Keywords:ATF7, cellular senescence, H3K9me2, longevity, NF-κB

Study subjects, RNA sequencing (RNA-seq), network construction, and centrality analysis

As described in our prior study [10], we sampled 135 females from longevous families in Hainan Province, southern China, and constructed 76 and 59 sequencing libraries using poly(A) capture and ribosomal RNA depletion methods, respectively. The RNA-seq reads were filtered to obtain clean reads [21], aligned to the human (hg19) andC. elegans (WBcel235) reference genomes [22], and quantified to determine gene abundance [23]. Differentially expressed gene (DEG) analysis was performed using the ‘limma’ package [24], with the effects of library type and cell composition considered (Gene expression ~ 0 + Group + Lymph + Neu + Mid). The ‘removeBatchEffect’ function in ‘limma’ was used to remove the effect of library type on longevous family transcriptome data. The DESeq2 function in the R platform was used to detect DEGs between different experimental groups. A parameter false discovery rate (FDR) cutoff of less than 0.05 was considered statistically significant. Pearson correlation coefficient (r) was used to determine correlations between differently expressed transcription factors (TFs) and immune-related genes, with a Benjamini-Hochberg adjustedP-value cutoff of less than 0.05. The immune-related gene list was downloaded from the ImmPort database (www.immport.org/home) and TFs were obtained from the footprintDB and TRRUST databases (http://floresta.eead.csic.es/footprintdb/index.php;www.grnpedia.org/trrust/). Co-expression network plots were constructed using Cytoscape (v3.9.0) [25] and centrality analysis was conducted using the cytoHubba program in Cytoscape. Functional enrichment analysis was performed using “Metascape” [26] and “DAVID” [27,28]. Raw RNA-seq data of replicative senescent human embryonic lung fibroblasts (WI-38), human neonatal foreskin fibroblasts (HFF and BJ), and oncogene-induced senescent human embryonic lung fibroblasts (IMR-90) were collected from the ‘‘GEO Repository’’ (www.ncbi.nlm.nih.gov/geo/) (accession numbersGSE63577,GSE64553,GSE61130).

Cell culture

Human dermal fibroblasts (HDFs) were obtained from circumcised foreskins of healthy human donors aged 5-20 years in Xiangya Hospital, Central South University, as described previously [29,30]. Human embryonic lung fibroblasts IMR-90 and HEL were purchased from the American Type Culture Collection (ATCC) and Kunming Cell Bank (Chinese Academy of Sciences), respectively. The HDF, HEL, and IMR-90 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, C11995500BT, Gibco, USA) or Minimum Essential Medium (MEM, C11095500BT, Gibco, USA) supplemented with 10% fetal bovine serum (35-076-CV, Gibco, USA) and 1% penicillin/streptomycin (15140-122, Gibco, USA) in a 37 °C/5% CO₂ humidified incubator. Human embryonic kidney HEK-293T cells were purchased from ATCC and cultured in DMEM (C11995500BT, Gibco, USA) supplemented with 10% fetal bovine serum (04-001-1ACS, BI, Israel) and 1% penicillin/streptomycin (15140-122, Gibco, USA) in a 37 °C/5% CO₂ humidified incubator.

Senescence-associated beta-galactosidase (SA-β-gal) staining

SA-β-gal staining was performed using a Senescence Cells Histochemical Staining Kit (CS0030-1kt, Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, cells were washed with phosphate-buffered saline (PBS) and fixed at room temperature for 7 min, then incubated in staining working solution overnight at 37 °C. Cells were visualized using a Nikon eclipse Ti inverted microscope and a minimum of 200 cells were counted.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIzol reagent (15596018, Invitrogen, USA) and reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific, USA) according to the manufacturer’s protocols. qRT-PCR was performed using 2 × Tsingke® Master qPCR Mix (TSE201, Tsingke, China), with actin as the endogenous normalization control. Gene expression levels were calculated using the 2^-ΔΔCt method [31]. All primers used are presented inSupplementary Table 1.

