CEST‐2.2 overexpression alters lipid metabolism and extends longevity of mitochondrial mutants
2022; Springer Nature; Volume: 23; Issue: 5 Linguagem: Inglês
10.15252/embr.202152606
ISSN1469-3178
AutoresAntonia Piazzesi, Yiru A. Wang, Joshua Jackson, Lena Wischhof, Viktoria V. Zeisler‐Diehl, Enzo Scifo, Ina Oganezova, Thorben Hoffmann, Pablo Gómez Martín, Fabio Bertan, Chester J. J. Wrobel, Frank C. Schroeder, Dan Ehninger, Kristian Händler, Joachim L. Schultze, Lukas Schreiber, Gerhild van Echten‐Deckert, Pierluigi Nicotera, Daniele Bano,
Tópico(s)Coenzyme Q10 studies and effects
ResumoArticle17 March 2022Open Access Source DataTransparent process CEST-2.2 overexpression alters lipid metabolism and extends longevity of mitochondrial mutants Antonia Piazzesi Antonia Piazzesi orcid.org/0000-0002-7591-6473 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Conceptualization, Supervision, Validation, Investigation, Writing - original draft Search for more papers by this author Yiru Wang Yiru Wang orcid.org/0000-0002-6522-9995 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Validation, Investigation Search for more papers by this author Joshua Jackson Joshua Jackson orcid.org/0000-0002-3515-5593 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Lena Wischhof Lena Wischhof orcid.org/0000-0002-2203-408X German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Viktoria Zeisler-Diehl Viktoria Zeisler-Diehl Institute of Cellular and Molecular Botany (IZMB), University of Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Enzo Scifo Enzo Scifo orcid.org/0000-0002-0450-6573 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Ina Oganezova Ina Oganezova orcid.org/0000-0001-8672-8497 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Thorben Hoffmann Thorben Hoffmann orcid.org/0000-0001-5532-6153 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Pablo Gómez Martín Pablo Gómez Martín German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Fabio Bertan Fabio Bertan German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Chester J J Wrobel Chester J J Wrobel Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA Contribution: Methodology Search for more papers by this author Frank C Schroeder Frank C Schroeder Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA Contribution: Methodology Search for more papers by this author Dan Ehninger Dan Ehninger German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Kristian Händler Kristian Händler German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany PRECISE Platform for Single Cell Genomics and Epigenomics, German Center for Neurodegenerative Diseases (DZNE), University of Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Joachim L Schultze Joachim L Schultze German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany PRECISE Platform for Single Cell Genomics and Epigenomics, German Center for Neurodegenerative Diseases (DZNE), University of Bonn, Bonn, Germany Department for Genomics and Immunoregulation, LIMES Institute, University of Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Lukas Schreiber Lukas Schreiber orcid.org/0000-0001-7003-9929 Institute of Cellular and Molecular Botany (IZMB), University of Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Gerhild van Echten-Deckert Gerhild van Echten-Deckert orcid.org/0000-0002-2816-7852 LIMES Institute for Membrane Biology and Lipid Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Pierluigi Nicotera Pierluigi Nicotera orcid.org/0000-0003-1175-7466 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Funding acquisition Search for more papers by this author Daniele Bano Corresponding Author Daniele Bano [email protected] orcid.org/0000-0002-9617-5504 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Antonia Piazzesi Antonia Piazzesi orcid.org/0000-0002-7591-6473 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Conceptualization, Supervision, Validation, Investigation, Writing - original draft Search for more papers by this author Yiru Wang Yiru Wang orcid.org/0000-0002-6522-9995 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Validation, Investigation Search for more papers by this author Joshua Jackson Joshua Jackson orcid.org/0000-0002-3515-5593 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Lena Wischhof Lena Wischhof orcid.org/0000-0002-2203-408X German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Viktoria Zeisler-Diehl Viktoria Zeisler-Diehl Institute of Cellular and Molecular Botany (IZMB), University of Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Enzo Scifo