Research Resource: A Reference Transcriptome for Constitutive Androstane Receptor and Pregnane X Receptor Xenobiotic Signaling

Abstract

The pregnane X receptor (PXR) (PXR/NR1I3) and constitutive androstane receptor (CAR) (CAR/NR1I2) members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors are well-characterized mediators of xenobiotic and endocrine-disrupting chemical signaling. The Nuclear Receptor Signaling Atlas maintains a growing library of transcriptomic datasets involving perturbations of NR signaling pathways, many of which involve perturbations relevant to PXR and CAR xenobiotic signaling. Here, we generated a reference transcriptome based on the frequency of differential expression of genes across 159 experiments compiled from 22 datasets involving perturbations of CAR and PXR signaling pathways. In addition to the anticipated overrepresentation in the reference transcriptome of genes encoding components of the xenobiotic stress response, the ranking of genes involved in carbohydrate metabolism and gonadotropin action sheds mechanistic light on the suspected role of xenobiotics in metabolic syndrome and reproductive disorders. Gene Set Enrichment Analysis showed that although acetaminophen, chlorpromazine, and phenobarbital impacted many similar gene sets, differences in direction of regulation were evident in a variety of processes. Strikingly, gene sets representing genes linked to Parkinson's, Huntington's, and Alzheimer's diseases were enriched in all 3 transcriptomes. The reference xenobiotic transcriptome will be supplemented with additional future datasets to provide the community with a continually updated reference transcriptomic dataset for CAR- and PXR-mediated xenobiotic signaling. Our study demonstrates how aggregating and annotating transcriptomic datasets, and making them available for routine data mining, facilitates research into the mechanisms by which xenobiotics and endocrine-disrupting chemicals subvert conventional NR signaling modalities.

Figure 1.

Chemical structures of CAR and PXR xenobiotic perturbants in transcriptomic datasets used to generate the reference transcriptome. CAR and PXR have overlapping specificity for Acmphn, Phenob, Chlorprom, and PCN, whereas TCPOBOP and CITCO are specific for CAR.

Figure 2.

Transcriptomine data processing and metadata biocuration pipeline. GEO, Gene Expression Omnibus; AE, ArrayExpress. Datasets were processed and annotated for inclusion in the NURSA database using a previously described protocol (11, 12). Experimental contrasts were annotated for RNA Source and regulatory molecule with reference to the original publication accompanying the dataset, and fold changes were crosschecked to ensure faithful recapitulation of the data points reported in the original manuscript.

Figure 3.

PANTHER GO biological process enrichment analysis identifies numerous cellular processes impacted by xenobiotic signaling. Biological processes enriched 9-fold or higher are shown (Supplemental Dataset 3 contains the full list of categories). The PANTHER Overrepresentation test identifies statistically significant over- or underrepresentation of genes mapped to a specific biological process in a user's gene list with that of a reference gene list representing the universe of genes from the appropriate species.

Figure 4.

GSEA heat map identifies convergence and divergence in the Acmphn, Phenob, and Chlorprom transcriptomes. Using the Transcriptomine API, we retrieved significantly differentially expressed genes for experiments in which Acmphn, Chlorprom, and Phenob were perturbants. To suppress differences between each of the treatments at the level of individual genes, we first represented each experiment as a collection of enriched pathways, based on GSEA and a collection of over 1900 MSigDB pathways, then used ANOVA parametric testing (P < .01) to determine significant pathways.