Western blotting

Cells were lysed using RIPA lysis buffer (P0013B, Beyotime, China) supplemented with 1% phenylmethylsulfonyl fluoride (ST506, Beyotime, China) on ice. Protein extract (30 μg) was separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (162-0177, Bio-Rad, USA). The membranes were incubated with appropriate primary antibodies at 4 °C overnight, then with horseradish peroxidase (HRP)-labeled goat anti-rabbit or anti-mouse immunoglobulin G (IgG) (H + L) secondary antibodies at room temperature for 1 h. Protein bands were visualized using an ECL Detection Kit (UEL-S6009L, US Everbright, China). Quantification of blots was performed using ImageJ software. Detailed information on primary antibodies used in western blotting is provided inSupplementary Table 2.

EdU incorporation assay

Cell proliferation was analyzed using a Cell-Light EdU Apollo567In Vitro Kit (C10310-1, Ribobio, China). Briefly, cells (8 × 10³) were seeded into each well of a 96-well plate and incubated for 24 h with 5 μM EdU at 37 °C. The cells were then added with 1 × reaction buffer for 30 min. Images were captured using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek).

Cellular reactive oxygen species (ROS) detection

Cellular ROS detection was performed using CellROX® Oxidative Stress Reagent (C10422, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Briefly, cells (8 × 10³) were seeded into each well of a 96-well plate, followed by the addition of CellROX® Reagent at a final concentration of 10 μM and incubation for 30 min at 37 °C. Images were captured using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Quantification was performed using ImageJ software.

Cytokine array

Cells were seeded into 6-cm dishes and washed and incubated in serum-free DMEM in a 37 °C/5% CO₂ humidified incubator. After 24 h, the conditioned medium (CM) was collected, filtered (0.2 μm), and frozen at -80 °C. The CM was analyzed using an antibody array (QAH-CYT-1, RayBiotech, USA) according to the manufacturer’s instructions. The array was used to detect 20 cytokines and chemokines, including IL1A, IL1B, IL2, IL4, IL5, IL6, IL8 (CXCL8), IL10, IL12, IL13, IFNγ, TNFα, GM-CSF, GROα/β/γ, MCP1 (CCL2), MIP1a (CCL3), MIP1b (CCL4), MMP9, VEGFA, and RANTES (CCL5). The quantity of secreted cytokines was measured by adding 90 μL of concentrated conditioned medium to the array, followed by overnight incubation at 4 °C. After washing with wash buffer, biotinylated primary antibodies were added and incubated at room temperature for 2 h. After washing again with wash buffer, Cy3 equivalent dye-conjugated streptavidin was added for 1 h. The chip was scanned using an InnoScan 300 Microarray Scanner (Innopsys, France). Cytokines were quantified according to the standard curve calibrated from the same array.

Chromatin immunoprecipitation (ChIP) analysis

ChIP analysis was performed using a SimpleChIP® Enzymatic Chromatin IP Kit (9003, Cell Signaling Technology, USA) according to the manufacturer’s instructions. Chromatin was crosslinked with 1% formaldehyde in cell medium at room temperature for 10 min. Glycine was then added to the medium to terminate the reaction. After washing in ice-cold PBS, cells were lysed and sonicated into 150-900-bp fragments. Immunoprecipitation was performed overnight at 4 °C with anti-histone H3K9me2 and anti-histone H3. Normal rabbit IgG was used as a negative control. The immunocomplexes were washed using ChIP-Grade Protein G Magnetic Beads (9006, Cell Signaling Technology, USA) and incubated at 65 °C overnight following the addition of 5 M NaCl and Proteinase K to reverse the crosslinks. Free precipitated DNA was further purified. The DNA samples were used for qRT-PCR. All primers are presented inSupplementary Table 3.

Co-immunoprecipitation

The HDF cells were lysed in IP-lysis buffer (50 mM Tris-HCl, 150 nM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.2% NP40, and 10% glycerol) for 30 min on ice. After centrifugation at 12 500 rpm and 4 °C for 15 min, the supernatants were incubated in anti-ATF7 or control IgG. The immunocomplexes were then collected using Protein-A-agarose beads (11134515001, Roche, Switzerland) and subjected to western blotting with anti-G9a antibodies.

