In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression.

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish <i>sox9b</i> has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of <i>sox9b</i> expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced <i>sox9b</i> expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (<i>slincR</i>) that is upregulated by strong AHR ligands and is located adjacent to the <i>sox9b</i> gene. We hypothesize that <i>slincR</i> is regulated by AHR2 and transcriptionally represses <i>sox9b.</i> The <i>slincR</i> transcript functions as an RNA macromolecule, and <i>slincR</i> expression is AHR2 dependent. Antisense knockdown of <i>slincR</i> results in an increase in <i>sox9b</i> expression during both normal development and AHR2 activation, which suggests relief in repression. During development, <i>slincR</i> was expressed in tissues with <i>sox9</i> essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of <i>slincR</i> resulted in altered neurologic and/or locomotor behavioral responses. Our results place <i>slincR</i> as an intermediate between AHR2 activation and the reduction of <i>sox9b</i> mRNA in the AHR2 signaling pathway.