Enzo Scifo orcid.org/0000-0002-0450-6573 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Ina Oganezova Ina Oganezova orcid.org/0000-0001-8672-8497 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Thorben Hoffmann Thorben Hoffmann orcid.org/0000-0001-5532-6153 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Pablo Gómez Martín Pablo Gómez Martín German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Fabio Bertan Fabio Bertan German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Chester J J Wrobel Chester J J Wrobel Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA Contribution: Methodology Search for more papers by this author Frank C Schroeder Frank C Schroeder Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA Contribution: Methodology Search for more papers by this author Dan Ehninger Dan Ehninger German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Investigation Search for more papers by this author Kristian Händler Kristian Händler German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany PRECISE Platform for Single Cell Genomics and Epigenomics, German Center for Neurodegenerative Diseases (DZNE), University of Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Joachim L Schultze Joachim L Schultze German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany PRECISE Platform for Single Cell Genomics and Epigenomics, German Center for Neurodegenerative Diseases (DZNE), University of Bonn, Bonn, Germany Department for Genomics and Immunoregulation, LIMES Institute, University of Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Lukas Schreiber Lukas Schreiber orcid.org/0000-0001-7003-9929 Institute of Cellular and Molecular Botany (IZMB), University of Bonn, Bonn, Germany Contribution: Investigation Search for more papers by this author Gerhild van Echten-Deckert Gerhild van Echten-Deckert orcid.org/0000-0002-2816-7852 LIMES Institute for Membrane Biology and Lipid Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Pierluigi Nicotera Pierluigi Nicotera orcid.org/0000-0003-1175-7466 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Funding acquisition Search for more papers by this author Daniele Bano Corresponding Author Daniele Bano [email protected] orcid.org/0000-0002-9617-5504 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Antonia Piazzesi1,†, Yiru Wang1,†, Joshua Jackson1,†, Lena Wischhof1, Viktoria Zeisler-Diehl2, Enzo Scifo1, Ina Oganezova1, Thorben Hoffmann1, Pablo Gómez Martín1, Fabio Bertan1, Chester J J Wrobel3, Frank C Schroeder3, Dan Ehninger1, Kristian Händler1,4, Joachim L Schultze1,4,5, Lukas Schreiber2, Gerhild Echten-Deckert6, Pierluigi Nicotera1 and Daniele Bano *,1 1German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 2Institute of Cellular and Molecular Botany (IZMB), University of Bonn, Bonn, Germany 3Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA 4PRECISE Platform for Single Cell Genomics and Epigenomics, German Center for Neurodegenerative Diseases (DZNE), University of Bonn, Bonn, Germany 5Department for Genomics and Immunoregulation, LIMES Institute, University of Bonn, Bonn, Germany 6LIMES Institute for Membrane Biology and Lipid Biochemistry, University of Bonn, Bonn, Germany † These authors contributed equally to this work *Corresponding author. Tel: +49 228 43302 510; Fax: +49 228 43302 689; E-mail: [email protected] EMBO Reports (2022)23:e52606https://doi.org/10.15252/embr.202152606 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Mitochondrial dysfunction can either extend or decrease Caenorhabditis elegans lifespan, depending on whether transcriptionally regulated responses can elicit durable stress adaptation to otherwise detrimental lesions. Here, we test the hypothesis that enhanced metabolic flexibility is sufficient to circumvent bioenergetic abnormalities associated with the phenotypic threshold effect, thereby transforming short-lived mitochondrial mutants into long-lived ones. We find that CEST-2.2, a carboxylesterase mainly localizes in the intestine, may stimulate the survival of mitochondrial deficient animals. We report that genetic manipulation of cest-2.2 expression has a minor lifespan impact on wild-type nematodes, whereas its overexpression markedly extends the lifespan of complex I-deficient gas-1(fc21) mutants. We profile the transcriptome and lipidome of cest-2.2 overexpressing animals and show that CEST-2.2 stimulates lipid metabolism and fatty acid beta-oxidation, thereby enhancing mitochondrial respiratory capacity through complex II and LET-721/ETFDH, despite the inherited genetic lesion of