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Chromatin-modifying genetic interventions suppress SEE COMMENTARY age-associated transposable element activation and extend span in

Jason G. Wooda, Brian C. Jonesa, Nan Jianga, Chengyi Changa, Suzanne Hosiera, Priyan Wickremesinghea, Meyrolin Garciaa, Davis A. Hartnetta, Lucas Burhenna, Nicola Nerettia, and Stephen L. Helfanda,1

aDepartment of Molecular Biology, Biology, and Biochemistry, Brown University, Providence, RI 02912

Edited by Nancy M. Bonini, University of Pennsylvania, Philadelphia, PA, and approved August 5, 2016 (received for review March 28, 2016) Transposable elements (TEs) are , highly in regions of heterochromatin (reviewed in ref. 9). Because TEs enriched in heterochromatin, that constitute a large percentage of function by either excising or copying themselves from the ge- the DNA content of eukaryotic . Aging in Drosophila mel- nome and integrating into new genomic locations, there is a anogaster is characterized by loss of repressive heterochromatin high potential for deleterious effects on cellular function. Over- structure and loss of silencing of reporter in constitutive expressing TE is sufficient to cause cytotoxicity, and heterochromatin regions. Using next-generation sequencing, we inhibiting expression of these TEs causes reversal of both se- found that transcripts of many genes native to heterochromatic nescence and cytotoxic (10, 11). TE activation has regions and TEs increased with age in fly heads and fat bodies. A been observed with age in mice (8, 12), as well as C. elegans (13), dietary restriction regimen, known to extend life span, repressed yeast (14), Drosophila (15, 16), and senescent tissue culture the age-related increased expression of genes located in heterochro- cells (7). matin, as well as TEs. We also observed a corresponding age-associ- To combat the genomic threat of activation of TEs and loss of ated increase in TE transposition in fly fat body cells that was delayed heterochromatic silencing, somatic cells have evolved mecha- by dietary restriction. Furthermore, we found that manipulating nisms to suppress TE , primarily through establish- genes known to affect heterochromatin structure, including overex- ment and maintenance of repressive heterochromatin at TE sites pression of Sir2, Su(var)3–9,andDicer-2, as well as decreased expres- (17). In Drosophila, the small interfering RNA (siRNA) pathway, sion of Adar, mitigated age-related increases in expression of TEs. through the proteins -2 and Argonaute 2 (AGO2), recruits the Increasing expression of either Su(var)3–9 or Dicer-2 alsoledtoan histone H3K9 methyltransferase Su(var)3–9 to catalyze formation increase in life span. of Dicer-2 ledtoanincreaseinDNA – double-strand breaks. Treatment with the inhib- of repressive heterochromatin at TE sites (18 20). Mutation of Dicer- itor 3TC resulted in decreased TE transposition as well as increased 2, the double-stranded RNase responsible for generating siRNAs, life span in TE-sensitized Dicer-2 . Together, these data sup- results in TE reactivation and heterochromatin disorganization (19, port the theory of aging, which hypothesizes that 20). The RNA-editing enzyme ADAR (adenosine deaminase act- epigenetically silenced TEs become deleteriously activated as cel- ing on RNA) is thought to modify the effectiveness of siRNA- lular defense and surveillance mechanisms break down with age. directed heterochromatinization, and therefore the efficacy of Furthermore, interventions that maintain repressive heterochroma- TE silencing, by editing the dsRNA substrates used for siRNA tin and preserve TE silencing may prove key to preventing damage biogenesis, thereby preventing proper processing by Dicer-2 caused by TE activation and extending healthy life span. (21). Sir2/Sirt1, the founding member of the sirtuin family of proteins, is a protein deacetylase important in maintaining gene aging | heterochromatin | transposable elements | dietary restriction | silencing Significance

here is mounting evidence that preservation of the epigenetic Most eukaryotic genomes contain abundant transposable ele- Tstructure of chromatin is important to maintaining healthy ments (TEs), mobile DNA elements that can replicate and move aging, and loss of this chromatin structure may be a hallmark of within the . Because of the deleterious nature of active the aging process (reviewed in ref. 1). In aging yeast, cells lose TEs, cells have mechanisms to suppress and prevent TE activation, including formation of repressive heterochromatin. In this report,

