A conserved role of CBP/p300 in mitochondrial stress response and longevity

Organisms respond to mitochondrial stress by activating multiple defense pathways including the mitochondrial unfolded protein response (UPR mt ). However, how different layers of UPR mt regulators are orchestrated to transcriptionally activate the stress responses remains largely unknown. Here we identified CBP-1, the worm ortholog of the 5 mammalian acetyltransferases CBP/p300, as an essential regulator for UPR mt activation, as well as for mitochondrial stress-induced immune response, reduction of amyloid-β aggregation and lifespan extension in Caenorhabditis elegans . Mechanistically, CBP-1 acts downstream of histone demethylases, JMJD-1.2/JMJD-3.1, and upstream of UPR mt transcription factors including ATFS-1, to systematically induce a broad spectrum of 10 UPR mt genes and execute multiple beneficial functions. In mouse and human populations, transcript levels of CBP/p300 positively correlate with UPR mt transcripts and longevity. Furthermore, CBP/p300 inhibition disrupts, while forced expression of p300 is sufficient to activate, the UPR mt in mammalian cells. These results highlight an evolutionarily conserved mechanism that determines mitochondrial stress response, and promotes 15 health and longevity through CBP/p300.


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Mitochondria not only contribute to the harvesting of energy, but also serve as key signaling hubs connecting numerous metabolic processes to essential cellular and organismal functions [1][2][3] . It is therefore not surprising that the dysfunction of mitochondria is tightly associated with ageing, as well as diverse human age-related diseases, including those affecting metabolic, cardiovascular and neuromuscular systems, as well as cancer 2,[4][5][6][7] . Moreover, mitochondria 5 function as platforms to regulate programmed cell death and innate immune responses 1,8,9 . Multiple mitochondrial stress response (MSR) pathways have evolved to adapt mitochondrial function to the ever-changing cellular milieu and to a variety of extra-cellular cues 10,11 .
However, aberrant activation of these MSR pathways may also be maladaptive and contribute to disease and ageing 2 , underscoring the importance of the tight control of these regulatory 10 circuits.
The mitochondrial unfolded protein response (UPR mt ), one of these MSR pathways, is triggered by mitochondrial-to-nuclear communication, leading to an adaptive transcriptional response that promotes repair and recovery of the cell or organism from transient mitochondrial dysfunction [10][11][12][13] . It recently emerged that the activation of the UPR mt provides resistance to 15 pathogen infections (e.g. Pseudomonas aeruginosa) in C. elegans, and animals that failed to activate UPR mt during P. aeruginosa infection died earlier, suggesting that the UPR mt is a bona fide component of the innate immune response 14, 15 . In mammalian cells, mitochondrial perturbations also lead to cellular stress responses closely associated with innate immunity 9,16 ; e.g., herpesvirus infections induce a mitochondrial DNA (mtDNA) stress response, which 20 enhances antiviral signaling and type I interferon responses and thereby confer viral resistance 17 .
The regulation of the UPR mt is complex and pleiotropic, and includes control at the level of transcription and chromatin organization. At the transcriptional level, the transcription factor ATFS-1 in C. elegans 18 , and its functional orthologues, ATF4, ATF5 and CHOP in mammals 19-25 21 , seem to be key regulators of the UPR mt . Two recent studies suggest that an OMA1-DELE1-HRI signaling pathway relays the mitochondrial stress from mitochondria to the cytosol in mammals 22,23 . On the epigenetic level, the MET-2/LIN-65 histone methyltransferase complex and two histone demethylases JMJD-1.2/PHF8 and JMJD-3.1/KDM6B, regulate the UPR mt and mitochondrial stress-induced longevity in both C. elegans and mammals 24,25 , whereas in 30 yeast the histone demethylase, Rph1p, is the key modulator 26 . However, how these different layers of UPR mt regulators are systematically coordinated to induce the expression of various UPR mt genes and execute different biological functions is still poorly understood. 4 Here, we demonstrate that CBP-1 acts as an essential link to translate the mitochondrial stress signal from the demethylases, JMJD-1.2/JMJD-3.1, to the UPR mt transcription factors (e.g.

