to S.Z.), a Clem Jones Centre for Ageing Dementia flagship grant (to S.Z.), a Stafford Fox senior research fellowship (to S.Z.), NHMRC fellowships APP1154646 (to A.F.) and APP1154932 (to O.R.), University of Queensland International Scholarships to C.-Y.D. and C.-C.N., and a DFG postdoctoral fellowship to I.K. A.F. and O.R. are investigators of the ARC Centre of Excellence in Synthetic Biology (CE200100029).

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Authors and Affiliations

  1. Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia

    Chuan-Yang Dai, Chai Chee Ng, Grace Ching Ching Hung, Ina Kirmes, Christopher A. Brosnan, Arnaud Ahier, Anne Hahn & Steven Zuryn

  2. Harry Perkins Institute of Medical Research, QEII Medical Centre, The University of Western Australia, Nedlands, Western Australia, Australia

    Laetitia A. Hughes, Oliver Rackham & Aleksandra Filipovska

  3. ARC Centre of Excellence in Synthetic Biology, QEII Medical Centre, The University of Western Australia, Nedlands, Western Australia, Australia

    Laetitia A. Hughes, Oliver Rackham & Aleksandra Filipovska

  4. Telethon Kids Institute, Northern Entrance, Perth Children’s Hospital, Nedlands, Western Australia, Australia

    Laetitia A. Hughes, Oliver Rackham & Aleksandra Filipovska

  5. Department of Molecular, Cell and Cancer Biology, UMass Chan Medical School, Worchester, MA, USA

    Yunguang Du & Cole M. Haynes

  6. Curtin Medical School and Curtin Health Innovation Research Institute, Curtin University, Bentley, Western Australia, Australia

    Oliver Rackham

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Contributions

C.-Y.D. carried out most experiments. C.-C.N., G.C.C.H., A.A., I.K., L.A.H., Y.D., C.A.B., A.H. and S.Z. contributed some experiments. C.M.H., O.R. and A.F. helped design and interpret experiments. C.-Y.D. and S.Z. designed and interpreted experiments and wrote the paper.

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Correspondence to Steven Zuryn.

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Extended data

Extended Data Fig. 1 Invoking mtDSBs in C. elegans.

a, Schematic of the C. elegans mtDNA and PstI cleavage sites (9,097 bp and 9,250 bp) within the COI (cytochrome c oxidase subunit 1) gene. b, Schematics of C. elegans constructs containing PstI used in this study. The mitochondrial targeting sequence (MTS) was obtained from the protein SDHB-1 (succinate dehydrogenase complex subunit B). The red fluorophore mKate2 was fused to PstI. A splice leading sequence (SL2) was used to separate this fusion protein from the nuclear marker HIS-58::GFP. Each individual myo-3p::PstI construct was integrated into the same chromosome III location (oxTi444) as a Mos1-mediated single-copy insertion (mosSCI)37 to obtain similar levels of expression. c, d, Representative photomicrographs of transgenic 1-day-old adult animals (outlined in white dashed line) before and after heat-shock (37 °C, 30 min). A close-up of intestinal cells after heat-shock (d) shows the induction of HIS-58::GFP (nuclei) and hsp-16.2p::MTSPstI::mKate2 (mitochondria) expression. Experiment was repeated twice independently. e, DNA binding and catalytic mutations made in PstI were derived from key residues identified from the alignment results between PstI and the family-related SdaI enzyme56. Colours are according to the ClustalX scheme in Jalview. Red asterisks indicate DNA binding residues and blue asterisks indicate active sites of the enzyme56. Residues denoted with red arrowheads were mutated in myo-3p::PstIinactive constructs (fig. S1B). f, Representative photomicrographs of body wall muscle cells outlined by white dashed lines in 1-day-old adult animals expressing myo-3p::GFPmit and each PstI variant expressed under the myo-3p promoter. MTSPstI and PstIinactive localize to mitochondria (white arrowheads), whereas PstI localizes to the cytosol (yellow arrowheads). Experiment was repeated three times independently. g, h, Fluorescence visualization (g) and immunoblots (h) of the hsp-6p::GFP reporter in L4 animals expressing variants of the myo-3p::PstI::mKate2 transgene. Experiments were repeated three times independently. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 ATFS-1 responds to mtDSBs by localizing to nuclei and inducing the UPRmt.

