The transcription factor ultraspiracle influences honey bee social behavior and behavior-related gene expression

Abstract

Behavior is among the most dynamic animal phenotypes, modulated by a variety of internal and external stimuli. Behavioral differences are associated with large-scale changes in gene expression, but little is known about how these changes are regulated. Here we show how a transcription factor (TF), ultraspiracle (usp; the insect homolog of the Retinoid X Receptor), working in complex transcriptional networks, can regulate behavioral plasticity and associated changes in gene expression. We first show that RNAi knockdown of USP in honey bee abdominal fat bodies delayed the transition from working in the hive (primarily "nursing" brood) to foraging outside. We then demonstrate through transcriptomics experiments that USP induced many maturation-related transcriptional changes in the fat bodies by mediating transcriptional responses to juvenile hormone. These maturation-related transcriptional responses to USP occurred without changes in USP's genomic binding sites, as revealed by ChIP-chip. Instead, behaviorally related gene expression is likely determined by combinatorial interactions between USP and other TFs whose cis-regulatory motifs were enriched at USP's binding sites. Many modules of JH- and maturation-related genes were co-regulated in both the fat body and brain, predicting that usp and cofactors influence shared transcriptional networks in both of these maturation-related tissues. Our findings demonstrate how "single gene effects" on behavioral plasticity can involve complex transcriptional networks, in both brain and peripheral tissues.

Figure 1. The transcription factor ultraspiracle (usp) influences honey bee behavioral maturation.

A) Knockdown of usp mRNA in the fat bodies of 3-day-old bees, 72 h after injection with dsUSP or control dsRNA (ds-pUC). qPCR; n = 10 bees; t-test: * P<0.05. B) usp RNAi does not knock down usp mRNA in the heads of these same bees. C) Knockdown of USP protein in the fat bodies 48, 72 and 96 h after injection was confirmed by immunoblotting with an antibody specific to honey bee USP (Figure S3). D) usp RNAi delays the age at onset of foraging. Pooled results from 9 independent trials. Cox Proportional Hazards: P = 0.03. Numbers in legend indicate how many bees were measured for each group.

Figure 2. Putative direct and indirect targets of USP in honey bee fat bodies.

Putative targets of USP in the fat bodies were characterized both by usp RNAi—deep mRNA sequencing (in combination with juvenile hormone analog, JHA, treatments) as well as by USP ChIP-chip with fat body tissue samples from nurses and from foragers. A. Genomic regions surrounding two putative target genes, the transcription factors SoxNeuro and Hr46. Units for mRNA-seq are read counts, and for ChIP-chip the ratio of a-USP to control. B. Venn diagram shows that many USP target genes identified by usp RNAi and USP ChIP-chip are differentially expressed between nurses and foragers (“Maturation”). Fold enrichment of overlap and its significance (hypergeometric test) are indicated for each comparison. C. The 1360 genomic binding sites of USP are enriched for conserved cis-regulatory sequences, putatively recognized by the TFs shown at left. D. USP binds genomic locations near 67 transcription factors (TFs), including members of the nuclear hormone receptor family (diamonds) and other TF families (circles). Some of these TFs were differentially expressed between nurses and foragers (blue, higher in nurse; yellow, higher in forager), and predicted targets for several of these TFs based on transcriptional regulatory network analysis were enriched for genes that are differentially expressed in maturation-related contexts (thick outlines). Some of these TFs were also identified as USP targets in D. melanogaster (red lines), and some binding sites contain the GGGGTCACS cis-regulatory sequence recognized by USP in D. melanogaster (solid lines).

Figure 3. USP mediates maturation-related gene expression responses to juvenile hormone.

Mechanisms linking USP's putative targets to maturation were examined by studying their expression dynamics during maturation and in response to juvenile hormone analog treatments (JHA) and diet manipulations. A. usp RNAi, JHA, and maturation influence many of the same genes. Fold enrichment of overlap between gene lists and its hypergeometric p-value are indicated for each comparison. B. Fold change responses to JHA and maturation are positively correlated. Data are shown for 97 genes that responded significantly to both JHA and maturation; genes that also responded to usp RNAi are represented by closed circles. C. 33 of the 42 genes that respond to both JHA and usp RNAi were activated by both factors. Genes that also responded to maturation are represented by closed circles. D. usp RNAi inhibited transcriptional responses to JHA. Fold responses to JHA in control (gfp RNAi) and usp RNAi conditions are shown for each of the 33 genes that were activated by both USP and JHA. E. Few usp RNAi-responsive genes are regulated by diet quality. F. Fold change responses to diet quality and maturation are weakly correlated (978 genes that responded significantly to both). G. Fold change responses to usp RNAi and diet quality are uncorrelated (19 genes that responded significantly to both; genes that also responded to maturation are represented by closed circles).