ATFS-1), into the coordinated transcriptional induction of a wide panel of UPR mt genes in C.
elegans. Importantly, the beneficial effects induced by mitochondrial perturbations, such as resistance to pathogen infection, improved proteostasis against amyloid-β aggregation, and 5 lifespan extension are almost completely blocked by cbp-1 silencing. Moreover, systematic correlation analysis in mouse and human populations, as well as genetic and pharmacological loss-of-function studies in mammalian cells, strongly suggest that the function of CBP/p300 in the regulation of the UPR mt , health and lifespan are also conserved in mammals. Collectively, these results highlighted that CBP/p300 is an evolutionarily conserved node for mitochondrial 10 stress signaling that defends mitochondrial function, and promotes health and longevity.
To determine the footprints of CBP-1 on the regulation of the UPR mt , we performed RNA sequencing (RNA-seq) on total RNA isolated from hsp-6p::gfp worms fed with cco-1 or mrps-5 RNAi, in combination with cbp-1 or atfs-1 RNAi (Extended Data Fig. 2a Table 1). The majority of transcripts induced by mrps-5 RNAi were also induced by cco-1 RNAi, but not the other way around (Extended Data Fig. 2b), which might due to the superior knockdown efficiency of cco-1 than that of mrps-5 (Extended Data Fig. 2c). We thus focused on the genes affected by cco-1 RNAi. 1,241 transcripts were significantly up-regulated after cco-1 RNAi (log2FC > 0.5, adjusted P < 0.05; defined here as UPR mt genes), among which 506 15 (40.8%) were CBP-1-dependent and 404 (32.6%) required ATFS-1 (Fig. 1f, g). The number of ATFS-1-dependent transcripts was similar to that found in a previous study 18 . Up to 259 genes induced by cco-1 RNAi were dependent on both CBP-1 and ATFS-1 (Fig. 1g). Gene ontology (GO) analysis revealed that a large number of "mitochondrion", "transmembrane transport" and "metabolic process"-related genes including hsp-6, timm-23 and gpd-2, required both CBP- 20 1 and ATFS-1 for induction (Fig. 1h, j and Extended Data Fig. 2d). In addition, many innate immune genes, such as the C-type lectin clec-65, were also included in this gene set ( Fig. 1f, h, j), supporting a role of UPR mt in regulating the immune response 14,15 .
It has been known that acetylation of H3K18 and H3K27, which transforms the condensed chromatin is into a more relaxed structure, is generally linked to active transcription 29,30,32,44 .
To examine if CBP-1-mediated histone acetylation contributes to the transcriptional activation of UPR mt genes, we performed chromatin immunoprecipitation sequencing (ChIP-seq) with 30 antibodies against H3K18Ac and H3K27Ac in worms fed with control or cco-1 RNAi. Among the 506 UPR mt genes regulated by CBP-1 (Fig. 1g), 203 had enriched H3K18Ac or H3K27Ac 7 peaks in the genome (Fig. 2d) Table 3). In contrast, no differences in H3K18Ac 5 or H3K27Ac marks were observed for the UPR ER markers hsp-3 and hsp-4, or the UPR CYT /heat shock response marker hsp-16.2, upon cco-1 RNAi treatment (Extended Data Fig. 3c-e). By analyzing the distribution of the 265 increased H3K18Ac/H3K27Ac peaks on the 134 UPR mt genes, we found that 54.0% (143/265) of them were located in promoter regions, 40.0% (106/265) were in coding regions, and 6.0% (16/265) were downstream of the coding region 10 (Extended Data Fig. 3f and Supplementary Table 3). Indeed, for some genes (e.g. hsp-60), acetylation peaks are restricted to the promoter (Fig. 2f); whereas for other genes (e.g. hsp-6 and timm-23), acetylation marks exist in both promoter and coding regions (Fig. 2e, g). It is also noteworthy that both gene sets up-regulated for the acetylation marks in response to cco-1 RNAi (4,639 genes for H3K18Ac, and 2,283 genes for H3K27Ac) were highly enriched for 15 GO terms including "metabolic pathways", "mitochondrion" and "determination of adult lifespan" (Fig. 2h, i and Supplementary Table 3), supporting a critical role of these epigenetic adaptations in the control of mitochondrial metabolism and lifespan upon mitochondrial stress.
Using ChIP-qPCR, we detected that the increased enrichment of H3K18Ac and H3K27Ac at the loci of UPR mt genes (e.g. hsp-6, hsp-60 and timm-23) in response to cco-1 knockdown was 20 completely blocked by cbp-1 RNAi (Fig. 2j-l). These results indicate that increased CBP-1dependent histone acetylation upon mitochondrial stress is closely associated with the transcriptional activation of a large set of UPR mt genes.

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Two histone demethylases, JMJD-3.1 and JMJD-1.2, have been reported to remove the repressive H3K27me3 mark from the promoter/coding regions of UPR mt genes, poising them for transcription, and overexpressing (OE) of jmjd-3.1 in worms is sufficient to activate the UPR mt 25 . RNAi for cbp-1 abolished the activation of the UPR mt in two independently generated jmjd-3.1 OE strains (Fig. 3a). Moreover, increased levels of H3K18Ac and H3K27Ac, but not 30 H3K9Ac, were detected in jmjd-3.1 OE worms, which was attenuated by cbp-1 RNAi (Fig. 3b).

