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. 2015 Dec 8;6(39):41418-33.
doi: 10.18632/oncotarget.6282.

Genome-wide endogenous DAF-16/FOXO recruitment dynamics during lowered insulin signalling in C. elegans

Neeraj Kumar  1   2 , Vaibhav Jain  1 , Anupama Singh  1 , Urmila Jagtap  1 , Sonia Verma  1 , Arnab Mukhopadhyay  1
Affiliations

Genome-wide endogenous DAF-16/FOXO recruitment dynamics during lowered insulin signalling in C. elegans

Neeraj Kumar et al. Oncotarget. .

Abstract

Lowering insulin-IGF-1-like signalling (IIS) activates FOXO transcription factors (TF) to extend life span across species. To study the dynamics of FOXO chromatin occupancy under this condition in C. elegans, we report the first recruitment profile of endogenous DAF-16 and show that the response is conserved. DAF-16 predominantly acts as a transcriptional activator and binding within the 0.5 kb promoter-proximal region results in maximum induction of downstream targets that code for proteins involved in detoxification and longevity. Interestingly, genes that are activated under low IIS already have higher DAF-16 recruited to their promoters in WT. DAF-16 binds to variants of the FOXO consensus sequence in the promoter proximal regions of genes that are exclusively targeted during low IIS. We also define a set of 'core' direct targets, after comparing multiple studies, which tend to co-express and contribute robustly towards IIS-associated phenotypes. Additionally, we show that nuclear hormone receptor DAF-12 as well as zinc-finger TF EOR-1 may bind DNA in close proximity to DAF-16 and distinct TF classes that are direct targets of DAF-16 may be instrumental in regulating its indirect targets. Together, our study provides fundamental insights into the transcriptional biology of FOXO/DAF-16 and gene regulation downstream of the IIS pathway.