complex I. Together, our findings unveil a metabolic pathway that, through the tissue-specific mobilization of lipid deposits, may influence the longevity of mitochondrial mutant C. elegans. SYNOPSIS Mitochondrial function influences survival and stress resilience in Caenorhabditis elegans. cest-2.2 overexpression promotes mobilization of lipid deposits and extends lifespan of complex I deficient mutants. cest-2.2 is a H3.3-target gene differentially expressed in long- versus short-lived mitochondrial mutant nematodes. CEST-2.2 O/E is sufficient to promote lifespan extension of short-lived, mitochondrial complex I deficient gas-1(fc21) mutants. CEST-2.2 O/E stimulates lipid catabolism and fatty acid beta-oxidation in complex I mutants. CEST-2.2 O/E enhances mitochondrial respiration of gas-1 mutants. Introduction Mitochondria contribute to energy production and host biosynthetic reactions that supply molecules for metabolic and signaling processes. Through iterative oxidation of nutrients (e.g., sugars, amino acids, and fatty acids), mitochondria generate reducing equivalents in the form of metabolic intermediates, NADH, and FADH2 (Spinelli & Haigis, 2018; Martinez-Reyes & Chandel, 2020). The exothermic transfer of electrons from these reduced molecules to the electron transport chain (ETC) and then to oxygen is used by the respiratory complexes (i.e., complex I, complex III, and complex IV) to drive protons from the matrix to the intermembrane space. The subsequent dissipation of the electrochemical gradient across the inner membrane promotes ATP biosynthesis and thermogenesis, ROS production, and ion transport (Martinez-Reyes & Chandel, 2020; Sies & Jones, 2020). All these processes influence cell division, differentiation, maintenance, and demise, thereby contributing to animal development, growth, and survival. A large literature has described an increasing number of genetic lesions and environmental toxins that alter mitochondrial oxidative phosphorylation (OXPHOS) and can elicit human metabolic disorders and neurodegenerative conditions, further emphasizing the relevance of mitochondria in human pathophysiology (Schon & Przedborski, 2011; Koopman et al, 2012; Gorman et al, 2016; Bano & Prehn, 2018; Wallace, 2018; Frazier et al, 2019). Despite the profound clinical importance, currently available therapeutic interventions are mostly symptomatic, since they may improve the life quality of the patients without substantially modifying the progression of the disease (Gorman et al, 2016; Russell et al, 2020). Other challenges include a better understanding of the molecular mechanisms that can counteract mitochondrial dysfunction, which may help to single out novel targets potentially relevant for therapeutic purposes. In this regard, preclinical studies in recent years have described innovative genetic and pharmacological approaches that improve organismal fitness by stimulating transcriptional programs that buffer toxic species and enhance the usage of available resources. Consistent with this concept, it was shown that inhibition of mTOR signaling ameliorates aberrant processes linked to mitochondrial dysfunction by shifting metabolism toward catabolism (Johnson et al, 2013; Ising et al, 2015; Peng et al, 2015; Zheng et al, 2016; Khan et al, 2017; Siegmund et al, 2017; Wischhof et al, 2018; Gioran et al, 2019). Similarly, chronic exposure to moderate hypoxia alleviates metabolic defects and triggers adaptative pathways that limit the damage due to impaired mitochondrial bioenergetics, thereby stimulating the survival of animals carrying mitochondrial genetic lesions (Jain et al, 2016, 2019, 2020; Ast et al, 2019; To et al, 2019; Grange et al, 2021). As additional examples, supplementation of nicotinamide mononucleotide, and pharmacological and genetic manipulations of the redox state improve the metabolic derangement due to NAD+ depletion in mitochondrial deficient cells (Canto et al, 2012; Karamanlidis et al, 2013; Mouchiroud et al, 2013; Cerutti et al, 2014; Pirinen et al, 2014; Cracan et al, 2017; Liu et al, 2021). Based on these converging lines of evidence, eukaryotic cells are capable of responding to defective mitochondrial energy production by establishing transcriptional activities and broad metabolic reprogramming that channel resources to build up protective mechanisms against long-lasting systemic changes. In the context of human pathophysiology, it is crucial to decode this stress response network, since it may provide molecular targets that are therapeutically valuable for chronic and/or inherited diseases