characteristic silencing in heterochromatic regions, resulting in deleterious age-related consequences such as sterility, and replica- we show that many TEs become activated with age in Dro- tive life span is regulated by the histone H4K16 acetylation mark (2, sophila, and this activation is prevented by dietary restriction, an 3). In Caenorhabditis elegans, genetic manipulation of chromatin intervention known to extend life span. We also show TE acti- factors affecting histone methylation and acetylation directly affect vation is blocked by genetic manipulations that stabilize hetero- life span (4). In Drosophila, the integrity of constitutive repressive chromatin and increase life span. This study provides evidence heterochromatin declines with age at both the genomic and cellular that a breakdown in TE silencing and repression may be a con- level (5), with one consequence of these changes being an age-re- tributing factor to aging, and preventing TE activation may be a lated loss of in repressive constitutive heterochro- significant method of ameliorating the diseases of aging. matin regions in multiple tissues of the fly (6). In mice, changes in Author contributions: J.G.W., B.C.J., and S.L.H. designed research; J.G.W., B.C.J., N.J., C.C., epigenetic chromatin marks are seen with age, and age-related S.H., P.W., M.G., D.A.H., L.B., and S.L.H. performed research; J.G.W., B.C.J., and N.N. analyzed phenotypes of behavior, memory, and learning are observed on data; and J.G.W. and S.L.H. wrote the paper. genetic manipulation of certain histone marks (1). A breakdown in The authors declare no conflict of interest. heterochromatin organization has also been observed during repli- This article is a PNAS Direct Submission. cative senescence of human cells in culture, with expression and See Commentary on page 11069. copy number of transposable elements (TEs) increasing (7, 8). 1To whom correspondence should be addressed. Email: [email protected]. TEs are mobile genetic elements that make up a large per- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. centage of eukaryotic genomes and that are particularly enriched 1073/pnas.1604621113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1604621113 PNAS | October 4, 2016 | vol. 113 | no. 40 | 11277–11282 Downloaded by guest on September 28, 2021 silencing in heterochromatin regions of yeast and flies through aging process is characterized by a failure to properly control and deacetylation of specific histone lysines required for establishment repress expression of heterochromatin-based genes and TEs in the and spreading of heterochromatin (3, 6, 22). Increasing Sir2 ex- soma. We show that genetic interventions positively modifying het- pression has been shown to repress the normal age-related loss of erochromatin structure or negatively modulating TE activity extend gene silencing in heterochromatin regions in both yeast and flies metazoan life span. Finally, we show that attenuating TE ac- (3, 6), as well as the loss of gene silencing seen in mammalian tivity pharmacologically decreases transposition and extends life span embryonic stem cells undergoing genotoxic stress (23). In addition in Dicer-2 mutants. This study offers the first comprehensive char- to their roles in heterochromatin formation and maintenance, these acterization of the effect of known life span-extending interventions pathways have also been linked to aging. Decreasing activity of the on heterochromatic regions and explores new interventions for siRNA pathway through Ago2 and Dicer-2 mutants leads to de- controlling age-associated TE reactivation. repression of TE activity and shortened life span (15, 24). Sir2 has a central role in aging, with increased gene dosage shown to extend Results life span in yeast (2) and in nine of 10 studies in nematodes and flies Expression of Genes Located Within Heterochromatin Increases with (25–28). Hypomorphs of Adar, a negative regulator of the siRNA Age and Is Attenuated Under Dietary Restriction. Because hetero- pathway, also exhibit an extension of life span (21). These pathways chromatic regions change in structure with age, specifically in share an ability to affect heterochromatin formation and life span. female heads and fat bodies (5), we reasoned that genes located Here we report a characterization of the heterochromatic tran- within these regions may also show changes in silencing or gene scriptome in adult Drosophila tissues and show activation of TEs expression with age. We used RNA-seq to assay the expression of the and increased transposition with age. We provide evidence that the ∼250 genes natively located within the Drosophila heterochromatin

