Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling

Abstract

FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild-type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo-independent effects. We demonstrate genome-wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.

Figure 1

Genome-wide dFOXO binding in whole adult flies. (A) ChIP-chip assays were carried out on 7-day-old females using anti-dFOXO antibody. ChIP-chip traces, showing the enrichment (log₂-transformed) of the dFOXO-immunoprecipitated DNA over total chromatin, are averages of three biological repeats after subtraction of the mock (pre-immune serum) control and are shown over a 3-Mbp region of chromosome 2R in wild-type flies (top) or dfoxo^Δ/Δ flies (bottom). Red dots denote the peaks identified in the ChIP-chip signal. Note that no peaks were identified in this region in the dfoxo^Δ/Δ flies. (B) qPCR was used to confirm the enrichment observed in dFOXO ChIP-chip in the three biological repeats of the wild-type chromatin. Relative enrichment was calculated as proportion of chromatin recovered in the IP for a single region divided by average recovered for all regions for that chromatin, with U6 enrichment set to one. The data are presented as means with standard errors. Red indicates regions that were expected to be enriched, white indicates those that were not. Significant difference was detected by ANOVA (P<10⁻⁴, n=3), and t-test revealed that the regions indicated in red were significantly different from the others (P<0.05).

Figure 2

dFOXO-binding sites in adults are distinct from those in larvae or S2 cells. (A) Overlap between the genomic sites bound by dFOXO in larvae and adults. The data for larvae were generated by Teleman et al (2008). The observed overlap was slightly smaller than expected by chance (P=0.02). Expected overlap of nine peaks was determined from simulation of 10³ random peak sets, of identical size, length and chromosomal distribution. (B) A schematic of the dInR locus is given with grey boxes representing exons, black marks the P1, P2 and P3 promoters (Casas-Tinto et al, 2007), red boxes the sites bound by dFOXO in adult flies (observed in ChIP-chip data) and green bars the location of amplicons (left—P1, right—coding region) used for ChIP-qPCR shown in (C). (C) dFOXO binding within the dInR locus in adults and S2 cells. The qPCR results show relative enrichment of the P1 promoter and the coding region (CDS) of dInR, or the U6 control, in three biological repeats of adult chromatin, or three IPs from a single chromatin sample from 2 h serum-starved S2 cells. The data are presented as in Figure 1B. ANOVA detected significant differences in enrichment (n=3, P<10⁻⁴), with P1 promoter being enriched in S2 cells and the coding region in adults (t-test, P<0.05).

Figure 3

Direct dFOXO targets in wild-type adult flies. (A) Overlap between the genes that neighbour a dFOXO-bound site and those with transcript levels altered in dfoxo^Δ/Δ flies relative to wild type. For consistency with later experiments, both dfoxo^Δ/Δ and wild-type flies also carried the daGAL4 driver. The probability of overlap was calculated based on hypergeometric distribution and an overlap significantly larger than expected by chance (P<10⁻³) is indicated with a red asterisk. Note that only the dFOXO-bound genes that were present on the expression arrays were taken into account. Representative biological functions enriched within the overlaps are shown. (B) dFOXO binding and regulation of IIS components. dFOXO binding and altered transcript levels in dfoxo^Δ/Δ flies were mapped onto a schematic of IIS. Note that PI3K denotes the p110 subunit. (C) The levels of Serine 505-phosphorylated AKT (pAKT) and the dually phosphorylated ERK (ppERK) were measured in wild-type and dfoxo^Δ/Δ females, as well as the levels of total AKT, ERK and dFOXO. dfoxo^Δ/Δ females had 70% (±10%) of the wild-type pAKT/AKT ratio, and 40% (±3%) of wild-type ppERK/ERK. In both cases, the difference to wild type was significant (P<0.05, n=3, t-test).

Figure 4

dFOXO binding under stress conditions or on downregulation of IIS. (A) Phosphorylation of dFOXO upon insulin injection. In all, 7-day-old females were injected with recombinant human insulin, mock-injected or not injected, and frozen after 5 min. The proteins were extracted, some treated with CIP and separated by SDS–PAGE. The phosphorylated (dFOXOppp) and unphosphorylated dFOXO is indicated. (B) dFOXO phosphorylation after 18 h of 20 mM paraquat administration or after 48 h of starvation. (C) Increased genome-wide enrichment of dFOXO-bound regions upon stress. Three biological repeats of the ChIP-chip assay were performed with anti-dFOXO antibody on flies treated with paraquat, starved or untreated controls. The intensity ratios of the peak probes (bound by dFOXO) to all probes, each taken at 0.75 quantile, is shown and was significantly greater for all treatment replicates (Wilcox rank sum test, n=3, P=0.024). (D) Increased region-specific enrichment of dFOXO-bound regions upon stress. The IPs were repeated on the same chromatin samples and the enrichment relative to U6 of Akt, dInR, TOR and the region between the Cat and Indy genes was determined by qPCR. The effect of treatment was found to be significant (two-way ANOVA, n=3, effect of treatment P<10⁻⁴, effect of genomic region P=0.02, no significant interaction of the two main effects). The same genome-wide (E) or region-specific (F) analysis was performed on daGAL4>UAS-dInR^DN flies or the driver alone control (daGAL4). This resulted in significant increase in the enrichment of dFOXO-bound regions, both on genome-wide scale (Wilcox rank sum test, n=3, P=0.05) and to the four target regions examined (two-way ANOVA on log-transformed data, n=3, effect of treatment P=7 × 10⁻⁴, no significant effect of genomic region).