associated with mitochondrial lesions (Lardenoije et al, 2015; Riera et al, 2016; Mottis et al, 2019). The mitochondrial threshold effect theory assumes that phenotypic presentations of mitochondrial dysfunction occur when a critical state is reached and functional compensation is irremediably compromised (Rossignol et al, 2003), however, it remains elusive, which are the key metabolic processes that dictate the trade-off between growth, reproduction, and somatic maintenance during adverse conditions. The nematode Caenorhabditis elegans has proven to be a useful in vivo model organism to investigate the mitochondrial threshold effect in stress resilience and lifespan-extending programs (Riera et al, 2016; Shpilka & Haynes, 2018). During C. elegans development (Feng et al, 2001; Dillin et al, 2002; Rea et al, 2007), mild disruption of the mitochondrial ETC activity impairs OXPHOS and evokes a profound transcriptional reprogramming that alleviates the chronic energy crisis. Along with a considerable shift toward catabolic processes (Gioran et al, 2019), transcriptional activation of unfolded protein response (UPR) and expression of detoxifying enzymes (e.g., SOD-3 and GST-4) prevent the detrimental collapse of cellular proteostasis (Lin & Haynes, 2016; Tian et al, 2016b; Shpilka & Haynes, 2018). In a cell-nonautonomous fashion, secretion of signals (e.g., ROS, neurotransmitters, and hormones) from mitochondrial deficient cells can communicate adverse conditions throughout the whole organism. In this regard, the enhanced responsiveness to stressful states presumably elicits global protective mechanisms that have beneficial effects on animal survival (Durieux et al, 2011). These broad physiological changes require a permissive chromatin landscape for the engagement and establishment of transcriptional programs that control homeostatic processes (Benedetti et al, 2006; Haynes et al, 2010; Nargund et al, 2012; Merkwirth et al, 2016; Piazzesi et al, 2016; Tian et al, 2016a). In this regard, we previously reported that aberrant chromatin remodeling due to the loss of replication-independent histone H3.3 (i.e., the main H3 variant that is expressed in postmitotic cells and accumulates during aging) impairs the establishment of lifespan-extending programs, including those dependent on mitochondria (Troulinaki & Bano, 2012; Piazzesi et al, 2016; Bano et al, 2017). Consistently, altered histone H3 methylation compromises the transcriptional activation of mitochondrial unfolded protein response (UPRmt) and, consequently, shortens the lifespan of mitochondrial C. elegans mutants (Merkwirth et al, 2016; Tian et al, 2016a, 2016b). Thus, these lines of evidence suggest that epigenetic plasticity can confer physiological fitness to multicellular organisms. If so, this concept raises a very basic question: can we transform sick, short-lived mitochondrial mutants into long-lived ones? Here, we report that overexpression of a single enzyme is sufficient to potentiate the residual respiratory capacity of complex I-deficient nematodes by redirecting lipid usage toward fatty acid beta-oxidation. Through multiomics analyses coupled with conventional epistatic studies, we demonstrate a previously unknown metabolic network that can extend C. elegans lifespan, with molecular aspects that may be partially conserved in some disease settings in higher metazoa. More broadly, our findings emphasize the strength of adaptive mechanisms in promoting epigenetic reprogramming that sustains survival of animals carrying mitochondrial lesions. Results CEST-2.2 expression influences the lifespan of complex I mutant nematodes We previously reported that H3.3 expression establishes lifespan-extending programs in C. elegans by supporting transcriptional plasticity underlying stress resilience (Piazzesi et al, 2016). Based on that, we hypothesized that H3.3 may be differently loaded onto chromatin in long-lived mitochondrial mutants compared with short-lived ones. To test this hypothesis, we employed animals carrying either nuo-6(qm200) or gas-1(fc21) alleles, both altering complex I activity, but extending or reducing C. elegans lifespan, respectively (Fig EV1A and Appendix Table S1). Using wild-type (wt) and mitochondrial mutant animals expressing GFP-tagged HIS-72 (Ooi et al, 2006; Piazzesi et al, 2016), we performed chromatin immunoprecipitation (ChIP) followed by deep sequencing (Fig 1A), focusing our analysis exclusively on promoter regions. We found that HIS-72::GFP was loaded onto the promoters of 996 genes and 917 genes in nuo-6(qm200) and gas-1(fc21) mutant