Interplay of dFOXO and Two ETS-Family Transcription Factors Determines Lifespan in <i>Drosophila melanogaster</i>

<div><p>Forkhead box O (FoxO) transcription factors (TFs) are key drivers of complex transcriptional programmes that determine animal lifespan. FoxOs regulate a number of other TFs, but how these TFs in turn might mediate the anti-ageing programmes orchestrated by FoxOs <i>in vivo</i> is unclear. Here, we identify an E-twenty six (ETS)-family transcriptional repressor, <i>Anterior open</i> (<i>Aop</i>), as regulated by the single <i>Drosophila melanogaster</i> FoxO (dFOXO) in the adult gut. AOP, the functional orthologue of the human Etv6/Tel protein, binds numerous genomic sites also occupied by dFOXO and counteracts the activity of an ETS activator, <i>Pointed</i> (<i>Pnt</i>), to prevent the lifespan-shortening effects of co-activation of dFOXO and PNT. This detrimental synergistic effect of dFOXO and PNT appears to stem from a mis-regulation of lipid metabolism. At the same time, AOP activity in another fly organ, the fat body, has further beneficial roles, regulating genes in common with <i>dfoxo</i>, such as the secreted, non-sensory, odorant binding protein (<i>Obp99b</i>), and robustly extending lifespan. Our study reveals a complex interplay between evolutionarily conserved ETS factors and dFOXO, the functional significance of which may extend well beyond animal lifespan.</p></div

<i>Aop</i> prevents the detrimental effects of <i>dfoxo</i> and <i>Pnt</i> co-activation.

<p><b>A</b> Survival of female flies expressing <i>dfoxo</i>, <i>Aop^RNAi</i> or both under the control of <i>S₁106</i> in the presence or absence or RU486. Log-rank test revealed significant effect of RU486 for <i>S₁106>dfoxo</i> (p<10⁻⁴; total dead/censored: − RU486 145/0, + RU486 139/6; median/maximum lifespan: − RU486: 77/87, + RU486 80/90) and <i>S₁106>dfoxo Aop^RNAi</i> (p<10⁻⁴; total dead/censored: − RU486 145/2, + RU486 155/2; median/maximum lifespan: − RU486: 74/90, + RU486 65/79) but not <i>S₁106>Aop^RNAi</i> (p = 0.5; total dead/censored: − RU486 142/0, + RU486 147/4; median/maximum lifespan: − RU486: 72/86, + RU486 74/87). CHP analysis showed that the response to RU486 in <i>S₁106>dfoxo Aop^RNAi</i> was significantly different from the response in <i>S₁106>dfoxo</i> (p<5×10⁻¹⁵) and <i>S₁106>Aop^RNAi</i> (p<10⁻⁷) females. <b>B</b> Survival of female flies expressing <i>dfoxo</i>, <i>PntP1 and Aop^ACT</i> under the control of <i>S₁106</i> in the presence or absence or RU486. Log-rank test revealed significant effect of RU486 for <i>S₁106>Pnt^P1 dfoxo</i> (p<10⁻⁴; total dead/censored: − RU486 138/6, + RU486 145/2; median/maximum lifespan: − RU486: 77/90, + RU486 63/83) but not <i>S₁106>Pnt^P1</i> (p = 0.06; total dead/censored: − RU486 147/0, + RU486 143/2; median/maximum lifespan: − RU486: 84/92, + RU486 79/90) or <i>S₁106>Pnt^P1 dfoxo Aop^ACT</i> (p = 0.1; total dead/censored: − RU486 147/3, + RU486 142/3; median/maximum lifespan: − RU486: 77/90, + RU486 77/83). CHP analysis showed that the response to RU486 in <i>S₁106>Pnt^P1</i> was significantly different from the response in <i>S₁106>Pnt^P1 dfoxo</i> (p<10⁻¹⁵) but not in <i>S₁106>Pnt^P1 dfoxo Aop^ACT</i> (p = 0.5) females. <b>C</b> Survival of <i>TIGS>dfoxo</i> female flies in the presence or absence of RU486. Log-rank test detected no significant differences (p = 0.3; total dead/censored: − RU486 152/1, + RU486 149/2; median/maximum lifespan: − RU486: 77/87, + RU486 77/86). <b>D</b> Survival of female flies expressing <i>dfoxo</i>, <i>Pnt^P1 and Aop^ACT</i> under the control of <i>TIGS</i> in the presence or absence or RU486. Log-rank test revealed significant effect of RU486 for <i>TIGS>Pnt^P1</i> (p<10⁻⁴; total dead/censored: − RU486 140/2, + RU486 136/7; median/maximum lifespan: − RU486: 74/86, + RU486 35/50), <i>TIGS>Pnt^P1 dfoxo</i> (p<10⁻⁴; total dead/censored: − RU486 146/3, + RU486 148/1; median/maximum lifespan: − RU486: 74/85, + RU486 32/45) and <i>TIGS>Pnt^P1 dfoxo Aop^ACT</i> (p = 0.1; total dead/censored: − RU486 143/1, + RU486 155/1; median/maximum lifespan: − RU486: 72/85, + RU486 35/55). CHP analysis showed that the response to RU486 in <i>TIGS>Pnt^P1</i> was significantly different from the response in <i>TIGS>Pnt^P1 dfoxo</i> (p = 2×10⁻⁴) but not in <i>TIGS>Pnt^P1 dfoxo Aop^ACT</i> (p = 0.8) females.</p

<i>Aop</i> extends lifespan.