Cleavage Under Targets and Tagmentation (CUT&Tag) assay

CUT&Tag assay was performed using a Hyperactive^® Universal CUT&Tag Assay Kit for Illumina (TD903, Vazyme, China) according to the manufacturer’s instructions. Briefly, 1 × 10⁵ cells were collected, washed in wash buffer, and added to 10 μL of activated ConA Beads. Bead-bound cells were incubated overnight at 4 °C with anti-histone H3K9me2. Normal rabbit IgG was used as a negative control. The cells were washed and incubated with rabbit secondary antibodies at room temperature for 1 h. After washing, the cells were incubated with pA/G-Tnp at room temperature for 1 h, then with TruePrep Tagment Buffer L (TTBL) and Dig-300 buffer for fragmentation at 37 °C for 1 h. The released chromatin fragments were extracted, and libraries were generated using TruePrep® Index Kit V2 for Illumina (TD202, Vazyme, China). Reads were aligned to the human reference genome hg19 using Bowtie2 (v.2.4.1) [32]. The ‘picard.jar’ program in GATK (v4.1.8.0) was used to remove duplicated reads [33]. For each replicate, peaks were first called using MACS2 (v.2.1.2) [34] with the parameters: -q 0.05 -B. Differential binding analysis of peaks was performed using the Bioconductor package ‘DiffBind’. The ‘annotatedPeak’ function in the R package ‘CHIPseeker’ [35] was used to annotate peaks. Peaks were visualized using Integrative Genomics Viewer (IGV) [36].

Lifespan assay

AllC. elegans lifespan experiments were conducted at 20 °C in accordance with standard protocols using HT115/RNA interference (RNAi) (with addition of Amp). Briefly, worms were branched to obtain synchronized eggs, which were cultured to hatch and used for lifespan assays. At L4 molting, animals were transferred to plates containing 20 M 5-fluoro-2’-deoxyuridine (FUDR), which kills their progeny at the embryo stage but does not significantly affect lifespan. The plates were refreshed daily. Approximately 50 worms were used for each treatment. The first day of adulthood was recorded as day 1 and dead worms were counted from day 5. Worms that did not respond to nose touch were picked out of the bacterial lawn, and worms that did not move were scored as dead.

Body bending assay

Worms were pretreated as in the lifespan assays. On the day of examination, 100 μL of M9 buffer was pipetted onto the surface of an empty 35-mm NGM plate. A single worm was added to the buffer and allowed 30 s to recover from the transfer. The number of thrashing movements over a 30-s period was counted (four times per worm, 15-20 worms per treatment). One worm from one treatment was counted, then one worm from another treatment was counted, and so on, with the cycle repeated to avoid environmental or operational interference.

Lipofuscin accumulation

Autofluorescence of intestinal lipofuscin was measured as an index of senescence in day 10 adults. Worms were randomly selected from each bacterial lawn and washed three times with M9 buffer. The worms were then placed on 5% agar pads coated with 10 mM sodium azide in M9 buffer to initiate paralysis. Lipofuscin autofluorescence images were taken using blue excitation light (405-488 nm) with 4’,6-diamidino-2-phenylindole (DAPI) and laser confocal scanning microscopy. Fluorescence was quantified using ImageJ software to determine lipofuscin levels. Three independent experiments were performed with more than 30 worms for each bacterial species each day.

Pharyngeal pumping

Wild-type andATF7-overexpressing worms were synchronized by timed oviposition onto an empty vector (control) andATF7 double-stranded RNA (dsRNA)-expressing HT115. Day 1 adult worms were transferred to control and RNAi plates with FUDR. Pumping rates were determined on day 10 of adulthood. Terminal pharyngeal bulb pumps were counted at 30-s intervals for each worm on the bacterial lawn. The pumping rate per minute was averaged for 10 worms per treatment. Pumping rates were assessed in three independent replicate experiments.