ABheads fat body Cheads Dfat body

CG17018 CG40053 Copia_I PLACW_DM CG6675 Copia_LTR CG17167 Copia_LTR Copia_I Rfabg Syt7 Copia2_I Copia2_I MAX_LTR CG17167 CG42402 Gypsy1−I_DM Gypsy6_LTR CG3107 sv Gypsy6_LTR TLD2 Kif3C CG31997 Gypsy1−LTR_DM Bica_LTR BARI_DM Gprk1 Gypsy3_I Pur−alpha ACCORD_I Yeti Spf45 ACCORD_I Gypsy7_LTR plexA Gypsy1−I_DM CG41378 R2_DM alpha−Cat DMTOM1_LTR Caps BATUMI_LTR ZAM_LTR CG40006 zfh2 ACCORD_LTR ACCORD_LTR gus Gypsy3_LTR I_DM alpha−Cat Gypsy12_LTR CG33978 Gfat1 PLACW_DM BURDOCK_LTR CG32230 ACCORD2_I Alg−2 DOC6_DM Tif−IA DM176_LTR cals MDG1_I Gypsy_LTR Rad23 MDG1_LTR CG1674 OSVALDO_I CG2316 NOMAD_I Invader3_LTR Cht3 G7_DM CG31997 BS CG17082 Copia1−LTR_DM CG17494 BLOOD_LTR HELENA_RT Rfabg Gypsy8_LTR CG32350 DMTOM1_LTR FW3_DM CG9935 FucTC Gypsy12_LTR Gypsy3_I CG41320 CG40191 BLOOD_I Chouto_I CG40263 Atf6 FW2_DM TRANSPAC_LTR DM1731_I fold change(old/young) Asator 2 CG32016 BEL_I POGO CG12567 CG40498 Mariner2_DM log QUASIMODO2−LTR fog CG12547 Invader5_LTR DOC BEL_LTR RhoGAP102A Ephrin BURDOCK_I IDEFIX_I CaMKI MDG1_LTR CG11360 BATUMI_I Actbeta NINJA_LTR CG17082 DIVER_I DM1731_LTR CG11360 CG32017 DIVER_LTR TABOR_I CG2177 Sox102F TOM_I HETA DM176_I CG3262 CG40178 G6_DM Gypsy_DS Fig. 1. DR attenuates age-related loss of silencing −N1_DM eIF4G p120ctn BEL_LTR DOC6_DM in heterochromatin genes and TEs. (A and B) Heat CG17486 CG40263 I_DM P−1_DY map shows log2 fold change (old/young) values for CG10465 Nipped−B TART_B1 FROGGER_I heterochromatin genes in both (A) heads and (B)fat myoglianin pan DOC Gypsy10_LTR G6_DM bodies. (Left)Log2 fold change under HC conditions. CaMKI tio Invader4_LTR TRANSPAC_I (Right)Log2 fold change under DR. Heat map shows CG17163 Ephrin TABOR_I BATUMI_LTR genes with log fold changes greater than 0.263 Transib5 2 HC DR HC DR HC DR Gypsy1−LTR_DM (1.2× fold change) under HC conditions that are lo- HMSBEAGLE_I cated within annotated heterochromatin regions ACCORD2_LTR QUASIMODO2−LTR (29). Ninety-five percent of shown genes in heads −2 0 2 −4 0 4 −4 0 4 Gypsy8_I and 89% in fat bodies are suppressed by DR. (C and Value HETA D) Heat map shows log fold change (old/young) of Value Value TRANSIB3 2 DOC5_DM TE expression for both HC (Left) and DR (Right) diet HC DR in both (C) heads and (D) fat bodies. Heat map shows TEs with log2 fold changes greater than 0.322 (1.25× fold change) under HC conditions. Eighty- −3 0 3 seven percent of shown TEs were suppressed by DR Value in both heads and fat bodies.