nematodes, respectively (Fig 1B). Of these, 711 genes overlapped between the two strains, with 591 of those also being shared with wt nematodes (Fig 1B). We investigated the expression profiles of the 179 genes in which H3.3-enriched promoters were exclusively detected in the long-lived nuo-6(qm200) mutants. We employed the respective RNA-seq datasets from these mutants (Piazzesi et al, 2016; Gioran et al, 2019) and found that 10 of these genes were consistently downregulated in gas-1(fc21) mutants, upregulated in nuo-6(qm200) mutants but downregulated in nuo-6(qm200), his-72(tm2066), and his-71(ok2289) triple mutant animals, consistent with their transcription being modulated by the presence of H3.3 (Fig 1C and D). Of these 10 genes, 5 of them encode evolutionarily conserved proteins (Fig 1D). One of these proteins is ZC376.2/CEST-2.2, which is a carboxylesterase phylogenetically related to the serine hydrolase family and orthologous to human carboxylesterases and carboxyl ester lipases (Chen et al, 2019; Le et al, 2020). As recently described (Le et al, 2020), CEST-2.2 localizes to intestinal granules and contributes to the biosynthesis of ascarosides, including ascr#8, ascr#81, and ascr#82. Since CEST-2.2 is the most likely to play a role in metabolism, we decided to make this candidate the focus of our study. We performed quantitative real-time PCR (qRT-PCR) and confirmed that cest-2.2 was downregulated in gas-1(fc21) mutants compared with wt animals, whereas it was upregulated in a H3.3-dependent manner in nuo-6(qm200) mutant nematodes (Figs 1E and EV1B). To test the role of cest-2.2 in longevity, wt and nuo-6 mutant nematodes were grown on bacteria expressing double-strand RNA against cest-2.2. We observed that cest-2.2 RNAi inhibited nuo-6(qm200) longevity (Fig 1F and G and Appendix Table S1) as well as also slightly (yet significantly) reduced the survival of wt nematodes (Fig 1H and I, Appendix Table S1). To further explore CEST-2.2 contribution to survival, we ablated the cest-2.2 encoding region using CRISPR/Cas9-based gene editing (Fig EV1C). We found that cest-2.2 loss-of-function (lf) mutants had a minor tendency toward a decreased median lifespan compared with wt animals, whereas cest-2.2(lf) inhibited the lifespan extension due to the RNAi against complex IV subunit CCO-1 (Figs 1J and EV1D, Appendix Table S1). When we attempted to generate nuo-6, cest-2.2 double mutants, we could obtain only hermaphrodites carrying the two mutant alleles in heterozygosity, further highlighting the contribution of CEST-2.2 to the survival of complex I-deficient nematodes. However, cest-2.2 downregulation had no effect on the survival of long-lived sod-2(ok1030) and complex III isp-1(qm150) mutants (Fig EV1E and F, Appendix Table S1). Consistent with the metabolic heterogeneity of mitochondrial diseases (Lake et al, 2016; Wallace, 2018; Frazier et al, 2019; Russell et al, 2020), these data suggest that CEST-2.2 influences the lifespan of mitochondrial mutant nematodes depending on the genetic lesions in question. Click here to expand this figure. Figure EV1. (relative to Fig 1). Characterization of cest-2.2 mutant nematodes A. Representative survival curve of wt, gas-1(fc21) and nuo-6(qm200) mutant nematodes. B. qRT-PCR of cest-2.2 mRNA expression in dissected intestines that were isolated from wt, nuo-6(qm200); his-72(tm2066); his-71(ok2289) and nuo-6(qm200) mutants (one-way ANOVA with the Tukey's multiple comparisons test, n = 3 biological replicates). C. CRISPR/Cas9 strategy for the generation of cest-2.2(bon52)V, with the resulting edited sequence below. In yellow: exon 1 fragment. In green: exon 11 fragment. In red: extra nucleotides inserted during CRISPR/Cas9 gene editing. D. Representative survival curves of wt and cest-2.2(lf) nematodes fed with either control (solid lines) or cco-1 (dashed lines) RNAi. E, F. Representative lifespan assays of (E) sod-2(ok1030) and (F) isp-1(qm150) fed with control and cest-2.2 RNAi. G. qRT-PCR of cest-2.2 mRNA expression in wt, gas-1(fc21), gas-1(fc21); cest-2.2 O/E and cest-2.2(lf); gas-1(fc21); cest-2.2 O/E (n = 2 biological replicates). H, I. qRT-PCR of cest-2.2 expression in (H) gas-1(fc21); cest-2.2 O/E and (I) gas-1(fc21) animals few with control and cest-2.2 RNAi (n = 2 biological replicates). J. Lifespan assay of gas-1 mutants grown on control and cest-2.2 RNAi bacteria. K, L. Representative survival analyses of (K) nuo-6(qm200) versus nuo-6(qm200); cest-2.2 O/E and (L) mev-1(kn1) versus mev-1(kn1); cest-2.2 O/E nematodes. M. Quantification of normalized body area of wt, cest-2.2 