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, 113 (40), 11277-11282

Chromatin-modifying Genetic Interventions Suppress Age-Associated Transposable Element Activation and Extend Life Span in Drosophila

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Chromatin-modifying Genetic Interventions Suppress Age-Associated Transposable Element Activation and Extend Life Span in Drosophila

Jason G Wood et al. Proc Natl Acad Sci U S A.

Abstract

Transposable elements (TEs) are mobile genetic elements, highly enriched in heterochromatin, that constitute a large percentage of the DNA content of eukaryotic genomes. Aging in Drosophila melanogaster is characterized by loss of repressive heterochromatin structure and loss of silencing of reporter genes in constitutive heterochromatin regions. Using next-generation sequencing, we found that transcripts of many genes native to heterochromatic regions and TEs increased with age in fly heads and fat bodies. A dietary restriction regimen, known to extend life span, repressed the age-related increased expression of genes located in heterochromatin, as well as TEs. We also observed a corresponding age-associated increase in TE transposition in fly fat body cells that was delayed by dietary restriction. Furthermore, we found that manipulating genes known to affect heterochromatin structure, including overexpression of Sir2, Su(var)3-9, and Dicer-2, as well as decreased expression of Adar, mitigated age-related increases in expression of TEs. Increasing expression of either Su(var)3-9 or Dicer-2 also led to an increase in life span. Mutation of Dicer-2 led to an increase in DNA double-strand breaks. Treatment with the reverse transcriptase inhibitor 3TC resulted in decreased TE transposition as well as increased life span in TE-sensitized Dicer-2 mutants. Together, these data support the retrotransposon theory of aging, which hypothesizes that epigenetically silenced TEs become deleteriously activated as cellular defense and surveillance mechanisms break down with age. Furthermore, interventions that maintain repressive heterochromatin and preserve TE silencing may prove key to preventing damage caused by TE activation and extending healthy life span.