11278 | www.pnas.org/cgi/doi/10.1073/pnas.1604621113 Wood et al. Downloaded by guest on September 28, 2021 compartments (29) from young (10 d) and old (40 d) female fly heads endogenous gypsy retrotransposon jumps into an engineered site and fat bodies. The vast majority of cells in the head are neurons and containing known hotspot sequences from the ovo locus (31). We SEE COMMENTARY glial cells. The fly fat body is the functional homolog of mammalian monitored flies as they aged and visually sorted them into either adipose tissue and liver, and a central organ in the fly immune sys- low, medium, or high categories based on the number of GFP- tem. We found a number of genes located in heterochromatic re- positive cells observed in the fat body (Fig. 2A). Longitudinal fly gions increased expression with age in both wild-type heads and fat cohorts were assayed for transposition every 7 d on either HC bodies (Fig. 1 A and B). Gene ontology analysis reveals no sig- or DR foods (Fig. 2B). We observed a clear age-associated in- nificantly enriched pathways among these genes, suggesting the crease in transposition, and DR delayed this increase relative to HC age-related increase in expression of these genes is likely to be a controls (Fig. 2 B and C). Similar results were seen in two additional consequence of loss of heterochromatic silencing, rather than a genetic backgrounds (Fig. S3 A–D). We examined the life spans of specific compensatory or correlative response tied to aging. these three different genetic background gypsy-TRAP lines and Dietary restriction (DR) is an intervention known to extend observed that life span correlated with the timing of increased life span and delay numerous age-related phenotypes. We pre- transposition. Lines exhibiting earlier transposition showed shorter viously observed that DR can oppose age-related loss of gene life span, and those with later transposition longer life span silencing of reporter genes in constitutive heterochromatin (6). (Fig. S3E). Similarly, the delay in age-related transposition On this basis, we examined whether the age-dependent increase resulting from DR correlated with the ability of DR to extend life in expression of native genes in the constitutive heterochromatin span in each line. To ensure the increased GFP expression we regions were also suppressed under a DR regimen. The vast ma- observed with age was caused by transposition rather than age- jority (95% in heads, 89% in fat bodies) of heterochromatin genes related disruption in the GAL80/GAL4/GFP reporter system, we that increased expression with age on high-calorie food increased examined a gypsy-TRAP line with a ovo locus not sus- less or decreased in expression with age on DR food [high calorie ceptible to transposition and observed no significant increase in (HC) vs. DR] in both heads (Fig. 1A) and fat bodies (Fig. 1B). GFP expression with age (Fig. S3F). Rather than a constant linear Although we observed a similar fraction of euchromatin genes that increase in GFP expression with time, the gypsy-TRAP assay increased expression with age, this pattern of suppression by DR showed a clear age-associated increase in TE mobilization, consis- was not maintained in the most highly up-regulated euchromatin tent with the TE theory of aging, which posits an age-related loss of genes (Fig. S1 A and B), suggesting DR has a selective effect on normal cellular surveillance mechanisms and consequent increase in heterochromatin genes. Similar to previous studies using reporter deleterious TE activity. The relationship between the timing of in- genes in both yeast (2) and flies (6), these data suggest that gene creased gypsy transposition and life span further supports TE mo- expression in heterochromatic regions becomes dysregulated with bilization as an age-associated phenomenon. age, and that a known life span-extending intervention, DR, can prevent this.

TE Expression Increases with Age and Is Attenuated Under DR. One of High calorie A B 100 the most notable attributes of heterochromatin is the abundant enrichment of TEs within these regions. The Drosophila genome 75 is rich in retroviral-like (LTR) and long 50 interspersed nuclear elements (LINEs) (30), and activation of 25 Staining

Low 0 level both LINEs and LTRs has been observed in the fly nervous system 5 12 19 26 33 42 49 and fat body during aging (15, 16). Because heterochromatin in- Dietary restriction High 100 tegrity declines with age in multiple model systems (1), we hy- Med

Percent staining 75 pothesized that a loss of heterochromatin in regions highly enriched Low with TEs would show a correlated increase in TE expression with 50 age. To test this hypothesis, we examined the expression profile of 25 0 TEs with age and diet in female fly heads and fat bodies, using 5 12 19 26 33 42 49 whole RNA-seq. Similar to our observations for Age (days) native heterochromatin genes, we also found many TEs that in- creased their expression with age, in both heads and fat bodies Medium C (Fig. 1 C and D). We observed a high degree of overlap in the TEs 100 High Calorie that increased expression most with age between these two tissues, DR most of which were (Fig. S2A). In both tissue 75 types, as observed with the heterochromatin genes, DR attenu- GENETICS ated the age-related increase in expression observed in HC food 50 (Fig. 1 C and D, right “DR” columns). TEs that had the highest

increase in expression with age on HC food tended to increase High 25 less or even decrease in expression with age under DR conditions

(87% lower in DR relative to HC in both heads and fat bodies; Percent medium or high 0 0 10 20 30 40 50 Dataset S1). These data suggest that, similar to reporter genes in Age (days) heterochromatin (2, 6) and native heterochromatin genes (Fig. 1 A and B), TE silencing is lost with age, and DR opposes this. Fig. 2. DR delays age-related increase in TE transposition. (A) Gypsy-TRAP flies (: w1118; If/+; r4-GAL4, UAS-GFP/tub-ovo-GAL80) were categorized as TE Transposition Increases with Age and Is Delayed by DR. An age- low (<20 GFP-positive cells), medium (20–100), or high (>100) based on number dependent increase in TE expression and transposition in the of GFP-positive cells observed in fat body (representative images shown). The demarcation between low and medium categories is clear; most flies have ei- Drosophila brain associated with decline in neural function has μ been reported (15). Similar increases in TE expression levels and ther fewer than 10 cells staining, or more than 50. (Scale bar, 200 m.) (B) Percentage of observed flies falling into each category at indicated points for copy numbers have been observed in aging mouse tissue (8). To HC (Top)andDR(Bottom)diets(n = ∼120 per cohort). (C) Transposition (de- assay for age-related transposition in our flies, we used the gypsy- fined here as medium- or high-category GFP staining) increases with age and is TRAP reporter system (15). This system is designed such that delayed by DR. Percentage of flies falling into either medium or high categories green fluorescent protein (GFP) becomes expressed when an is displayed with time for both HC and DR diets. Log-rank P < 10−10.