RNAi in C. elegans

RNAiATF7 was generated using primers GAATTCC TGCAGCCCCGCGTCCAATTGATGGGC and ACGC GTGGATCCCCCCACAGATAACAATTTGGAAATGTCC and linked to L4440 by infusion (SmaI). TheE. coli strain HT115 (DE3) expressingATF7 dsRNA was grown overnight at 37 °C in Luria-Bertani (LB) broth containing 100 μg/mL ampicillin, then spread on nematode growth medium (NGM) plates containing 100 μg/mL ampicillin and 1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). The RNAi-expressing bacteria were then grown at 25 °C overnight. Synchronized L4 larvae were placed on the plates at 20 °C. RNAi was induced from L4 unless otherwise noted. L4 adult animals were used for further experiments.

ATF7 overexpression in C. elegans

ATF7 cDNA expression was driven by anATF7 promoter andATF7 cDNA insertion was verified by sequencing. The primers used for cloningATF7 were:ATF7 promoter-F: GTCGACTCTAGAGGATCCCCTT TTGGAGCCG CCAAAGATCG;ATF7 promoter-R: CGACCGACATA ATTGTGTTGGACGATTGCTGA;ATF7 cDNA -F: CAACACAATTATGTCGGTCGTAACAACGA;ATF7 cDNA-R: TGGGTCCTTTGGCCAATCCCTTGGAGT TT GGGTAATTTCAGTTGC. The cDNA and promoter ofATF7 were then linked to pPD95_75 by infusion (SmaI). The plasmid was injected at a concentration of 50 ng/μL into N2 worms using a Pmyo-3::mCherry::unc-54 (pCFJ104) expressing plasmid as a co-injection marker.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism v9 (GraphPad Software). Data are presented as means ± standard error of the mean (SEM). Normal distribution of data was determined using the Shapiro-Wilk test. Comparisons were performed using two-tailed Student’st-test or Mann-Whitney U test. Survival curves were compared, andP-values were calculated using log-rank (Mantel-Cox) analysis.P < 0.05 was defined as statistically significant.

Centenarians display lower inflammation with possible ATF7 involvement

Peripheral blood mononuclear cells (PBMCs) from centenarians show reducedIL6 mRNA levels [13]. To determine overall gene expression changes in proinflammatory factors in LLIs, we analyzed RNA-seq data in a cohort of longevous families [10]. In total, 2 870 significant DEGs were identified between LLIs and younger controls (YCs) (P < 0.05) (Fig. 1A). Among the DEGs, a portion of major proinflammatory factors, includingIL6,CXCL8 (IL8),TNF,IGFBP4, andIGFBP6 [37-39], were down-regulated in LLIs compared to YCs (Fig. 1B). Consistent with the RNA-seq results, cytokine array showed that LLIs had significantly lower levels of MMP9 and CCL4 (MIP1b) and lower levels of proinflammatory factors, including IL6, IL1A, and TNFα, compared to the sex-matched YCs (Fig. 1C). As proinflammatory factors are primarily regulated at the transcriptional level, we focused on differentially expressed TF genes that may have both anti-inflammatory and potentially anti-aging functions. We performed network centrality analysis to prioritize candidate TF genes [40,41], and identified five TF genes related to inflammatory regulation (Fig. 1D). Among them,ELF1,IRF3,ZNF410, andARNT may promote the development of inflammation or senescence [42-45].ATF7 not only plays a repressive role in innate immunity [18], but also responds to intrinsic and extrinsic stresses [17], and thus was chosen as the candidate gene (Fig. 1B, D). We confirmed the expression level ofATF7 in LLIs and YCs using qRT-PCR (Supplementary Fig. 1A) and explored its potential roles and mechanisms in the following experiments.