a, Representative photomicrographs of TOMM-20MTS::RFP and ATFS-1::GFP localization in a body wall muscle cell. Right panel, fluorescence intensity of RFP (localized to the outer mitochondrial membrane) and GFP in a cross-section of a mitochondrial puncta showing that GFP localizes to the mitochondrial matrix. Experiment was repeated three time independently. b, c, Representative photomicrographs (b) and quantification (c) of ATFS-1::GFP localization in body wall muscle cells in the heads of animals. Columns represent mean ± SEM; Control, n = 32; myo-3p::MTSPstI, n = 23; myo-3p:: PstI, n = 26; myo-3p:: PstIinactive, n = 24; all n values represent biologically independent animals. One-way ANOVA with Tukey’s post hoc test. In (a) and (b), muscle cells are outlined by a white dashed line and nuclei are outlined by a yellow dashed line. d, Representative photomicrographs of hsp-6p::GFP reporter fluorescence restricted to body wall muscle cells (outlined in yellow dashed line) in animals expressing myo-3p::MTSPstI::mKate2. Experiment was repeated twice independently. e, f, Fluorescence visualization (e) and immunoblots (f) of the hsp-6p::GFP, hsp-60p::GFP, hsp-4p::GFP, and hsp-16.2p::GFP reporters in animals +/- myo-3p::MTSPstI. Experiments were repeated twice independently. g, h, Fluorescence visualization (g) and immunoblots (h) of hsp-60p::GFP reporter in atfs-1(tm4525) mutants +/- myo-3p::MTSPstI. Immunoblots were repeated twice independently. i – k WormLab quantification of body bends in the atfs-1 mutant L4 animals (alleles tm4525, tm4919 and cmh15). Columns represent mean ± SEM; (i) myo-3p::MTSPstI, n = 100; myo-3p::MTSPstI; atfs-1(tm4525), n = 101; (j) myo-3p::MTSPstI, n = 30; myo-3p::MTSPstI; atfs-1(tm4919), n = 36; (k) myo-3p::MTSPstI, n = 118; myo-3p::MTSPstI; atfs-1(cmh15), n = 99; all n values represent biologically independent animals examined over two independent experiments. Two-way Student’s t test. l, Schematic representation of the atfs-1 gene and mutant alleles. MTS, mitochondrial targeting sequence. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 Dissection of ATFS-1 subcellular activities in body wall muscle cells.

a, Schematics of the ATFS-1::Cerulean constructs used in this study. Each construct was integrated into the same chromosome I location (oxTi185) as a mosSCI insertion to obtain comparable expression levels between the transgenic strains. b, c, Representative photomicrographs (b) and quantification (c) (n = 35 L4 animals for each condition) of the subcellular localization patterns of ATFS-1::Cerulean variants +/- myo-3p::MTSPstI in body wall muscle cells. Experiment was repeated twice independently. d - f, Fluorescence visualization (d), fluorescence intensity quantification (e) (n = 29 biologically independent animals for each genotype) and immunoblots (f) of the hsp-60p::GFP reporter in body muscle cells in myo-3p::MTSPstI;atfs-1(tm4525) L4 animals expressing variants of ATFS-1. For (e), columns represent mean ± SEM; n = 29 biologically independent animals for each genotype; one-way ANOVA with Tukey’s post hoc test. Immunoblots (f) repeated twice independently. g, WormLab quantification of body bends in L4 animals expressing ATFS-1 under the control of tissue-specific promoters for the intestine (ges-1p), all neurons (rgef-1p), hypodermis (dpy-7p), or body wall muscles (myo-3p). -, non-transgenic animals; +, transgenic animals. Columns represent mean ± SEM; intestine (-), n = 55; intestine (+), n = 73; pan-neuronal (-), n = 94; pan-neuronal (+), n = 64; hypodermis (-), n = 99; hypodermis (+), n = 72; body wall muscle (-), n = 60; body wall muscle (+), n = 47; all n values represent biologically independent animals examined over two independent experiments. One-way ANOVA with Šidák’s multiple comparision test. h, CRISPR genome editing of the endogenous atfs-1 gene to ATFS-1mit partially restores body wall muscle function in myo-3p::MTSPstI animals. Columns represent mean ± SEM; n = 60 biologically independent animals examined over two independent experiments for each genotype; one-way ANOVA with Tukey’s post hoc test. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 ATFS-1 does not decrease mtDSB levels but reduces the accumulation of age-accumulated mtDNA damage in a dose-dependent manner.

