A transient, homeostatic transcriptional response can result in transcriptional memory, programming subsequent transcriptional outputs. Transcriptional memory has great but unappreciated potential to alter animal aging as animals encounter a multitude of diverse stimuli throughout their lifespan. Here we show that activating an evolutionarily conserved, longevity-promoting transcription factor, dFOXO, solely in early adulthood of female fruit flies is sufficient to improve their subsequent health and survival in midlife and late life. This youth-restricted dFOXO activation causes persistent changes to chromatin landscape in the fat body and requires chromatin remodelers such as the SWI/SNF and ISWI complexes to program health and longevity. Chromatin remodeling is accompanied by a long-lasting transcriptional program that is distinct from that observed during acute dFOXO activation and includes induction of Xbp1. We show that this later-life induction of Xbp1 is sufficient to curtail later-life mortality. Our study demonstrates that transcriptional memory can profoundly alter how animals age.
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Raw RNA-seq and ATAC-seq data are available from the GEO under accession number GSE183542. Metabolomics data are available from the MetaboLights repository (study identifier MTBLS3251). All other data are available as Supplementary Data or can be made available by the corresponding author on reasonable request.
Publicly available data obtained and used in the study were: (1) Drosophila female fat body gene expression in different ages (GSE130158) and gene expression of Xbp1 mutant flies at larval stage 2 (GSE99676) from the GEO (https://www.ncbi.nlm.nih.gov/geo/); (2) raw gene count data from mouse gene expression datasets of different ages and tissues from the Tabula Muris Senis project (https://twc-stanford.shinyapps.io/maca/); (3) 5,176 processed peak files of the publicly available Drosophila ChIP–seq datasets following the uniform processing protocol as well as the annotations of each peak file from ChIP-Atlas (https://chip-atlas.org/); (4) fly genome release and annotation from FlyBase (https://flybase.org/).
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We thank H. D. Ryoo, L. Partridge and T. Niccoli for providing fly stocks; C. Regnault from Glasgow Polyomics for assistance with Metabolomics analysis; The University of Cambridge Department of Genetics Fly Facility for generating fly stocks; Y. Zhao for help with Tn5 synthesis; A. Vieira and J. Uriach for technical assistance; and the IHA members for support, comments and discussion throughout this project. Fly stocks were obtained from the Bloomington Drosophila Stock Center. This work was supported by a Biotechnology and Biological Sciences Research Council grant (BB/R014507/1) to T.D.S. and N.A. and a Medical Research Council grant (MR/S033939/1) to A.J.D. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. N.A. dedicates this work to the memory of his mother, E. Hodžić.
The authors declare no competing interests.
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Extended Data Fig. 1 Experimental setup – expression.
a Expression pattern of UAS-n8-GFP, driven with the S106 driver in 7 day-old female guts and fat bodies. All images were taken at exactly the same laser conditions under the confocal microscope, to allow the comparison of GFP levels between the two tissues. Scale bars are 50 µm. b representative example of a S106 > n8-GFP fat body, induced or not with RU486, under different imaging conditions to (a), allowing a better view of GFP induction in fat body cells. Scale bars are 50 µm. c Quantification of dfoxo mRNA levels in S106 alone and S106 UAS-dfoxo both uninduced. Boxplots – quantiles; whiskers – extremes; overlay – individual data points. N = 4, 3 (left to right), p = 1, unpaired two-sided t-test. d Uncropped images of the western-blot membranes shown in Fig. 1c.
Extended Data Fig. 2 dfoxo-switch – additional lifespans, and climbing ability measurements.