Figure 5

Enrichment of dFOXO-bound genes within IIS transcriptional response. Overlaps between the genes regulated in whole daGAL4>UAS-dInR^DN flies relative to driver only controls (daGAL4) and genes bound by dFOXO. A red asterisk denotes an overlap significantly larger than expected by chance (P<10⁻³), as computed from a hypergeometric distribution.

Figure 6

Direct dFOXO targets in an IIS mutant. (A) Overlap between the genes that neighbour a dFOXO-bound site and those with transcript levels altered in dfoxo^Δ/Δ daGAL4>UAS-dInR^DN flies relative to daGAL4>UAS-dInR^DN. Representative biological functions enriched within the overlaps are shown. (B) Comparison of direct dFOXO targets in wild-type and in daGAL4>UAS-dInR^DN flies. The Venn diagram on the left compares genes that directly require dFOXO for activation of transcription in the two genetic contexts (i.e. genes bound by dFOXO and downregulated upon deletion of dfoxo), while the one on the right compares genes that directly require dFOXO for repression in the two genetic contexts (i.e. genes bound by dFOXO and upregulated upon deletion of dfoxo). In both (A, B), the probability of overlap was calculated based on hypergeometric distribution and an overlap significantly larger than expected by chance (P<10⁻³) is indicated with a red asterisk. Note that only the dFOXO-bound genes that were present on the expression arrays were taken into account. (C) dFOXO binding and regulation of IIS components. dFOXO binding and altered transcript levels in dfoxo^Δ/Δ daGAL4>UAS-dInR^DN flies were mapped onto a schematic of IIS. Note that PI3K denotes the p110 subunit.

Figure 7

dfoxo-dependent part of the transcriptional response to altered IIS. (A) Sets of genes that are transcriptionally upregulated in response to over-expression of dInR^DN (daGAL4>UAS-dInR^DN vs daGAL4; left) or downregulated upon deletion of dfoxo in a fly over-expressing dInR^DN (dfoxo^Δ/Δ daGAL4>UAS-dInR^DN vs daGAL4>UAS-dInR^DN; right) were compared in the Venn diagram above. The overlap is composed of the genes that require dfoxo for upregulation in daGAL4>UAS-dInR^DN flies. The Venn diagram below shows the reciprocal situation, and the overlap is composed of the genes that require dfoxo for downregulation in daGAL4>UAS-dInR^DN flies. The probability of overlap was calculated based on hypergeometric distribution and an overlap significantly larger than expected by chance (P<10⁻³) is indicated with a red asterisk. The proportion of the genes in the overlaps that are direct dFOXO targets (bound by dFOXO) is indicated with colour. (B) Non-redundant functional categories that are enriched (P<0.05) in the genes within the overlaps in (A). Those that are enriched within the direct dFOXO targets within the same overlaps are highlighted in red. Note that ‘proteolysis’ included predominantly extracellular proteases.

Figure 8

dfoxo-independent part of the transcriptional response to altered IIS. Sets of genes that are transcriptionally upregulated in response to over-expression of dInR^DN in the wild-type fly (daGAL4>UAS-dInR^DN vs daGAL4; left) or upregulated upon over-expression of dInR^DN in an dfoxo null fly (dfoxo^Δ/Δ daGAL4>UAS-dInR^DN vs dfoxo^Δ/Δ daGAL4; right) were compared in the Venn diagram above. The overlap is composed of the genes that do not require dfoxo for upregulation in daGAL4>UAS-dInR^DN flies. The Venn diagram below shows the reciprocal situation, and the overlap is comprised of genes that do not require dfoxo for downregulation in daGAL4>UAS-dInR^DN flies. The probability of overlap was calculated based on hypergeometric distribution and an overlap significantly larger than expected by chance (P<10⁻³) is indicated with a red asterisk.

Figure 9

dFOXO-bound genes are conserved between the fly and the worm. (A) Overlap between the fly orthologues of the genes that are downregulated by reduced function of daf-2 and upregulated by reduced function of daf-16 in the worm (Murphy et al, 2003), on the one hand, and those downregulated in daGAL4>UAS-dInR^DN and upregulated in dfoxo^Δ/Δ daGAL4>UAS-dInR^DN in the fly, on the other (hypergeometric distribution, P=7 × 10⁻⁶). (B) Overlap between the fly orthologues of the genes that are upregulated by reduced function of daf-16 in a daf-2 background in the worm (McElwee et al, 2007), on the one hand, and upregulated in dfoxo^Δ/Δ daGAL4>UAS-dInR^DN in the fly, on the other (hypergeometric distribution, P<10⁻¹⁵). (C) Overlap between the genes bound by dFOXO in the fly and the fly orthologues of the genes bound by DAF-16 in the worm (Oh et al, 2006; Schuster et al, 2010). (D) Representative functional categories enriched within the overlap shown in (C).