a, qPCR analysis of mtDNA cleavage (primer set 1, crossing the PstI cleavage sites) and total mtDNA copy numbers (primer set 2) in glp-4(bn2) L4 animals +/- hsp-16.2p::MTSPstI::mKate2::SL2::HIS-58::GFP and atfs-1p::ATFS-1mit::GFP. N = 3 biological replicates for each condition; one-way ANOVA with Tukey’s post hoc test. b, Quantification of mtDNA damage in the somatic tissues of L1 to 7 days-adult glp-4(bn2) animals using long-template semi-qPCR as an alternative to long-template qPCR (Fig. 2A). c, qPCR quantifications of mtDNA damage (left side of graph) and the average copy number of the atfs-1p::ATFS-1mit transgene (right side of graph) in color-matched individuals from individual samples of glp-4(bn2) animals. Reductions in mtDNA damage were dependent on transgene copy-number. N = 6 biological replicates for each condition; two-way Student’s t test. d, Representative photomicrographs of mitochondrial swelling (white arrowheads) in the body wall muscle cell (outlined in white dashed line) of live animals. e, Quantification of the fraction of animals with mitochondrial swelling in their body wall muscle cells. N = 3 independent experiments for each data point. One-way ANOVA with Dunnett’s post hoc test. f, Representative photomicrographs of GFP-labelled myosin filament structures in muscle cells. g, Quantification of the fraction of animals with MYO-3::GFP puncta in their muscle cells. N = 3 independent experiments for each data point. One-way ANOVA with Tukey’s post hoc test. The exact animal numbers used in figure (e) and (g) are shown in the source data. h, i, WormLab quantification of body bends in L1 and L4 animals expressing variants of the myo-3p::ATFS-1 transgene. N values are shown in source data and they represent biologically independent animals examined over two independent experiments. One-way ANOVA with Tukey’s post hoc test. Columns in all panels represent mean ± SEM. Source numerical data are available in source data.

Source data

Extended Data Fig. 5 NTH-1::YFP and TWNK-1::YFP localize to mitochondria in HEK293T cells.

a, Representative photomicrographs of NTH-1::YFP (top panels) and TWNK-1::YFP (bottom panels) in HEK293T cells. Mitochondria were co-stained with MitoTracker (red) and nuclei with DAPI (blue). Experiments were repeated twice independently. b, Fluorescence intensity of YFP and MitoTracker in a cross section of a mitochondrial puncta (white diagonal line in merge panels in (a)) shows an overlap in signals. c, ATP content of 7-day-old animals. e.v., empty vector. Columns represent mean ± SEM; for the tests on twnk-1 and nth-1 mutants, control, n = 8; twnk-1 (ok3198), n = 8; nth-1 (ok724), n = 5; for the test on ung-1 RNAi, control, n = 6; ung-1 (RNAi), n = 7; all n values represent biologically independent samples. For mutants, one-way ANOVA with Tukey’s post hoc test; for RNAi, Student’s t test. d, qPCR detection of somatic mtDNA damage in L4 animals +/- atfs-1p::ATFS-1mit::GFP (labeled as atfs-1p::ATFS-1mit in the figure) after ultraviolet C (300 J m-2) exposure. e, the results of (d) normalized to control (non-UVC treatment) levels of mtDNA damage. Columns represent mean ± SEM; (d) without UVC treatment, wild type, n = 26; atfs-1p::ATFS-1mit, n = 27; with UVC treatment, wild type, n = 27; atfs-1p::ATFS-1mit, n = 27; (e) wild type, n = 26; atfs-1p::ATFS-1mit, n = 27; all n values represent biologically independent samples. Two-way Student’s t test. For (c) and (d), glp-4(bn2) animals were raised at the non-permissive temperature, 25 °C. Source numerical data are available in source data.