Wood et al. PNAS | October 4, 2016 | vol. 113 | no. 40 | 11279 Downloaded by guest on September 28, 2021 Age-Related Increases in TE Expression Are Attenuated by Genetic observed significant overlap in the TEs that increased with age Interventions That Affect Heterochromatin Structure. Our data dem- among the different genetic backgrounds (Fig. S2B). Together, onstrate a correlation between changes in heterochromatin struc- these data suggest that increased expression of Sir2, Dicer-2,orSu ture with age and expression and transposition of TEs during normal (var)3–9, as well as decreased gene dosage of Adar, all prevent the aging. This led us to examine whether genetically manipulating age-related loss of silencing of TEs observed in wild-type and con- heterochromatin structure and maintenance, either directly or trol lines, suggesting a common link between heterochromatin fac- indirectly, could also affect the age-related changes in expres- tors and proper maintenance of TE silencing. sion of TEs. To test our hypothesis, we genetically increased the TE activation is known to disrupt gene expression and destabilize activity of two important systems that maintain repressive het- the genome. Increased DNA damage has been observed in aging fat erochromatin in somatic cells: the siRNA pathway and Sir2. Using body nuclei, as assayed by γH2Av immunostaining (16). Our finding transgenic fly lines, we increased gene dosage of Sir2, Dicer-2,and of a repression of TE expression with increased Dicer-2 expression Su(var)3–9 andalsousedanAdar hypomorph, interventions we led us to investigate whether genomic integrity was similarly com- expect to stabilize heterochromatin. Each of these genes offers a promised in with disrupted RNAi/heterochromatin path- unique and complementary approach to simultaneously manipu- ways and increased TE activity. Similar to depletion of AGO2 in fat lating heterochromatic regions and enhancing TE silencing. body cells (16), we observed an increase in double-strand breaks in We assayed age-dependent loss of silencing of TEs in each of Dicer-2 mutants compared with controls in fat body nuclei (Fig. these various transgenic [Sir2, Su(var)3–9,andDicer-2]orhypo- S4A), suggesting deleterious genomic consequences when proper morphic (Adar) fly lines compared with their respective controls. TE silencing is disrupted. The TEs that showed the greatest increase in expression with age in the control lines were overwhelmingly attenuated or decreased Genetic Interventions That Affect Heterochromatin Structure Also Affect with age by these genetic interventions [Fig. 3 A–D; Sir2,72%; Life Span. In addition to their effects of delaying age-related het- Adar,84%,Su(var)3–9,92%;Dicer-2, 68%]. Interestingly, we again erochromatin gene and TE silencing loss, both increased Sir2