Figure 4. USP binds to similar genomic locations in nurse and forager fat bodies.

The genomic binding sites of USP were assessed in 3 biological replicates each from nurse and forager fat bodies. A. Comparison of the intensity with which USP bound each of its 1360 binding sites in nurses and foragers. Normalized, log2-transformed fold differences in binding intensity and the significance of these differences (ANOVA) are shown. We observed no dramatic differences in USP binding at any of these sites between nurse and forager samples. B. Differences in USP binding between nurses and foragers were uncorrelated with the expression of nearby genes in nurses and forager fat bodies. Data are shown for 116 genes located near genomic binding sites of USP that had >1.25-fold difference in binding intensity between nurses and foragers. C. Fold change responses to usp RNAi and maturation are weakly correlated. Data are shown for the 29 genes that responded significantly to both. Each circle represents a single gene. Genes represented by closed circles also responded to juvenile hormone analog treatments.

Figure 5. cis-regulatory sequences predict behaviorally-related responses of USP targets.

A. The GRCACGCKVS motif enriched at USP binding sites (Figure 2C) matches a Juvenile Hormone Response Element (JHRE) recognized by MET (and other bHLH TFs) , . B. Predicted binding sites of USP and MET (the GGGGTCACS and GRCACGCKVS motifs, respectively) were consistently overlapping, with start positions located 3 bp apart. All adjacent pairs of Patser-predicted matches to these motifs in USP-binding loci (ChIP peaks) were considered; shown is the histogram of spacing between start positions of each pair, with negative numbers indicating that the MET site is on the left. Inset shows a zoomed-in view of the histogram for spacings in the range of 1–10 bp. C. Spacing constraints between sites of GGGGTCACS (USP) and potential cofactor motifs. Each of the 10 most significantly overrepresented motifs in USP-binding loci was tested for a constraint on spacing (< = 25 bp vs. >25 bp) between sites for USP and that motif. The Y-axis shows the −log10 of the p-value of this test, at five different statistical thresholds (X-axis) for defining motif matches.

Figure 6. Transcriptional co-expression analysis reveals that maturation involves overlapping, hormonally regulated gene modules in the fat bodies and brain.

A. CoherentCluster revealed 85 maturation-related “coherent” gene modules that were preserved between the fat bodies and brain (colored circles). These modules were enriched for genes that were differentially expressed between nurses in foragers in either the fat bodies, the brain, or both, as indicated by Venn Diagram categories. The number within each circle indicates the number of genes in the module; a bold outline indicates that the module contains at least one TF; underlined numbers indicate modules containing at least one usp-related gene (identified empirically by either RNAi or ChIP-chip experiments). Circles are colored based on their enrichment (P<0.05) for genes that are differentially expressed in response to usp RNAi, vg RNAi, or JHA. One module (f-15-2-1) is selected for demonstration (inset). Nodes in this inset represent individual genes, and edges indicate co-expression. Yellow nodes indicate genes that were responsive to (all three of) usp RNAi, vg RNAi, and JHA in the fat bodies. In addition, we show enriched Gene Ontology processes for genes within this module. B. Hormones regulate the expression of genes within many coherent modules. Left: contingency table for the number of coherent modules that were enriched for maturationally-regulated genes and for hormonally-regulated genes (USP targets, JHA-responsive genes, and Vg RNAi–responsive genes). Right: Proportion of coherent modules and of fat body-specific modules that were enriched for JHA-responsive genes and Vg RNAi-responsive genes.

Figure 7. Model for usp regulation of behavioral maturation.

According to this model, USP mediates responses to JH as part of a complex of proteins – pre-assembled at the promoters of JH-responsive genes – that likely includes USP, MET, EcR, Chd64, and FKBP39; all of these TFs have been shown to physically interact with one another in vitro , . Low JH titers (nurse bees) lead to target gene repression. High JH titers (foragers) cause target gene activation. This might occur via ligand-dependent conformational changes in the protein complex and recruitment of general transcriptional machinery, both of which are known for USP in other contexts (not shown). JH most likely binds MET, the only TF in this complex known to have strong affinity for JH . The model also suggests the presence of a feed-forward loop that stabilizes responses to JH; this role could be played by two components of the JH signaling complex – usp and Chd64 – themselves USP targets. Other TFs among USP targets – including TFs previously implicated in JH signaling such as E75 and Hr46 – are available to propagate these responses to indirect targets of USP and JH. This model can explain differential gene expression caused by both USP and JH despite the fact that USP was found to bind the same genomic locations in the fat bodies of nurses and foragers. It also is consistent with findings that these genomic locations are enriched for two very closely located (3 bp apart) cis-regulatory motifs, one recognized by USP and the other recognized by bHLH TFs, including MET.