a Driver-alone (S106) RU486-switch control lifespan. Control n = 150 dead/1 censored fly, switch n = 148 dead/0 censored flies, p = 0.69, log-rank test. b UAS-dfoxo alone RU486-switch control lifespan. Control n = 135 dead/2 censored flies, switch n = 136 dead/12 censored flies, p = 0.86, log-rank test. c Male driver-alone (S106) RU486-switch control lifespan. Control n = 126 dead/12 censored flies, RU486-switch n = 143 dead/6 censored flies, p = 0.49, log-rank test. d Male dfoxo-switch lifespan. Control n = 154 dead/6 censored flies, dfoxo-switch n = 142 dead/8 censored flies, p = 0.72, log-rank test. e TiGS > dfoxo-switch lifespan. Control n = 128 dead/3 censored flies, dfoxo-switch n = 141 dead/4 censored flies, p = 0.85, log-rank test. f S106 > aopAct-switch lifespan. Control n = 145 dead/7 censored flies, aopAct-switch n = 147 dead/2 censored flies, p = 0.00274, log-rank test. g Experimental trials of the negative geotaxis assays of dfoxo-switch and control that were combined for the analysis presented in Fig. 1e. N = individual flies, boxplots – quantiles; whiskers – extremes; overlay – individual data points. Experiment 1: effect of dfoxo-switch p = 0.008, age p < 10−4, age-by- dfoxo-switch interaction p = 0.0008, mixed-effects linear model (LM). Experiment 2: effect of dfoxo-switch p > 0.05, age p < 10−4, age-by-dfoxo-switch interaction p = 0.033, mixed-effects LM. Experiment 3: effect of dfoxo-switch p > 0.05, age p < 10−4, age-by-dfoxo-switch interaction p = 0.07, mixed-effects LM. h Experimental trials of S106 > dfoxo-switch lifespans used for the analyses presented in Fig. 1f. Experimental trial 1: control n = 61 dead/0 censored flies, dfoxo-switch n = 76 dead/0 censored flies, p = 0.009, log-rank test. Experimental trial 2: shown in Fig. 1d. Experimental trial 3: control n = 145 dead/1 censored fly, dfoxo-switch n = 145 dead/1 censored fly, p = 1.72189 ×10-5, log-rank test. Experimental trial 4: control n = 125 dead/0 censored flies, dfoxo-switch n = 145 dead/2 censored flies, p = 6.05 ×10-9, log-rank test.
Extended Data Fig. 3 ATAC-Seq – additional information.
a Schematic distribution of the ATAC peaks on the four main Drosophila chromosomes. Additional peaks were detected on contigs and are not shown. b Violin plots showing the size of all ATAC peaks detected in the gut and fat body, and in the significantly differentially accessible peaks in the fat body. c Distribution of the distance of ATAC peaks from a transcriptional start site (TSS). Proportion of peaks in each distance category are presented from 5’ to 3’ relative to the TSS. d ATAC-qPCR of the levels of sequences near the MED1 locus where an ATAC-Seq peak opened by dfoxo-switch was detected, near Prosap where a peak unaltered by dfoxo-switch was detected, and Sox21b region which contained no peak in ATAC-Seq. Boxplots – quantiles; whiskers – extremes; overlay – individual data points. N = 3 biologically independent samples; effect of dfoxo-switch: MED1 p = 0.00024, Prosap p = 0.59, Sox21b p = 0.76, pairwise comparisons with two-sided unpaired t-tests with pooled SD. e ATAC-qPCR signal intensity for regions near the Xbp1 locus within a peak detected by ATAC-Seq (3’end of the gene) or two regions within the promoter of Xbp1. The levels were normalized to Prosap. Boxplots – quantiles; whiskers – extremes; overlay – individual data points. N = 3 biologically independent samples, effect of region p = 0.0011, effect of dfoxo-switch p > 0.05, mixed effects LM.
Extended Data Fig. 4 dfoxo-switch dependance on chromatin remodelers – lifespan.
Lifespan curves of the switch in S106 > dfoxo & RNAi (a) or S106 > RNAi (b) with indicated RNAi lines. These were used to generate the analysis shown in Fig. 2e. P values are obtained comparing control vs RU486-switch conditions (after day 23, log-rank test). Detailed statistical analyses including number of flies per experiment are shown in Supplementary Data 2 and 3.
Extended Data Fig. 5 dfoxo-switch dependance on chromatin remodelers – climbing ability.
a Negative geotaxis assay of dfoxo-switch + mor RNAi at all ages, combining two independent trials. Effect of the switch p = 0.6, age p < 10−4, age-by-switch interaction p = 0.36, mixed-effects LM. b Negative geotaxis assay of dfoxo-switch + iswi RNAi at all ages. Effect of the switch p = 0.12, age p < 10−4, age-by-switch interaction p = 0.14, mixed-effects LM. c Negative geotaxis assay of mor RNAi switch at all ages. Effect of the switch p = 0.02, age p < 10−4, age-by-switch interaction p = 0.006, mixed-effects LM. d Negative geotaxis assays of iswi RNAi switch at all ages. Effect of the switch p = 0.62, age p < 10−4, age-by-switch interaction p = 0.49, mixed-effects LM. N - individual flies, boxplots – quantiles; whiskers – extremes; overlay – individual data points. The negative geotaxis assays of dfoxo-switch that were performed at the same time are shown in Extended Data Fig. 2g, experiments 2 and 3.
