Cross-species transcriptomic analysis elucidates constitutive aryl hydrocarbon receptor activity

Abstract

Background: Research on the aryl hydrocarbon receptor (AHR) has largely focused on variations in toxic outcomes resulting from its activation by halogenated aromatic hydrocarbons. But the AHR also plays key roles in regulating pathways critical for development, and after decades of research the mechanisms underlying physiological regulation by the AHR remain poorly characterized. Previous studies identified several core genes that respond to xenobiotic AHR ligands across a broad range of species and tissues. However, only limited inferences have been made regarding its role in regulating constitutive gene activity, i.e. in the absence of exogenous ligands. To address this, we profiled transcriptomic variations between AHR-active and AHR-less-active animals in the absence of an exogenous agonist across five tissues, three of which came from rats (hypothalamus, white adipose and liver) and two of which came from mice (kidney and liver). Because AHR status alone has been shown sufficient to alter transcriptomic responses, we reason that by contrasting profiles amongst AHR-variant animals, we may elucidate effects of the AHR on constitutive mRNA abundances.

Results: We found significantly more overlap in constitutive mRNA abundances amongst tissues within the same species than from tissues between species and identified 13 genes (Agt, Car3, Creg1, Ctsc, E2f6, Enpp1, Gatm, Gstm4, Kcnj8, Me1, Pdk1, Slc35a3, and Sqrdl) that are affected by AHR-status in four of five tissues. One gene, Creg1, was significantly up-regulated in all AHR-less-active animals. We also find greater overlap between tissues at the pathway level than at the gene level, suggesting coherency to the AHR signalling response within these processes. Analysis of regulatory motifs suggests that the AHR mostly mediates transcriptional regulation via direct binding to response elements.

Conclusions: These findings, though preliminary, present a platform for further evaluating the role of the AHR in regulation of constitutive mRNA levels and physiologic function.

Figure 1

Overview of significantly altered genes. The number of genes statistically associated with AHR-status (q < 0.05) in each individual tissue (A) and across multiple tissues (B). Variations in constitutive mRNA levels were visualized in a heatmap of log₂ fold changes, using significant genes common to at least two tissues (C).

Figure 2

Assessment of transcriptomic similarity. Hypergeometric testing was conducted on all pairs of tissues to identify similar transcriptomic profiles (A). Spot size represents the magnitude of the calculated gene enrichment ratio while the background shade denotes q-values obtained from hypergeometric testing. Genes not probed on the array are represented by “X”. Venn diagrams of commonly altered genes between all tissues (B), rat tissues (C) and liver tissues (D).

Figure 3

Transcriptomic profiles of exogenous and endogenous AHR activation. Transcriptional profiling of AHR core-response genes shows little association with AHR-status in the absence of exogenous ligands (A). By contrast, 13 genes were observed to significantly differ based on AHR-status between animals in at least four tissues, with most of these demonstrating higher levels in AHR-less-active animals (B). All fold changes shown are in log₂ scale, with the magnitude represented by spot size and q-values are denoted by background shade. Only rat gene names are shown. Genes are ordered alphabetically for AHR core-response genes (A) and by decreasing average absolute magnitude of change across studies for AHR constitutive genes (B).

Figure 4

Assessment of pathway similarity. Hypergeometric testing was conducted on all pairs of tissues to identify commonly enriched biological pathways (A). Magnitude of enrichment is represented by spot size while background shade represents q-values from hypergeometric testing. “X” denotes absence of significant enrichment. Assessment of common GO terms across all species and tissues revealed localized overlap (B). Three general ontologies, biological process, lipid metabolic process and molecular function, are represented among the ten commonly enriched pathways (C).

Figure 5

Evaluation of AHR binding: presence and effects. A higher fraction of genes that were significantly affected by AHR-status across multiple tissues were found to possess AHRE-I (Full) motifs in the upstream 5′-regulatory region, compared to genes that were AHR-status independent (A). At several count thresholds, the fractions of significant and non-significant genes were contrasted for the presence of both AHRE-I (Full) and AHRE-II motifs (B). Similar to findings of the transcription factor binding site analysis, a higher fraction of genes significantly altered across multiple tissues were found to exhibit AHR binding in vitro (C). The AHR appears to largely exert an upregulating effect on genes in rat liver in the absence of exogenous ligands, but a smaller fraction of downregulation is present as well (D). Genes that were differentially-abundant in the constitutive condition were also observed to be altered in greater proportions following TCDD-induced AHR activation in the mouse liver (E) and rat liver (F).