ABCSir2 Adar Su(var)3-9 DDicer-2 Copia_I BATUMI_LTR Copia_LTR Copia_LTR Copia_I Copia_I R2_DM Copia_I Gypsy12_LTR Copia_LTR ACCORD_I Gypsy6_LTR BATUMI_LTR Copia2_I BATUMI_I Gypsy1−LTR_DM ZAM_I Chouto_I Gypsy9_I R1_DM Copia2_I ACCORD2_I CIRCE Copia_LTR PLACW_DM Stalker2_LTR Bica_LTR HETA MDG1_LTR Gypsy11_I BS3_DM TABOR_I Chimpo_LTR Gypsy1−I_DM DOC5_DM MAX_LTR BATUMI_LTR BATUMI_LTR I_DM R1_DM DM297_I BARI_DM DIVER2_LTR Bica_LTR MAX_I MAX_I BS MAX_I MAX_LTR TART BS Gypsy5_LTR Ulysses_I BURDOCK_LTR BATUMI_I Gypsy_LTR Gypsy12_LTR TRANSIB3 DMRT1B Copia2_I G7_DM TRANSIB2 G_DM TABOR_I ROO_I MDG3_I Gypsy2−LTR_DM Gypsy1−I_DM Gypsy5_I G2_DM DM176_I ZAM_LTR BEL_LTR DM1731_LTR MDG1_I Gypsy_I TRANSPAC_LTR FW_DM Gypsy5_I Bica_LTR Gypsy6_LTR Gypsy1−LTR_DM ROO_LTR ROVER−I_DM ACCORD2_LTR Gypsy6_LTR R2_DM Gypsy8_I DMRT1A Chimpo_LTR Gypsy3_I Gypsy6_LTR Gypsy12A_LTR BATUMI_I Gypsy6A_LTR BATUMI_I Copia2_I Baggins1 TAHRE ACCORD_LTR BARI_DM Gypsy8_LTR DOC5_DM Gypsy1−I_DM DOC ACCORD2_LTR TV1I DMRT1C I_DM TART Gypsy5_LTR TIRANT_LTR TABOR_LTR NOMAD_LTR Invader1_LTR DMCR1A Ulysses_I MDG3_LTR Gypsy4_I Gypsy2_LTR G6_DM MDG1_I DM412 Copia1−I_DM Gypsy5_I ZAM_LTR Baggins1 DMLTR5 BLOOD_I NOMAD_I Gypsy9_I ACCORD_LTR IVK_DM Gypsy5_I IDEFIX_LTR Gypsy3_I Gypsy3_I TIRANT_I Gypsy7_LTR DMRT1B TIRANT_LTR Gypsy4_I Copia1−LTR_DM Gypsy10_LTR TRANSPAC_LTR QUASIMODO_I Stalker2_LTR DIVER_I ROO_I NINJA_I TOM_I Fig. 3. Manipulation of genes in heterochromatin BS2 Gypsy4_LTR Stalker2_LTR STALKER4_I pathways affects TE expression. Heat maps of log2 MAX_LTR DIVER2_LTR BEL_LTR DIVER2_LTR EtOH RU wt hyp wt SV2 FB4_DM fold change (old/young) of TEs for the following gen- DM412B_LTR otypes and their respective controls: (A) elav-Gen- G6_DM > ACCORD2_LTR eSwitch UAS-EP2300 (Sir2 overexpression); (B) Adar FW_DM hypomorph vs. wild-type; (C) Su(var)3–9 overexpression M4DM [SV2 and SV3 lines overexpress Su(var)3–9 from a log2 fold change (old/young) TART_B1 Chouto_LTR basally expressed UAS 160–180% above EtOH RU control; Fig. S4B]; and (D) tubulin-GeneSwitch > −3 0 3 UAS-Dicer-2 (Dicer-2 overexpression). (Left)Control Value condition. (Right) Transgenic, hypomorph, or RU486- induced condition. The percentage of shown TEs that log2 fold change (old/young) are suppressed by the indicated genotype are (A)72%,(B)84%,(C)92%,and(D) 68%. All TEs with

−4 0 4 log2 fold change (old/young) greater than 0.322 (1.25× Value fold change) in the control condition are shown.

11280 | www.pnas.org/cgi/doi/10.1073/pnas.1604621113 Wood et al. Downloaded by guest on September 28, 2021 expression and decreased Adar expression have been shown to ex- AB 100 SEE COMMENTARY tend life span in flies (21, 25), whereas Dicer-2 and Ago2 mutants 60 Control are short-lived (15, 24). Given our findings presented earlier, we 50 3TC examined whether increased expression of Su(var)3–9 or Dicer-2 75 40 L811fsX also have a positive effect on life span. We found that two different Dcr-2 Su(var)3–9 transgenic lines, SV2 and SV3, which constitutively in- 30 50 – – crease Su(var)3 9 expression to 160 180% of the wild-type level in 20