Extended Data Fig. 6 RNA-Seq – additional information.
a qPCR quantification of transcripts detected as differentially expressed in our RNA-Seq data (HDAC6, Pfk, Pepck1) in fat bodies after dfoxo-switch. Effect of dfoxo p = 0.0055, transcript p = 0.0386, and dfoxo-switch-by-transcript interactions p = 0.032, mixed effects LM. b qPCR quantifications of dfoxo-switch targets (MED1, HDAC6, Xbp1s, Pfrx) at week 7 in dfoxo-switch females. Effect of dfoxo-switch p = 0.0133, effect of transcript or dfoxo-switch-by-transcript interaction p > 0.05, mixed effects LM. N – biologically independent samples; boxplots – quantiles; whiskers – extremes; overlay – individual data points. c and d Overlaps of sets of differentially expressed genes between dfoxo-switch (red circles) and dfoxo-acute (grey circles) in the fat body and gut employing previously published gene lists. Overlap p-values from one-sided hypergeometric test. e Bar plot comparing the log2 fold change of the transcripts in common between the sets of genes differentially regulated by dfoxo-acute (meta analysis) and by dfoxo-switch. f qPCR quantifications of transcripts during acute induction of dfoxo in the fat body. HDAC6, Pfk, Xbp1s, Xbp1u were examined as they are all differentially expressed after dfoxo-switch in the fat body (RNA-Seq analysis and qPCR confirmation shown elsewhere). N – biologically independent samples; boxplots – quantiles; whiskers – extremes; overlay – individual data points. Effects of RU486, transcript or RU486-by-transcript interaction p > 0.05, mixed effects LM. g Activating/Repressing Function prediction in BETA applying one-tailed Kolmogorov-Smirnov test. Differentially accessible ATAC peaks explained transcriptional activation (p = 2.97x10-46) and repression (p = 0.028) after dfoxo-switch.
Extended Data Fig. 7 Additional GO terms enrichment analysis.
Top 5 GO terms and KEGG pathways for genes differentially expressed (DE) a exclusively after dfoxo-switch and b exclusively during dfoxo-acute induction in the fat body. Note that no significant GO enrichment was observed in the set of genes that are differentially expressed in both dfoxo-switch and dfoxo-acute. BP - biological process, CC - cellular component, MF - molecular function.
Extended Data Fig. 8 Involvement of Xbp1 in the effects of dfoxo-switch – additional information.
a qPCR quantification of transcripts whose levels are increased after dfoxo-switch in fat bodies (hsc70-4, Eip75b, kay) in female S106 > Xbp1s fat bodies, with or without RU486 induction. Effect of RU486 p = 0.0088, effect of transcript or RU486-by-transcript interaction p > 0.05, mixed effects LM. b qPCR quantifications of the same transcripts in fat bodies after dfoxo-switch. Effect of RU486 p = 0.0001, effect of transcript or RU486-by-transcript interaction p > 0.05, mixed effects LM. N – biologically independent samples; boxplots – quantiles; whiskers – extremes; overlay – individual data points. c Survival of dfoxo-switch flies challenged with tunicamycin after 1 week of recovery (day 30; control n = 142 dead/0 censored, dfoxo-switch n = 129 dead/0 censored, p = 0.001, log-rank test). d Same for driver-alone (control n = 114 dead/0 censored, switch n = 131 dead/0 censored, p = 0.21, log-rank test). e Starvation assay of 30-day-old dfoxo-switch flies (control n = 142 dead/0 censored, dfoxo-switch n = 151 dead/0 censored, p = 0.67, log-rank test). f Same for driver-alone (control n = 149 dead/0 censored, switch n = 149 dead/0 censored, p = 0.07, log-rank test). g-j. Survival in the presence of tunicamycin a week after the switch in: S106 > morRNAi (control n = 141 dead/1 censored, switch n = 154 dead/0 censored, p < 6x10-7, log-rank test), S106 > iswiRNAi (control n = 146 dead/0 censored, switch n = 145 dead/0 censored, p = 0.60, log-rank test), S106 > dfoxo morRNAi (control n = 140 dead/0 censored, switch n = 139 dead/0 censored, p < 8x10-7, log-rank test), S106 > dfoxo iswiRNAi (control n = 153 dead/0 censored, switch n = 148 dead/0 censored, p = 0.079 log-rank test).
Extended Data Fig. 9 Age-related expression changes in the mouse – additional information.
Relationship between the expression changes triggered by dfoxo-switch in the fly fat body and the expression changes caused by ageing of their mouse orthologues (FDR 10%) in the functionally equivalent organs in the mouse. Points – genes; lines with shading – line of best fit and 95% CI; grey – those that are not significantly changed with age, red – those that are significantly changed with age. None of the organs show significant correlation between age-related change and dfoxo-switch change (p > 0.05, LM).
Supplementary Table 1
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Martínez Corrales, G., Li, M., Svermova, T. et al. Transcriptional memory of dFOXO activation in youth curtails later-life mortality through chromatin remodeling and Xbp1. Nat Aging 2, 1176–1190 (2022). https://doi.org/10.1038/s43587-022-00312-x