Manipulation of ATF7 alters cellular senescence progression

AsATF7 was upregulated in LLIs compared to YCs, we investigated whether it affects cellular senescence, one of the major hallmarks of aging. Unlike the case in LLIs, the protein level of ATF7 was progressively down-regulated during serial passage in HDF cells, accompanied by the up-regulation of p16 (CDKN2A), a marker of cellular senescence (Fig. 2A). Similarly,ATF7 was down-regulated in senescent WI-38 fibroblasts, HFF and BJ fibroblasts, and IMR-90 fibroblasts compared to non-senescent cells (Supplementary Fig. 1B) (online published data) [46-48]. The downregulation of ATF7 was partly due to increased phosphorylation by p38MAPK (Supplementary Fig. 2A), which can cause instability and degradation [49,50]. Notably, we found that application of SB203580, an inhibitor of p38MAPK, decreased the degradation of the ATF7 protein (Supplementary Fig. 2B).

Next, we examined the effect ofATF7 on HDF senescence. Overexpression ofATF7 significantly decreased senescence markers, including p16 (CDKN2A) protein levels and SA-β-gal-staining positive cells, and increased H3K9me2 levels compared to control HDFs (Fig. 2B-C). In contrast, knockdown ofATF7 with shRNA reversed the above senescence markers (Fig. 2D-F) and inhibited DNA synthesis indicated by EdU staining (Fig. 2G). These results were validated in the IMR-90 fibroblasts (Supplementary Fig. 3A-B), suggesting a suppressive role ofATF7 in cellular senescence. As DNA damage and ROS are primarily responsible for cellular senescence, we tested ROS levels inATF7-overexpressed or down-regulated cells. As shown inFig. 2H, compared to untreated control HDFs, overexpression and down-regulation ofATF7 significantly decreased and increased ROS levels in HDFs, respectively.

ATF7 represses secretion of SASP

SnCs affect surrounding cells/tissues by secreting SASP, which consists of inflammatory factors, extracellular modifiers, and growth factors [51]. To test whetherATF7 regulates SASP and to identify the pathways involved in senescence followingATF7 manipulation, we performed transcriptome analysis ofATF7-overexpressing and control HDFs. In total, 827 DEGs were identified, including 534 down-regulated and 293 up-regulated DEGs in theATF7-overexpressing HDFs relative to the control cells. KEGG pathway enrichment analysis indicated that the down-regulated DEGs were enriched in inflammatory pathways, including cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, MAPK signaling pathway, JAK-STAT signaling pathway, and NF-κB signaling pathway (Fig. 3A). Up-regulated DEGs were enriched in the TGF-β signaling pathway (Fig. 3B), among whichID1,ID2,ID3,ID4,SMAD6,SMAD7, andSMAD9 are negative regulators of inflammation [52-55]. These findings suggest thatATF7 may play an inhibitory role in modulating HDF inflammation. SnC-secreted SASP is responsible for multiple age-related pathologies [56]. Based on transcriptome data, 13 down-regulated SASP factors, together with upstream inflammation-regulatory genes (e.g.,STAT3,NFKB2, andRELB), were confirmed using qRT-PCR (Fig. 3C, D). In addition,ATF7 overexpression significantly reduced the mRNA expression levels ofIL8 andGM-CSF (Fig. 3D). In contrast, compared to the control cells, knockdown ofATF7 up-regulated several SASP factors and down-regulated genes in the TGF-β signaling pathway, i.e.,ID1,ID2,SMAD6, andSMAD7 (Supplementary Fig. S4A-C). To verify the inhibitory role ofATF7 on SASP, we analyzed SASP release in culture medium using antibody arrays. As shown inFig. 3E, a panel of inflammatory factors, including IL6, IL8, TNFα, GM-CSF, IL2, IL5, and IFNγ, were significantly decreased inATF7-overexpressing HDFs compared to the controls.

Notably, the release of TNFα, GM-CSF, and IL2 was markedly inhibited (>50% reduction) and IL5 was completely inhibited in the HDFs withATF7 overexpression relative to the control cells (Fig. 3E). The effect ofATF7 on suppressing SASP in SnCs mimicked that in LLIs, in whichATF7 up-regulation was accompanied by lower levels of SASP factors compared to YCs (Fig. 1B-C). These findings suggest thatATF7 can repress cellular senescence and SASP secretion in SnCs and may contribute, at least partially, to lower inflammation in LLIs.