theabsenceofadriver(Fig. S4B), extend life span significantly Percent Alive 25 compared with their genetically matched control (Fig. 4A). Simi- 10 larly, increasing Dicer-2 expression in adult life in either all tissues 0 Percent GFP positive at 30 days Percent GFP 0 (Fig. 4B) or selectively in adult neurons (Fig. 4C), using the condi- Control 3TC 0 20 40 60 tional inducible GeneSwitch system, was able to extend life span, Age (days) with flies fed the gene-activating drug RU486 outliving a genetically identical control cohort fed only the diluent control EtOH. Together Fig. 5. Administration of lamivudine (3TC) suppresses retrotranposition and extends Dicer-2 mutant life span. (A) Percentage of gypsy-TRAP flies with with previously published data (21, 25), these observed life span more than 30 fat body cells positive for GFP expression at 30 d of age for extensions suggest a coordinated response integrating maintenance both untreated (control) and 10 μM 3TC treated fly cohorts. Fisher’s exact of heterochromatin structure, TE suppression, and longevity. test P < 0.0001. (B)10μM 3TC treated flies are long-lived compared with untreated controls in the Dicer-2L811fsX genetic background. Survivorship of Pharmacological Suppression of Transposable Element Activity Delays adult female flies is shown (n = 250). Control mean = 43.0 d, median = 45 d; Age-Related Transposition and Extends Life Span. To test whether 3TC mean = 46.4 d, median = 48 d, 6.6% median extension, log-rank P < the age-related effects we observed were a result of increased TE 0.0001. Life span was repeated with similar results. activity, we sought to directly modulate transposition pharmaco- logically. We used the reverse transcriptase inhibitor lamivudine (3TC) to directly block TE transposition. Using the gypsy-TRAP background, which exhibits high levels of TE activity because reporter system in the fat body, we observed that flies treated with of defective RNAi silencing (32), we found that administra- tion of 3TC was able to extend life span significantly compared 3TC had a significant delay in age-related transposition compared with controls (Fig. 5B). Together, these data suggest that het- with controls (Fig. 5A). This effect was observed whether the drug erochromatin-altering pathways that extend life span may do so was administered constantly throughout life (Fig. 5A)orstarted at least in part by suppressing TE activity to ensure healthy during midadulthood at 20 d of age (Fig. S4C). longevity, and confirm that suppression of TE activation is As our evidence suggests that maintenance of TE silencing is likely to be an attractive target mechanism for pharmacological important for ensuring longevity, we tested whether attenuating TE L811fsX intervention. activity directly could extend life span. In the Dicer-2 mutant Discussion Aging is characterized by a breakdown in homeostasis through- A B out multiple cellular and organismal systems. Genomic and epi- 100 100 tub-GS > UAS-Dcr-2 genomic stability is vital for somatic cells to maintain proper gene WT expression and silencing. A number of studies have shown changes 80 SV2 80 SV3 EtOH in chromatin structure during organismal aging in various model 60 60 RU486 systems, especially in heterochromatin (1, 2). Although hetero- 40 40 chromatin regions contain some genes, a likely reason for the

Percent Alive 20 Percent Alive 20 central importance of maintaining repressive heterochromatin is 0 0 that heterochromatin is replete with TEs, and unchecked TE ac- 0 20 40 60 80 0 20 40 60 80 100 Age (days) Age (days) tivation has particularly damaging ramifications for genomic sta- C bility. Indeed, the expression and mobilization of TEs is disruptive 100 elav-GS > UAS-Dcr-2 enough that cells have evolved RNAi pathways responsible for 80 silencing and preventing TE activation. These cellular defenses EtOH 60 against TE activation include siRNA-directed induction and RU486 maintenance of repressive heterochromatin to prevent TE ex- 40 pression, and degradation of target transcripts. siRNA pathway GENETICS Percent Alive 20 mutants exhibit both an activation of TEs and a shortening of life 0 span (15, 24). Here we provide evidence that many heterochro- 0 20 40 60 80 100 Age (days) matin genes and TEs do in fact increase expression with age in two different tissues. Increased transposition has been previously – Fig. 4. Increasing expression of Su(var)3 9 or Dicer-2 increases life span. reported in the aging fly brain (15). Similarly, we also observe an (A) SV2 and SV3 lines [overexpressing Su(var)3–9] are long-lived compared with controls. Survivorship of adult female flies is shown (n = 250). WT mean increase in somatic TE transposition with age in the fat body. life span = 40.6 d, median = 44 d; SV2 mean = 58.5 d, median = 60 d, 36% DNA double-strand breaks increase with age in the fat body (16), − median extension, log-rank P < 10 10; SV3 mean = 58.3 d, median = 60 d, and we show this DNA damage also accumulates at a younger age − 36% median extension, P < 10 10.(B) Whole-adult animal Dicer-2 over- in Dicer-2 mutant flies with high levels of TEs. The age-related expressing flies are long-lived compared with controls. Survivorship of tu- increase in TE transcript levels as well as TE mobilization were bulin-GeneSwitch > UAS-Dicer-2 adult females (n = 250) is shown, either suppressed by DR. Together, these data suggest that an increase in with EtOH (uninduced) or 200 μM RU486 (induced expression). EtOH mean = TE expression and transposition is an important hallmark of so- 52.3 d, median = 54 d; RU486 mean = 63.5 d, median = 66 d, 22% median < −10 matic aging, and preventing this increased TE activity is likely to extension, P 10 .(C) Flies expressing Dicer-2 in adult neurons are long-lived preserve genomic stability and cellular homeostasis. compared with controls. Survivorship of elav-GeneSwitch > UAS-Dicer-2 adult females is shown, for EtOH (uninduced) or 500 μMRU486(induced)(n = 250). We demonstrate in a whole model that aging is as- EtOH mean = 53.8 d, median = 56 d; RU486 mean = 61.7 d, median = 66 d, sociated with a loss of silencing of both TEs and genes natively − 18% median extension, P < 10 10. All life span experiments were repeated located in heterochromatin regions, with a resultant increase in with similar results; representative experiments shown. TE mobilization and an associated increase in DNA damage. It