O/E and cest-2.2 (lf) C. elegans (ordinary one-way ANOVA with the Dunnett's multiple comparison test, n = 10–11 animals). N. Locomotory activity (arbitrary units) of wt, cest-2.2 O/E and cest-2.2 (lf) nematodes (n. of animals > 6). Data information: In A, D, E, F, J, K, and L, the average median lifespan ± SEM across replicates is shown under the graphs, and additional information (e.g., n numbers) are reported in Appendix Table S1; in B, G, H, I, M, and N, bars represent average ± SEM). Across experiments, P-value summary is ns = not significant, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. CEST-2.2 expression influences the lifespan of mitochondrial mutant C. elegans Schematic representation of ChIP-Seq strategy. An antibody against GFP was used to immunoprecipitate HIS-72::GFP loaded onto chromatin. Subsequent DNA sequencing was performed onto immunoprecipitated chromatin. Venn diagram of genes with H3.3-enriched promoters between wt (blue), gas-1(fc21) (red), and nuo-6(qm200) (green) mutants. Heatmap of genes with H3.3-enriched promoters exclusively in nuo-6(qm200) mutants, downregulated in gas-1(fc21) versus wt, upregulated in nuo-6(qm200) versus wt, and downregulated in nuo-6(qm200); his-72(tm2066); his-71(ok2289) versus nuo-6(qm200). Table of genes with H3.3-enriched promoters exclusively in nuo-6(qm200) nematodes, whose gene expression was upregulated in nuo-6(qm200) versus wt, downregulated in gas-1(fc21) versus wt, and downregulated in nuo-6(qm200); his-72(tm2066); his-71(ok2289) versus nuo-6(qm200). Human orthologs and corresponding % homology are indicated. qRT-PCR of cest-2.2 in wt, gas-1(fc21), and nuo-6(qm200) mutants (one-way ANOVA with Dunnet's post-hoc correction, n = 9–12 technical replicates pooled from three independent experiments). qRT-PCR of cest-2.2 mRNA expression in nuo-6(qm200) animals grown on control and cest-2.2 RNAi (unpaired t-test, n = 3 biological replicates). Representative survival curves of nuo-6(qm200) mutants fed with control (C RNAi) and dsRNA against cest-2.2. qRT-PCR of cest-2.2 mRNA expression in wt animals grown on control and cest-2.2 RNAi (unpaired t-test, n = 3 biological replicates). Lifespan assay of wt animals grown on control and cest-2.2 RNAi. Representative survival curves of wt and cest-2.2 (lf) nematodes. qRT-PCR of cest-2.2 mRNA expression in wt, gas-1(fc21), gas-1(fc21); cest-2.2 O/E and cest-2.2 (lf) nematodes (one-way ANOVA with Dunnett's post-hoc correction, n = 3 biological replicates). SureQuant-based quantitative mass spectrometry analysis of CEST-2.2 protein levels. Light/heavy ratios (L/H) of the two unique CEST-2.2 peptides (LSTGTIEGR and HGEDFAYAFGTNR) used for quantitation are represented as violin plots (n = 3 for wt and gas-1; cest-2.2 O/E, n = 2 for gas-1; the n of analyzed samples per group refers to biological replicates of pooled nematodes from a single plate; the median is indicated with a dashed line). Statistical analysis was performed using the nonparametric t-test. Representative survival curves of wt and cest-2.2(lf) nematodes. Lifespan assay of wt, gas-1(fc21), gas-1(fc21); cest-2.2 O/E and cest-2.2(lf); gas-1(fc21); cest-2.2 O/E mutant nematodes. Representative lifespan assay of wt and gas-1(fc21); cest-2.2 O/E mutant animals grown on control (solid lines) and cest-2.2 (dashed lines) RNAi bacteria from hatching. Quantification of body area of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes, normalized to wt (one-way ANOVA with the Tukey's multiple comparison test, n = 18–30 biological replicates). Locomotor activity (arbitrary unit) of 10-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutants (one-way ANOVA with Dunnett's post-hoc correction, n = 8). Number of pharyngeal pumps over 60 s of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (one-way ANOVA with the Tukey's multiple comparison test, n = 10 biological replicates). Total number of eggs (left panel) and number of eggs laid each day (right panel) since hatching by wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (ordinary one-way ANOVA with the Tukey's multiple comparisons test, n = 8–12 biological replicates). Data information: In E, F, H, K, R, and S (left), mean ± SEM; in L, P, and Q, middle lines represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively; in G, I, J, M, N, and O, the median lifespan ± SEM is reported underneath the graphs and additional information (e.g., n numbers) are reported in Appendix Table S1; in S (right) points indicate mean ± SEM. Across experiments, P-value sum
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