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Extended Data Fig. 6 ATFS-1 physically interacts with HMG-5 and requires the bZIP domain for cytoprotection against mtDSBs.

a, b, qPCR quantification of mtDNA copy number in glp-4(bn2) mutants in the (a) +/- ATFS-1mit (CRISPR) animals, and (b) +/- atfs-1p::ATFS-1mit::GFP animals (labeled as atfs-1p::ATFS-1mit in the figure). Columns represent mean ± SEM; (a) n = 3 and (b) n = 6 biological replicates for each genotype; two-way Student’s t test. c, d, Representative photomicrograph (c) and quantification (d) (n = 46 animals) of the subcellular localization pattern of ATFS-1∆bZIP::Cerulean in a body wall muscle cell (outlined by a white dashed line) in L4 animals expressing myo-3p::MTSPstI. e, WormLab quantification of body bends in myo-3p::MTSPstI;myo-3p::ATFS-1mit L4 animals grown on hmg-5 RNAi or empty vector (e.v.). e.v. (RNAi), n = 32; hmg-5 (RNAi), n = 36; all n values represent biologically independent animals examined over two independent experiments. f, ATP content of L4 animals. e.v. (RNAi), n = 10; hmg-5 (RNAi), n = 9; all n values represent biologically independent samples. Two-way Student’s t test. g, WormLab quantification of body bends in myo-3p::MTSPstI animals +/- myo-3p::NTH-1::mCherry L4 animals. myo-3p::MTSPstI, n = 73; myo-3p::MTSPstI; myo-3p::NTH-1::mCherry, n = 67; all n values represent biologically independent animals examined over two independent experiments. For (e - g), columns represent mean ± SEM; two-way Student’s t test. For (a) and (b), glp-4(bn2) animals were raised at the non-permissive temperature, 25 °C. Source numerical data are available in source data.

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Extended Data Fig. 7 Co-immunoprecipitation experiments.

a, Tagged C. elegans proteins are separated by 2 A peptide sequences, allowing a single transcript RNA to produce all three proteins at approximately ratiometrically equal amounts. b, Alternative immunoblots of co-immunoprecipitation of HMG-5 by RPOM-1N corresponding to Fig. 4c, which were used for quantification purposes. Experiment was repeated three time independently. c, Representative immunoblots of co-immunoprecipitation of HMG-5 by ATFS-1. ATFS-1-HMG-5 interaction prevented detection of RPOM-1N interaction as demonstrated by the lack of an RPOM-1N signal. Experiment was repeated three time independently. d, Alternative immunoblots of co-immunoprecipitation of RPOM-1N by HMG-5 corresponding to Fig. 4d, which were used for quantification purposes. Arrowheads, bands corresponding to proteins of interest; *, unspecific background bands; FL, full-length ATFS-1; ∆bZIP, ATFS-1∆bZIP; †, HMG-5::HA detection by secondary antibody recognition of anti-HA and anti-myc primary antibodies. Experiment was repeated three time independently. Unprocessed blots are available in source data.