Wood et al. PNAS | October 4, 2016 | vol. 113 | no. 40 | 11281 Downloaded by guest on September 28, 2021 is highly likely that the pathways and processes of heterochro- kit (Ambion). Then, 100 ng total RNA was used as input for stranded matin maintenance, RNAi, and gene and TE silencing represent RNA-seq library construction, using the Ovation Universal RNA-seq kit potential homeostatic failure points that lead to the phenotypes (Nugen) with Drosophila rRNA depletion module. Libraries were sequenced × of aging. Here we have also presented genetic evidence that on an Illumina HiSEq. 2500 in 1 50 bp mode. All samples were prepared using at least three individually isolated biological replicates for each these pathways are linked to aging, as manipulating the expres- condition. sion of several genes involved in maintaining heterochromatin structure, TE silencing, and RNAi efficacy directly suppresses Bioinformatics. Standard RNA-seq analysis was performed using Tophat 2.0 age-related TE expression as well as extends life span. Further- for , followed by gene-based counting using the easy- more, we show that DR, an environmental intervention that RNASeq R package from Bioconductor. Finally, trimmed mean of M-values extends life span robustly and delays most age-related pheno- (TMM) normalization and fold change calculation was performed using types, also opposes the loss of silencing we observe during nor- edgeR, with a generalized linear model (GLM) correcting for batch effects, mal aging and delays age-dependent transposition, further as described in the edgeR vignette. To account for repetitive sequencing lending support to the idea that these processes are associated reads and perform TE quantification, RepEnrich (33) was used to generate a with and may be relevant to aging. We also demonstrate that count table of reads for each TE, and edgeR was used to normalize, calculate fold changes, and perform differential expression testing. The Repbase an- inhibition of retrotransposon activity is strongly correlated with notation was used as input for RepEnrich. extension of life span. Our data support the retrotransposon theory Heat maps were generated by ranking the TEs or genes in order of log2 of aging model, where proper maintenance of silent heterochro- fold change of expression with age (old/young) for the reference (control/

matin and prevention of TE activation are key to guaranteeing ge- HC) condition and then plotting log2 fold change with age for all conditions. nomic stability and homeostasis during aging and ensuring healthy To show consistent trends in our data and include genes/TEs that behaved longevity. Furthermore, our demonstration that dietary (DR), ge- consistently but did not meet the threshold of statistical significance, we × netic, and pharmacological interventions that reduce the age-related displayed genes or TEs where log2 fold change was greater than 0.322 (1.25 increases in TE activity can also extend life span suggests new and fold; TEs) or 0.263 (1.2x fold; heterochromatin genes). Plots were generated novel pathways for the development of interventions designed to using the heatmap.2 function in the gplots2 R package. extend healthy life span. See SI Materials and Methods for additional details on fly stocks and methods. Materials and Methods ACKNOWLEDGMENTS. We thank Will Lightfoot and Gillian Horwitz for technical assistance and Christoph Schorl and the Brown University Genomics Gypsy-TRAP Assay. Flies from the gypsy-TRAP line were aged for 5 d and Core Facility for sequencing. We also thank Joshua Dubnau, Robert Reenan, initially examined for the number of GFP-positive cells, then examined every Kristen Johansen, and the Bloomington Drosophila Stock center for fly 7 d until age 49 d, using a fluorescent dissecting microscope. Flies were stocks and Robert Reenan and John Sedivy for helpful discussions. This work separated into three categories based on visual inspection, according to how was supported in part by National Institute on Aging (NIA) T32 Training many fat body cells were GFP positive: low (<20 cells), medium (20–100 cells), Grant AG41688 and Fellowship Award AG47736 (to B.C.J.); NIA Grants and high (>100 cells). The same cohort of flies was examined longitudinally AG16667 and AG24353, a Glenn/American Federation for Aging Research (AFAR) Breakthroughs in Gerontology award, NIH Program Project Grant throughout each of the experiments. AG51449, and Grant P30AI042853 from the Providence/Boston Center for AIDS Research (to S.L.H.); Ellison/AFAR Postdoctoral Fellowship awards (to RNA-seq. Total RNA from the indicated was collected from J.G.W. and N.J.); a Nathan Shock Center Pilot Grant (to J.G.W.); and NIH ∼100 fly heads or ∼50 dissected fat bodies, using the miRvana RNA prep Grants AG28753 and AG50582 (to N.N.).

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