<p><b>A</b> Survival of <i>S₁106>Aop^ACT</i> female flies in the presence or absence or RU486. Log-rank test detected significant differences (p = 2×10⁻¹⁰; total dead/censored: − RU486 145/1, + RU486 129/3; median/maximum lifespan: − RU486: 74/87, + RU486 84/97). <b>B</b> Median lifespan extension achieved by RU486 feeding in <i>S₁106>Aop^ACT</i> (6 experiments) or <i>TIGS>Aop^ACT</i> (3 experiments) females. Log-rank test detected significant extension (p<0.05) of lifespan in 6 out of 6 <i>S₁106>Aop^ACT</i> and in 1 out of 3 <i>TIGS>Aop^ACT</i> trials. In one <i>TIGS>Aop^ACT</i> trial lifespan was shortened. <b>C</b> Haemolymph glucose and trehalose in <i>S₁106>Aop^ACT</i> females fed or not RU486, where RU486 had a significant effect in each case (t-test, p = 0.01 and 0.02 respectively; n = 8 where 10 measurements were made and the highest and lowest measurement removed from each group). <b>D</b> Survival of <i>S₁106>Aop^ACT</i> or dfoxoΔ/Δ <i>S₁106>Aop^ACT</i> female flies in the presence or absence or RU486. Log-rank test detected significant differences for both: <i>S₁106>Aop^ACT</i> (p<10⁻⁴; total dead/censored: − RU486 139/3, + RU486 136/7; median/maximum lifespan: − RU486: 77/87, + RU486 86/94) and dfoxoΔ/Δ <i>S₁106>Aop^ACT</i> (p<10⁻⁴; total dead/censored: − RU486 138/0, + RU486 136/4; median/maximum lifespan: − RU486: 56/81, + RU486 72/90). CHP analysis revealed significant effects of RU486 (p<10⁻¹⁵) and <i>dfoxo</i> (p<10⁻¹⁵) but no significant difference in the response to RU486 between the two lines (p = 0.2).</p

<i>dfoxo</i> and <i>Pnt</i> act synergistically on lipid metabolism.

<p><b>A</b> Top three most significantly enriched GO categories within genes bound by FLAG-AOP^ACT alone or FLAG-AOP^ACT and dFOXO, as determined by EASE analysis. The intensity of red indicates log-10 derived p value associated with the enrichment. The complete GO analysis is given in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004619#pgen.1004619.s010" target="_blank">Dataset S1</a></b>. <b>B</b><i>CG6296</i> mRNA was quantified relative to <i>Act</i> by qPCR in the females of the indicated genotypes, induced or not with RU486. Boxplots show log-10 derived relative expression with − RU486 values set to zero. Data were analysed with a linear model and the effects of RU486, genotype and their interaction were significant (n = 4, p<10⁻⁴), with <i>S₁106>dfoxo Pnt^P1</i> + RU486 condition being different to all others (t-test , p<10⁻⁴). <b>C</b> The levels of TAG were quantified under the same conditions as in B. Data were analysed with a linear model and the effects of RU486 (p = 2×10⁻⁴), genotype (p<10⁻⁴) and their interaction (p = 0.01) were significant (n = 8, where 10 measurements were made and the lowest and highest measurement removed from each group). The <i>S₁106>dfoxo Pnt^P1</i> + RU486 condition was different from all others (t-test , p<0.05). The genotypes are denoted with the same colours in B and C, with the legend given in B.</p

dFOXO targets in the adult gut and fat body.

<p><b>A</b> Proportional Venn diagram showing the sets of genes that were differentially regulated by <i>dfoxo</i> induction in the gut or the fat body. The number of genes that were bound by GFP-dFOXO within each differentially expressed-gene set are given in black. p values for significant set overlaps are indicated. <b>B</b> Biological process GO categories differentially regulated (p<10⁻¹⁰) in the fat body or gut upon induction of <i>dfoxo</i> as determined by Catmap analysis. Any redundant categories (overlap by more than 75%) were removed, retaining the most specific category. The full list is given in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004619#pgen.1004619.s010" target="_blank">Dataset S1</a></b>. The intensity of red shows the log₁₀-transformed p-value associated with differential regulation for each category.</p

dFOXO regulates expression of <i>Aop</i> in the adult gut.