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Extended Data Fig. 8 Mitochondrial ATFS-1 does not alter mitochondrial translation and reducing mitochondrial translation is not cytoprotective against mtDSBs.

a, b, Representative immunoblots (a) and ratiometric quantification (b) of the nuclear encoded mitochondrial protein ATPB (ATP synthase subunit beta) and the mtDNA-encoded protein MTCOI (cytochrome c oxidase subunit 1) in glp-4(bn2) animals carrying either atfs-1p::ATFS-1mit or atfs-1p::ATFS-1∆bZIP (labeled as ATFS-1mit or ATFS-1∆bZIP in the figure, respectively). Columns represent mean ± SD; n = 2 biological replicates. c, Representative immunoblots of HSP-60 (heat shock protein 60) in the same animals as in (a). d, e, Representative immunoblots (d) and ratiometric quantification (e) of ATPB and MTCOI in animals treated with doxycycline (30 μg ml-1), (e) Columns represent mean ± SEM, n = 2 biologically independent samples. f, WormLab quantification of body bends in L4 animals expressing myo-3p::MTSPstI treated with doxycycline. Columns represent mean ± SEM; wild type, n = 34; 0 doxycycline, n = 46; 30 doxycycline, n = 54; all n values represent biologically independent animals examined over two independent experiments. One-way ANOVA with Tukey’s post hoc test. Immunoblot (d) was repeated twice independently. g, WormLab quantification of body bends in L4 animals expressing myo-3p::MTSPstI with MRPS-12 mutations K89I and K90I. Columns represent mean ± SEM; wild type, n = 67; myo-3p::MTSPstI, n = 90; myo-3p::MTSPstI; CRISPRMRPS-12K89T, n = 107; myo-3p::MTSPstI; CRISPRMRPS-12K90I, n = 72; all n values represent biologically independent animals examined over 2 independent experiments. One-way ANOVA with Tukey’s post hoc test. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 9 Selective expression of mitochondrial ATFS-1 in the intestine protects against defects in growth caused by intestinal-specific mtDSBs.

a - c, Fluorescence visualization (a), fluorescence quantification (b), and immunoblots (c) of the hsp-6p::GFP reporter in L4 animals +/- ges-1p::MTSPstI::mKate2. Columns represent mean ± SEM; n = 11 biologically independent animals for each genotype; two-way Student’s t test. d, Representative photomicrograph of a CAL51 human cells expressing ATFS-1mit::mEGFP and the mitochondrial ribosomal protein MRPL12::mCherry. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 10 Mitochondrial ATFS-1 increases during aging and in response to mtDSBs.

a, b, Representative photomicrographs (a) and fluorescence intensity quantification (b) of atfs-1p::ATFS-1mit::GFP fluorescence in L4 and 7-day-old adult animals (outlined by a white dashed line). *, co-injection marker (odr-1p::GFP) expression was excluded from quantification. See materials and methods for quantification methods. Columns represent mean ± SEM; n = 48 biologically independent animals for both L4 and 7 days animals were examined over two independent experiments. Two-way Student’s t test. c, Representative Western blot detection of ATFS-1mit::GFP in 30 animals at L4 and 7-day-old adult stages. d, e, Representative photomicrographs (d) and fluorescence intensity quantification (e) of myo-3p::ATFS-1mit::Cerulean fluorescence in control and myo-3p::MTSPstI L4 animals. Columns represent mean ± SEM; control, n = 13; myo-3p::ATFS-1mit::Cerulean, n = 36; myo-3p::ATFS-1mit::Cerulean; myo-3p::MTSPstI, n = 24; all n values represent biologically independent animals. Two-way Student’s t test. Source numerical data and unprocessed blots are available in source data.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. A list of candidate screening, one-way ANOVA with Tukey’s post hoc test. Supplementary Table 2. Strains generated for this study. Supplementary Table 3. Oligonucleotides used in this study. Source numerical data and unprocessed blots are available in source data.

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Dai, CY., Ng, C.C., Hung, G.C.C. et al. ATFS-1 counteracts mitochondrial DNA damage by promoting repair over transcription. Nat Cell Biol 25, 1111–1120 (2023). https://doi.org/10.1038/s41556-023-01192-y

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