Aip mutation leads to significant alterations in gene expression, altered sensitivity to Ahr ligands, and early lethality in zebrafish

Humans and other organisms are regularly exposed to chemical compounds that elicit toxicity through the aryl hydrocarbon receptor (AHR) signaling pathway. AHR agonists include polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and some natural products, pharmaceuticals, and endogenous compounds. Interindividual differences in sensitivity to AHR agonists exists. Several populations of Atlantic killifish (Fundulus heteroclitus) have independently evolved resistance to high levels of pollution. Genome-wide association studies suggested that variation in the AHR interacting protein (AIP) was associated with resistance to AHR ligands. AIP binds to AHR in the cytosol where it plays a role in stability and nuclear localization; however, its exact function is poorly understood. Two CRISPR-Cas9 generated aip mutant zebrafish (Danio rerio) lines were utilized. Homozygous mutants (aip^-/- ) from either line died at 7-10-days post-fertilization (dpf), while no such effect is observed in ahr mutant lines. Larvae were exposed to four toxic AHR ligands from diverse chemical classes. aip^-/- larvae exposed to PCB126, 5-nitroacenaphthene, and benzo(k) fluoranthene showed reduced sensitivity compared to both heterozygous (aip^+/- ) and wildtype (aip^+/+ ) larvae. However, changes in sensitivity were ligand-dependent. In contrast, aip^-/- larvae demonstrated increased sensitivity to leflunomide. mRNA sequencing was performed at five dpf on unexposed larvae of all genotypes from both mutant lines. Approximately 1,000 differentially expressed genes and alternative splicing products were observed in aip^-/- larvae. Gene sets and transcription factor binding motifs involved in cellular stress, apoptosis, innate immune response, proteolysis, replication, and metabolism were enriched. Single-cell deconvolution of the bulk RNA-seq results was performed using MuSiC, which estimated transcriptomic contributions from 28 different cell types, with significant shifts in transcriptional abundance estimated for cell types from structural tissues of mesodermal origin, neural tissues, immune cells, and metabolic organs. Our findings highlight the context-dependent nature of Aip in Ahr modulation and the critical roles that Aip plays in regulating cellular homeostasis.