Beneficial effects of UPR mt requires CBP-1
We then explored the physiological functions of CBP-1 on MSR regulation. In line with the fact that mild mitochondrial stress protects against infection by pathogens, such as P. aeruginosa 14,15 , cco-1 or mrps-5 knockdown increased the survival rate of worms exposed to P. aeruginosa, an effect that was completely abolished by cbp-1 knockdown (Fig. 4a, b). To 20 further examine the vital role of CBP-1 in mitochondrial surveillance, we raised wild-type (N2) and the mitochondrial respiration mutants that have disruptions in one of the mitochondrial electron transport chain (ETC) components, isp-1(qm150) and clk-1(qm30) 46,47 , on control or cbp-1 RNAi. Compared to C. elegans fed with control RNAi, cbp-1 RNAi even at 10% led to severe synthetic growth defects of the isp-1(qm150) and clk-1(qm30) mutants, whereas the 25 development of wild-type worms was only slightly delayed (Fig. 4c). Similar effects were also observed in A-485 treated worms (Fig. 4c), indicating that mitochondrial mutants strongly rely on CBP-1 activity to maintain growth.
We then questioned whether cbp-1 is required for mitochondrial stress-induced lifespan extension in C. elegans 13,48 . RNAi for cbp-1 at 20%, which was enough to suppress the UPR mt 30 activation induced by cco-1 knockdown (Extended Data Fig. 1c), completely blocked the lifespan extension induced by cco-1 RNAi (Fig. 4d). Likewise, cbp-1 RNAi at 10% fully 9 abolished mrps-5 knockdown-induced lifespan extension (Fig. 4e), in line with its capacity to block mrps-5 RNAi-induced UPR mt activation (Extended Data Fig. 1d). Meanwhile, consistent with the finding in another study 49 , cbp-1 knockdown alone shortened the lifespan of C. elegans ( Fig. 4d, e), potentially due to the attenuated basal expression of diverse nuclear-encoded MSR transcripts (Fig. 1j). 5 We have previously shown that humans with Alzheimer's disease (AD), as well as mouse and C. elegans models of AD, are all typified by the induction of a cross-species conserved MSR transcript signature 50 . Strikingly, further activation of these MSR pathways reduced amyloidβ (Aβ) proteotoxicity in cells, worms and in transgenic mouse models of AD 50 . The GMC101 strain is a worm AD model that expresses the human Aβ 1-42 peptide in body wall muscle 10 cells 51 . Adults of GMC101 develop age-progressive paralysis and amyloid deposition after a temperature shift from 20 to 25 °C. In these worms, cbp-1 RNAi at 10% caused a severe developmental delay even in the absence of the disease-inducing temperature shift, phenocopying mitochondrial respiration mutants that rely on cbp-1 for adaption, whereas the control CL2122 strain was not affected (Extended Data Fig. 4). Similar to atfs-1 RNAi, cbp-1 15 RNAi exacerbated Aβ aggregation in the GMC101 strain (Fig. 4f). In addition, cbp-1 knockdown in GMC101 worms prominently repressed not only the classical UPR mt transcripts (e.g. hsp-6), but also many UPR mt genes involved in "proteolysis" that only depend on CBP-1, but not ATFS-1 (e.g. asp-10) (Figs. 1i, j and 4g). Interestingly, the transcripts of another branch of the MSR, i.e. autophagy/mitophagy (e.g. sqst-1, dct-1), were conversely increased during 20 cbp-1 RNAi, suggesting a specific role of CBP-1 in regulating the UPR mt branch of the MSR.
Finally, cbp-1 RNAi worsened the paralysis and completely blocked the beneficial effect of Dox, an antibiotic that inhibits mitochondrial translation and activates the MSR 13 , on the reduction of Aβ aggregates in GMC101 worms 50 (Fig. 4h, i). Together, these results indicate that CBP-1 is essential for mitochondrial stress-induced immune response, lifespan extension 25 and amyloid-β aggregation reduction in C. elegans.