Keywords: aging; dietary restriction; heterochromatin; silencing; transposable elements.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
DR attenuates age-related loss of silencing in heterochromatin genes and TEs. (A and B) Heat map shows log2 fold change (old/young) values for heterochromatin genes in both (A) heads and (B) fat bodies. (Left) Log2 fold change under HC conditions. (Right) Log2 fold change under DR. Heat map shows genes with log2 fold changes greater than 0.263 (1.2× fold change) under HC conditions that are located within annotated heterochromatin regions (29). Ninety-five percent of shown genes in heads and 89% in fat bodies are suppressed by DR. (C and D) Heat map shows log2 fold change (old/young) of TE expression for both HC (Left) and DR (Right) diet 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-seven percent of shown TEs were suppressed by DR in both heads and fat bodies.
Fig. S1.
Fig. S1.
Effect of DR on age-related loss of silencing in euchromatin genes. (A and B) Heat map shows log2 fold change (old/young) of top 50 euchromatin genes up-regulated with age in heads (A) and fat bodies (B). (Left) HC diet. (Right) DR. Genes located in euchromatin regions do not show the same consistent DR suppression of age-related increase in expression observed in heterochromatin genes (Fig. 1 A and B).
Fig. S2.
Fig. S2.
Spectrum of up-regulated TEs shows significant overlap among different tissues and genotypes. (A) Venn diagram indicating overlap of TEs that increase >0.322 log2 fold change in head and fat body libraries (P = 0.0395, Fisher’s exact test). The LTR retrotransposons copia, gypsy6, and accord were among the most highly up-regulated TEs with age in both backgrounds. Copia is the most highly expressed TE in both our head and fat body datasets, and additionally, copia is known to be active in D. melanogaster. (B) Four-way Venn diagram showing overlap of TEs displayed in four genotypes of Fig. 3 A–D. TEs shown are those that increase with age in the control or uninduced condition. Five TEs (seven sequences including Copia_LTR, Copia_I, Gypsy6_LTR, Copia2_I, BATUMI_LTR, BATUMI_I, Gypsy5_I; internal and LTR portions of sequences are annotated separately) appear in all four genetic backgrounds. The fact that there are also differences between the genotypes indicates that effects on TE expression are likely dependent on genetic background and strain-specific genomic location of TEs.
Fig. 2.
Fig. 2.
DR delays age-related increase in TE transposition. (A) Gypsy-TRAP flies (genotype: w1118; If/+; r4-GAL4, UAS-GFP/tub-ovo-GAL80) were categorized as low (<20 GFP-positive cells), medium (20–100), or high (>100) based on number of GFP-positive cells observed in fat body (representative images shown). The demarcation between low and medium categories is clear; most flies have either fewer than 10 cells staining, or more than 50. (Scale bar, 200 µm.) (B) Percentage of observed flies falling into each category at indicated points for HC (Top) and DR (Bottom) diets (n = ∼120 per cohort). (C) Transposition (defined here as medium- or high-category GFP staining) increases with age and is delayed by DR. Percentage of flies falling into either medium or high categories is displayed with time for both HC and DR diets. Log-rank P < 10−10.
Fig. S3.
Fig. S3.
DR delays age-related increase in TE transposition in alternate genetic backgrounds. (A and C) Percentage of gypsy-TRAP flies in two independently derived CyO lines (w1118; CyO/+; r4-GAL4, UAS-GFP/tub-ovo-GAL80; see SI Materials and Methods for further details) falling into each GFP staining category at indicated points, for both HC (Top) and DR (Bottom) diets. Data are presented analogously to Fig. 3B (n = 120 HC, 80 DR). (B and D) Percentage of flies falling into medium or high staining categories in these two gypsy-TRAP lines with time, for both HC and DR diets, analogous to Fig. 3C (n = 123 HC, 129 DR). Log-rank P < 10−10 for both lines. (E) Life span of individual gypsy-TRAP lines correlates with onset of increased transposition. Survivorship curves for the three independent gypsy-TRAP genetic backgrounds shown in A–D, as well as Fig 2 B and C. Adult females (n = 250) are shown for each line on HC (solid lines) and DR (dotted) food types. If line: HC mean = 72.3, median = 76; DR mean = 90.9, median = 92. CyO line 1: HC mean = 77.5, median = 76; DR mean = 83.1, median = 82. CyO line 2: HC mean = 82.4, median = 86; DR mean = 88.6, median = 90. All DR life spans are significantly different compared with their respective HC cohort at P < 10−6 (log-rank). All HC life spans are also significantly different from each other (P < 10−6 for If/CyO1 and If/CyO2; P = 0.03 for CyO1/CyO2). (F) Observed increases in GFP expression are not a result of nonspecific age-related changes in GAL80, GAL4, or GFP expression. Percentage of fat body cells staining positive for GFP with age in the normal gypsy-TRAP line (black) as well as a gypsy-TRAP line containing a mutated ovo locus that is not sensitive to gypsy transposon integration (red). Note the mutant gypsy-TRAP line shows no increase in GFP expression with age, indicating GFP expression is reporting increased transposition, rather than general age-related changes.
Fig. 3.
Fig. 3.
Manipulation of genes in heterochromatin pathways affects TE expression. Heat maps of log2 fold change (old/young) of TEs for the following genotypes and their respective controls: (A) elav-GeneSwitch > UAS-EP2300 (Sir2 overexpression); (B) Adar hypomorph vs. wild-type; (C) Su(var)3–9 overexpression [SV2 and SV3 lines overexpress Su(var)3–9 from a basally expressed UAS promoter 160–180% above control; Fig. S4B]; and (D) tubulin-GeneSwitch > UAS-Dicer-2 (Dicer-2 overexpression). (Left) Control condition. (Right) Transgenic, hypomorph, or RU486-induced condition. The percentage of shown TEs that are suppressed by the indicated genotype are (A) 72%, (B) 84%, (C) 92%, and (D) 68%. All TEs with log2 fold change (old/young) greater than 0.322 (1.25× fold change) in the control condition are shown.
Fig. S4.
Fig. S4.
(A) Dicer-2 mutants have increased double-strand breaks in nuclei compared with controls. Relative nuclear γH2Av fluorescence intensity in fat body cells from w1118 controls or Dicer-2L811fsX mutants. Dicer-2 mean fluorescence is 23% higher than control (n = 50; t test P = 0.027). Representative images from each genotype are shown. (B) Expression level of Su(var)3–9 relative to control for SV2 and SV3 lines, assayed by quantitative PCR, using GAPDH1 as a reference. Error bars represent SD. (C) 3TC treatment delays age-related increase in transposition. Time course of GFP expression in gypsy-TRAP flies either untreated (control) or treated with 10 µM 3TC, starting at day 20 of adulthood. Percentage of GFP-positive (>100 cells) fat bodies for each condition is shown.
Fig. 4.
Fig. 4.
Increasing expression of Su(var)3–9 or Dicer-2 increases life span. (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 life span = 40.6 d, median = 44 d; SV2 mean = 58.5 d, median = 60 d, 36% median extension, log-rank P < 10−10; SV3 mean = 58.3 d, median = 60 d, 36% median extension, P < 10−10. (B) Whole-adult animal Dicer-2 overexpressing flies are long-lived compared with controls. Survivorship of tubulin-GeneSwitch > UAS-Dicer-2 adult females (n = 250) is shown, either with EtOH (uninduced) or 200 µM RU486 (induced expression). EtOH mean = 52.3 d, median = 54 d; RU486 mean = 63.5 d, median = 66 d, 22% median extension, P < 10−10. (C) Flies expressing Dicer-2 in adult neurons are long-lived compared with controls. Survivorship of elav-GeneSwitch > UAS-Dicer-2 adult females is shown, for EtOH (uninduced) or 500 µM RU486 (induced) (n = 250). EtOH mean = 53.8 d, median = 56 d; RU486 mean = 61.7 d, median = 66 d, 18% median extension, P < 10−10. All life span experiments were repeated with similar results; representative experiments shown.
Fig. 5.
Fig. 5.
Administration of lamivudine (3TC) suppresses retrotranposition and extends Dicer-2 mutant life span. (A) Percentage of gypsy-TRAP flies with more than 30 fat body cells positive for GFP expression at 30 d of age for both untreated (control) and 10 µM 3TC treated fly cohorts. Fisher’s exact test P < 0.0001. (B) 10 µM 3TC treated flies are long-lived compared with untreated controls in the Dicer-2L811fsX genetic background. Survivorship of adult female flies is shown (n = 250). Control mean = 43.0 d, median = 45 d; 3TC mean = 46.4 d, median = 48 d, 6.6% median extension, log-rank P < 0.0001. Life span was repeated with similar results.

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