<p><b>A</b> Schematic of the <i>Aop</i> locus with black boxes representing exons, red boxes – regions detected as bound by GFP-dFOXO in the ChIP-chip experiment on induced <i>S₁106>GFP-dfoxo</i> females (dFOXO gut/fat body), yellow box - region detected as bound by dFOXO in wild-type females (dFOXO whole fly, data obtained from reference <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004619#pgen.1004619-Alic2" target="_blank">[17]</a>), and black bars – position of amplicons used for qPCR in B. <b>B</b> The enrichment of 5′ or 3′ end of the <i>Aop</i> locus, relative to <i>U6</i>, after anti-GFP IP of chromatin from RU486-induced <i>S₁106>dfoxo</i> females (mock), anti-GFP IP of chromatin from RU486-induced <i>S₁106>GFP-dfoxo</i> females (gut/fat body) or anti-dFOXO IP of wild-type female chromatin (whole fly). Means ± SEM of three biological repeats are shown, with enrichment in the mock control set to one. ANOVA on log-transformed data detected significant differences (p = 0.03 per region) and the enrichment of the 5′ region was different in gut/fat body from the mock (one-tailed t-test, p = 6×10⁻³), while the 3′ region was enriched in the whole fly (one-tailed t-test, p = 5×10⁻³). <b>C</b><i>Aop</i> mRNA was quantified relative to <i>Act</i> by qPCR in guts or fat bodies of <i>S₁106>dfoxo</i>, or <i>TIGS>dfoxo</i> flies induced or not with RU486. Boxplots show log-10 derived relative expression with - RU486 values set to zero. Data for <i>S₁106>dfoxo</i> females were analysed with a mixed-effects linear model with dissection batch as a random effect. The effects of RU486, tissue and their interaction was significant (p<0.05) and RU486 caused significant up-regulation of <i>Aop</i> in the gut (one-tailed t-test, n = 3–4, p = 2×10⁻³) but not the fat body (one-tailed t-test, n = 4, p>0.05). Significant changes were observed in <i>TIGS>dfoxo</i> guts (t-test, n>3, p = 0.02).</p

<i>Aop</i> and <i>dfoxo</i> both regulate a humoral factor, Obp99b, in the fat body.

<p><b>A</b> AOP was visualised by immunofuorescence in fat bodies of <i>S₁106>Aop^ACT</i> flies induced or not with RU486. In the merged image, AOP is shown in green, DAPI-stained nuclei in blue. AOP accumulates in nuclei of 26±6% of fat body cells. <b>B</b> Genes regulated by RU486 induction in fat bodies of <i>S₁106>Aop^ACT</i> flies were determined by microarray analysis and compared to changes in the fat body upon induction of <i>S₁106>dfoxo</i>. Mean log₂ fold change caused by induction of <i>Aop^ACT</i> (y-axis) is plotted against that caused by induction of <i>dfoxo</i> (x-axis). Genes with significant differential expression (at 20% FDR) upon induction of <i>Aop^ACT</i> are shown in red, <i>dfoxo</i> in green and both <i>Aop^ACT</i> and <i>dfoxo</i> in blue. The location of the <i>Obp99b</i> gene on the graph is indicated. <b>C</b> The levels of <i>Obp99b</i> mRNA in abdominal fat body mRNAs were determined relative to <i>actin</i> by qPCR upon RU486 induction in <i>S₁106>Aop^ACT</i> (n = 4) or <i>S₁106>dfoxo</i> (n = 8) female flies. Boxplots show log-10 derived ratios scaled to set the −RU486 condition to 0. Data were analysed using a mixed-effects linear model with genotype and RU486 as main effects and dissection batch as a random effect. Both main effects as well as their interaction were significant (p<0.01), and one-tailed t-test indicated the +RU486 condition had more Obp99b transcript for each genotype (p<0.05). <b>D</b> Obp99b-V5 was detected in haemolymph (1 µl) of <i>S₁106>Obp99b-V5</i> female flies, fed or not RU486, or in whole-fly extracts (equivalent of half a fly) using anti-V5 antibody. The secreted IMP-L2 protein was used as a loading control.</p

dFOXO binding sites in adult gut and fat body.

<p><b>A</b> Enrichment of the P3 promoter sequences of the <i>dInR</i> locus in the chromatin samples prepared from RU486-fed <i>S₁106>GFP-dfoxo</i>, RU486-fed <i>S₁106>dfoxo</i> (mock for the anti-GFP IP), wild-type 7-day old females or 2-h serum-starved S2 cells, after IP with either anti-GFP antibody or anti-dFOXO antibody, as indicated. Enrichment is expressed relative to <i>U6</i>, as means ± SEM of three biological replicates of chromatin, except for S2 cells where three IPs were performed from the same chromatin sample. ANOVA on log-transformed data detected significant differences (p<10⁻³), and enrichment in <i>S₁106>GFP-dfoxo</i>, after IP with anti-GFP antibody, was greater than all others (t-test, p<10⁻³). <b>B</b> ChIP-chip traces, showing the enrichment (log₂-transformed) of the GFP-dFOXO-immunoprecipitated DNA over total chromatin, are averages of three biological repeats after subtraction of the mock and are shown over a region of chromosome 3R. Red denotes the enrichment associated with peak regions. <b>C</b> Summary of the regulatory relationships between dFOXO and the five TFs it directly induces in the adult gut. Arrows indicate transcriptional activation.</p