CBP/p300 expression correlates with UPR mt transcripts and lifespan
Next, we examined if the role of CBP-1 in UPR mt activation and MSR-associated beneficial effects is conserved in mammals. CBP expression in spleen, pituitary, adrenal and eye 30 positively correlated with p300 expression in the BXD mouse genetic reference population (GRP) 43,52 (www.genenetwork.org, Fig. 5a), confirming a complementary function of the two 10 acetyltransferases 35-37 . Their expression levels also correlate with transcript levels of Kdm6b and Phf8, the murine homologs of jmjd-3.1 and jmjd-1.2 (Fig. 5a). Moreover, in these tissues, CBP/p300 expression overall positively correlated with transcripts of UPR mt -related genes 10-12 , including the mitochondrial proteases (Lonp1, Yme1l1 and Spg7), the DNA-binding proteins (Satb1 and Ubl5), the mitochondrial chaperones (Hspe1, Hspd1 and Hspa9), and asparagine 5 synthetase Asns (Fig. 5a). Similar correlation networks were also found in the hippocampus and hypothalamus of BXD mice (Extended Data Fig. 5a), and in the brain and prefrontal cortex of mice from a different GRP, the LXS cohort 53 (Extended Data Fig. 5b). In accordance with the indispensable role of CBP-1 in MSR-associated health and lifespan regulation in C. elegans Finally, in the human Genotype-Tissue Expression (GTEx) database 54 , mRNA levels of CBP and p300 positively correlated with KDM6B, PHF8 and UPR mt transcripts in many tissues including brain, hypothalamus, liver, heart, stomach, pancreas, kidney and small intestine, 15 forming a systematic network (Fig. 5d). These observations suggest that CBP/p300 likely play an evolutionarily conserved role in MSR regulation across species from worms to human.
These Dox-induced and CBP/p300-dependent transcripts were enriched for "aminoacyl-tRNA 30 synthetase", confirming a close link between mRNA translation and the UPR mt 21,55 ; for "serine biosynthesis", including Phgdh, Psat1, Psph and Shmt2 19,56 ; and for metabolic and 11 mitochondrial pathways (e.g. Eno1b, Timm10) ( Fig. 6c and Extended Data Fig. 6c). Similar gene sets were also recently reported to be induced by other MSR inducers, such as CCCP (carbonyl cyanide m-chlorophenyl hydrazone) and oligomycin in different mammalian cells 22,23 . It is also noteworthy that Dox-induced expression of both Atf4 and Atf5, two key transcriptional regulators of the UPR mt 19,20 , was heavily dependent on CBP/p300 (Extended 5 Data Fig. 6c), suggesting a commanding role of CBP/p300 in UPR mt activation. In addition, reconstitution of WT-p300, but not a p300 acetyltransferase activity-defective mutant, restored Dox-induced UPR mt activation in CBP/p300 -/-MEFs (Fig. 6d, e), confirming that the catalytic activity of CBP/p300 is indispensable for this stress response.
In line with increased CBP/p300-mediated histone acetylation during mitochondrial  Table 6). Notably, in 25 addition to the GO terms found in MEFs (e.g. "aminoacyl-tRNA synthetase" and "Mitochondrion"), the Dox-induced transcripts in HepG2 cells were also enriched for "Innate immunity" and "Response to exogenous dsRNA", containing 12 genes (two genes belonged to both terms) and 7 of them were dependent on CBP/p300 activity for induction ( Fig. 6i and Supplementary Table 6). Finally, forced expression of WT-p300, but not the KAT activity-30 defective mutant of p300, is sufficient to induce the expression of UPR mt and Dox-induced immune response genes (e.g. DDX21, SLC3A2) in HepG2 cells (Fig. 6j). Taken together, these results point to a conserved and central role of CBP/p300 in MSR regulation in mammals. 12