Keywords: C. elegans; ChIP-seq; DAF-16; FOXO; Gerotarget; transcription.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Genome-wide recruitment profile of DAF-16/FOXO
A. A DAF-16 peak on sod-3 promoter in daf-2(−) is absent in daf-16(−);daf-2(−). B. Distribution of DAF-16 peaks with respect to TSS. C. Enrichment of ranked normalized reads at the DAF-16 peak summits (left panel) and its heat map representation (right panel) in daf-2(−) that is absent in daf-16(−);daf-2(−). D. DAF-16 enrichment in daf-2(−) normalized to input samples. Enrichment was calculated using MACS in DAF-16 peaks that were common between this study (Endogenous DAF-16) and that of Riedel et al. (2013) (Overexpressed DAF-16). E. Recruitment profiles of DAF-16 isoforms in daf-2(−) compared to daf-16(−);daf-2(−) as determined by ChIP-Q PCR. DAF-16a, DAF-16b or DAF-16f represents transgenic lines where only one of the DAF-16 isoforms is expressed in a daf-16(−);daf-2(−) background [9].
Figure 2
Figure 2. Promoter-proximal binding of DAF-16 ensures optimal transcriptional response
A. Genes activated (N=667) or repressed (N=1213) in daf-2(−) in a DAF-16-dependent manner were overlapped with genes in which DAF-16 binds within 2.5 kb promoter region upstream of TSS (Grey circle; lower panel) to obtain 223 significant (Hypergeometric test) DAF-16 direct targets that are transcriptionally relevant. B. DAF-16-dependent genes that have peaks located within 0.5 kb of TSS are more likely (Hypergeometric test) to be activated in daf-2(−). C. Average mRNA fold change of genes activated or repressed in daf-2(−). The genes were categorized based on distance of DAF-16 peaks from TSS. D. Gene annotation enrichment analysis highlights biological functions of genes directly activated by DAF-16.
Figure 3
Figure 3. DAF-12 and EOR-1 may bind chromatin at close proximity to DAF-16 during low IIS
A. Upper panel shows the frequency of DAF-16 motif (red) within the DAF-16 peaks as compared to random sequences (blue). Lower panel contains the consensus DAF-16 motif identified by RSAT. B. Correlation of best PSSM hit scores of DAF-16, EOR-1, DAF-12, GATA or an unknown motif with DAF-16 peak heights. C. Upper panel shows the frequency of DAF-12 motif (red) within the DAF-16 peaks as compared to random sequences (blue). Lower panel contains the consensus DAF-12 motif identified by RSAT. D. Distribution of DAF-12 motifs with respect to DAF-16 motifs in daf-2(−). E. Upper panel shows the frequency of EOR-1 motif (red) within the DAF-16 peaks as compared to random sequences (blue). Lower panel contains the consensus EOR-1 motif identified by RSAT. F. Distribution of EOR-1 motifs with respect to DAF-16 motifs in daf-2(−). P values calculated using unpaired student's t test.
Figure 4
Figure 4. DAF-16 binds to variants of core FOXO consensus sequence on genes that it recruits to exclusively under low IIS
A. Overlap of DAF-16 binding peaks in WT and daf-2(−). Many new peaks (2385) appear in daf-2(−). P calculated using Hypergeometric test. B. FOXO consensus sequence was identified by RSAT de novo motif finding tool in DAF-16 binding peaks (within ± 250 bp of summit) for genes that the transcription factor recruits to in WT, daf-2(−) or both. Percentage occurrence of each consensus sequence is shown. C.-D. Gene annotation enrichment analysis highlights biological functions of genes that DAF-16 recruits to exclusively in WT C. or daf-2(−) D.
Figure 5
Figure 5. Genes that are activated under low IIS have higher DAF-16 recruitment in WT
A. Genes that are activated in daf-2(−) have higher DAF-16 recruitment on their promoters in WT compared to genes whose expression remain unchanged or are repressed. Ranked normalized read counts in WT were plotted against the distance from the peak summits (± 0.5 kb). Coding genes that are DAF-16-bound both in WT and daf-2(−) (2506) were considered. They were categorised as activated, repressed or no change based on their expression in daf-2(−) compared to WT. P calculated using Mann Whitney test. B. Ranked normalized read counts in daf-2(−) were plotted against the distance from the peak summits (± 0.5 kb) similar to A. P calculated using Mann Whitney test. C. RPKM of genes that are activated under low IIS condition as in daf-2(−) compared to WT (lower panel). Genes are categorized based on the fact that DAF-16 binds to the promoters exclusively in daf-2(−) or commonly in both WT as well as in daf-2(−). P between WT and low IIS calculated by Wilcoxon signed rank test. P between low IIS (DAF-16 peaks only in Low IIS) and WT (DAF-16 peaks both WT and Low IIS) in calculated by Mann Whitney test. The corresponding ranked normalized read counts are provided in the upper panel. P calculated using Mann Whitney test. n.b. indicates no binding peaks observed.
Figure 6
Figure 6. DAF-16 “core” direct targets contribute robustly towards IIS-regulated phenotypes
A. Comparison of DAF-16 ChIP-seq with transcriptomics data from multiple studies [6, 19, 29] reveals 37 “core” direct DAF-16 targets that are activated in daf-2(−). Each coloured square represents a RNA-seq or microarray data taken from the indicated studies. Genes that were found to be common with our ChIP-seq data are highlighted by dotted circles (direct DAF-16 targets). The numbers adjoining the dotted circles represent genes that overlap with our transcriptomics data. B. No significant overlap was observed in case of repressed genes. C. IIS pathway-dependent phenotypes are differentially affected when the “core” DAF-16 targets are knocked down by RNAi. The P values (obtained either by Student's t test or log rank test) of significantly affected genes are plotted. Details of the phenotypic analysis experiments provided in Table S7. D. DAF-16 core direct target genes are co-expressed with each other as determined by STRING database analysis (upper panel). No such co-expression was observed in case of a randomly chosen set of 37 genes.
Figure 7
Figure 7. FOXO recruitment to its target genes is conserved
Orthologous genes between a pair of species is shown outside the triangle. Common orthologous genes were overlapped with the binding data (direct targets) from either human FOXO, dFOXO or DAF-16 (this study) as indicated. Species-specific direct targets were overlapped and are shown in the centre of the triangle along with P values calculated using Hypergeometric test. B. Relative enrichment of different types of transcription factors that are directly activated or repressed by DAF-16. Numbers indicate R = representation score. *P ≤ 0.05 by Hypergeometric test.
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