ATF7 restrains SASP by inhibiting NF-κB signaling and modulating H3K9me2

To determine howATF7 inhibits SASP, we focused on the inflammatory pathways enriched in KEGG analysis. Based on immunoblotting, we found that the phosphorylation level of P65, a canonical indicator of NF-κB signaling activity, was significantly decreased inATF7-overexpressing HDFs compared to control HDFs (Supplementary Fig. S5A). NF-κB is a recognized regulator of SASP production [6,57]. Therefore, we investigated upstream signals in this pathway and found that the phosphorylation levels of P65 and IKKα/β were diminished inATF7-overexpressed cells compared to the control cells, whereas the total protein levels of P65 and IKKα/β remained unchanged (Fig. 4A, B).

In addition to its role in regulating NF-κB signaling,ATF7 is also reported to regulate inflammatory gene expression epigenetically [18,50]. As shown inFig. 2B,ATF7 overexpression up-regulated H3K9me2, which negatively regulates SASP factorsIL6 andIL8 [58]. To investigate whetherATF7 regulates SASP through H3K9me2, we performed chromatin immunoprecipitation plus qRT-PCR analysis using H3K9me2 antibodies. As shown inFig. 4C andSupplementary Fig. 5B, co-immunoprecipitation revealed that ATF7 could directly bind G9a, which regulates H3K9 dimethylation. These results suggest that ATF7 may recruit G9a to regulate H3K9me2 and silence its target genes, as reported previously [18,50]. The ChIP-qRT-PCR analysis also showed thatATF7 overexpression up-regulated H3K9me2 levels around the promotor regions of several SASP factors, includingIL8,IL6,CXCL1,CXCL2,CXCL3,CCL8, andTNF, as well as the upstream regulatorSTAT1, but not p16 (Fig. 4D). These data were validated using the CUT&Tag assay, which showed a high signal-to-noise ratio (Supplementary Fig. 6) [59]. Thus, these results suggest thatATF7 suppresses SASP via both NF-κB and H3K9me2.

Manipulation of ATF7 affects lifespan and survival in C. elegans

As overexpression ofATF7 delayed cellular senescence, suppressed SASP productionin vitro, and was associated with longevity in LLIs, we wondered whether it may function to promote lifespan. We tested this usingC. elegans, a model species widely used to determine molecular mechanisms of lifespan [60]. As shown inFig. 5, transgenicATF7 overexpression extended theC. elegans lifespan by 10.3% compared to the control, whereas RNAi knockdown ofATF7 decreased lifespan by 17.2% (Fig. 5B). Consistently, compared to the control, overexpression ofATF7 reduced intestinal lipofuscin and promoted body locomotion and pharyngeal pumping (Fig. 5C, D), applied as aging markers inC. elegans [61]. In contrast, knockdown ofATF7 accelerated aging inC. elegans compared to the control group (Fig. 5C, D).

To explore howATF7 regulates lifespan and aging inC. elegans, we performed RNA-seq analysis ofATF7-overexpressed or silencedC. elegans and controlC. elegans. Gene Ontology (GO) enrichment analysis showed that the up-regulated genes inATF7-overexpressedC. elegans relative to controlC. elegans were mainly enriched in innate immune response, whereas the down-regulated genes inATF7-silencedC. elegans were mainly enriched in innate immune response and defense responses against gram-positive bacteria (Fig. 5E, F). The downregulation and up-regulation of innate immune-related genes inATF7-silenced and overexpressing worms support the regulatory role ofATF7 in innate immunity [18,62,63]. Moreover, most up-regulated DEG orthologs in humans play roles in modulating immune function, depending on different physiological/pathophysiological contexts (Supplementary Table 4). Importantly, asATF7 is closely associated with innate immunity, we suspect it may influence the survival of pathogen-challengedC. elegans. Indeed, in the presence ofPseudomonas aeruginosa (PA14), transgenicATF7 overexpression enhancedC. elegans survival, whereas RNAi knockdown ofATF7 reduced survival (Fig. 5G, H). Overall, these results suggest thatATF7 can extend the lifespan and promote healthy aging inC. elegans, in part by regulating innate immunity genes.

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