Discussion
Here, by employing multilayered genetic and pharmacological approaches applied to C. elegans, mouse and human populations and cell lines, we provided strong evidence that CBP-1 or the mammalian CBP/p300 act downstream of demethylases JMJD-3.1/JMJD-1.2 or 5 mammalian KDM6B/PHF8, switching the transcription-repressive histone methylation marks (e.g. H3K27Me3) to the transcription-active acetylation marks (e.g. H3K27Ac), and thereby relays the mitochondrial stress signal to the transcriptional induction of diverse UPR mt genes in C. elegans as well as in mammals (Fig. 7). Notably, many of the CBP-1-or CBP/p300dependent UPR mt effectors positively contribute to mitochondrial function recovery, improved 10 immune response, enhanced proteostasis against amyloid-β aggregation, and lifespan extension.
In support of these findings, changes in CBP/p300 function tightly associate with multiple ageing/mitochondrial-related diseases, including Alzheimer's and Huntington's diseases 57-59 , and forced expression or pharmacological activation of CBP/p300 is sufficient to ameliorate neurodegenerative phenotypes in both mice and Drosophila AD models 60-62 . 15 How CBP-1 or CBP/p300 as well as the histone demethylases, sense mitochondrial stress remains an important direction for future work. One possibility is that CBP-1 itself is a downstream target that is activated in response to mitochondrial stress, as evidenced by increased cbp-1 expression after cco-1 and mrps-5 silencing (Extended Data Fig. 2c), and after jmjd-3.1 OE (Fig. 3e). Changes in mitochondrial metabolism may also modulate the levels of 20 acetyl-CoA, which acts as a substrate for the acetyltransferase activity of KATs including CBP/p300 30,32,63 . Of note, CBP/p300 may also affect mitochondrial function and stress resistance by targeting proteins besides histones, through mechanisms dependent or independent of their acetyltransferase activity. For instance, it has been reported that the PPARγ coactivator-1 (PGC-1α) can be acetylated by p300 and deacetylated by Sirt1, serving 25 as an important switch controlling mitochondrial biogenesis and function 64,65 . In another study, p300 was identified as a binding partner for ATF4, and could enhance ATF4-mediated transcriptional activation through a mechanism independent of its acetyltransferase activity 66 .
In addition to the indispensable role of CBP-1 or CBP/p300 in MSR, we have noticed that the basal expression of some UPR mt transcripts also decreased after cbp-1 silencing, CBP/p300 30 KO or CBP/p300 activity inhibition (Figs. 1j, 6a, h; Supplementary Tables 1 and 5), suggesting that CBP/p300 functions in maintaining "basal UPR mt activity" as well. Nevertheless, the 13 distinction between "basal" and "stress" conditions is somehow artificial, especially considering that organisms and cells are constantly exposed to multiple cues, and different wild C. elegans strains differ with respect to the level of UPR mt activation at "basal" condition 67 .
Moreover, it is likely that some UPR mt genes controlled by CBP-1 or CBP/p300 may also contribute to basal mitochondrial function. For example, the chaperone hsp-60 or its mouse 5 ortholog Hspd1, which demonstrated decreased "basal" H3K18Ac/H3K27Ac enrichment, ATFS-1 binding and mRNA expression upon cbp-1 RNAi or CBP/p300 suppression (Figs. 2k, 3h, 6a, g, h), have been reported to be essential for mitochondrial homeostasis even at basal state 27,68 .
Altogether, by applying genetic and pharmacological LOF approaches, combined with 10 bioinformatic and mechanistic studies, we identified the acetyltransferase CBP-1, as an essential regulator for the activation of the MSR and in particular the UPR mt . The beneficial effects on pathogen infection resistance, protein aggregation reduction and lifespan extension caused by mitochondrial perturbations are almost completely dependent on CBP-1 in C.
elegans. Furthermore, systematic correlation analysis in mouse and human populations, as well 15 as LOF studies in mammalian cells, indicate that functions of CBP/p300 in UPR mt regulation and longevity are also conserved in mammals. Our results thus reveal an evolutionarily conserved mechanism that coordinates the multiple layers of UPR mt regulators to systematically activate the stress responses, defend mitochondrial function, and promote health and longevity. Further studies will have to define whether genetically or pharmacologically 20 targeting these CBP/p300-driven MSR pathways can have therapeutic applications against mitochondrial-related diseases, pathogen infections as well as ageing. were then down-regulated by more than 25% of the log2FC after cbp-1 or atfs-1 RNAi cotreatment, compared to the log2FC of cco-1 RNAi condition, were considered as CBP-1-or ATFS-1-dependent. Genes whose expressions were significantly down-regulated with log2FC < -0.5 (adjusted P < 0.05) were defined as the down-regulated genes. For MEFs or human 20 HepG2 cell samples, similar analysis procedure was used, except that the "Mus_musculus.GRCm38.95" genome or the "Homo_sapiens.GRCh38.95" was used for mapping. Genes whose expressions were up-regulated with log2FC > 0.5 (adjusted P < 0.05) in Dox treatment condition; and were then down-regulated by more than 25% of the log2FC Western blotting. For worm samples, proteins were extracted as described previously 13 .

C. elegans strains
Western blotting was performed with antibodies against GFP (Cat. 2956, CST), Actin (Cat.  Pseudomonas aeruginosa infection assay. The P. aeruginosa PA14 slow killing assay was 5 performed as described 84 . Briefly, P. aeruginosa overnight cultures were seeded onto slowkilling NGM agar plates with 0.35% peptone. Plates were allowed to dry for 20 min at room temperature, and then incubated at 37 C for 24 h and allowed to equilibrate at 25 C for another

Competing interests
Authors declare no competing interests.
Correspondence and requests for materials should be addressed to J.A.