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

Abstract

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-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 sox9b 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 sox9b 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 sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway.

AHR2 required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish

“…In response to TCDD exposure, expression of cytochrome p450 cyp3a65 and slincR transcripts were significantly increased in the wild type line. AHR2 is required for the TCDD-induced increase of cyp3a65 and slincR transcripts, providing further support for the hypothesis that AHR2 is not functional in the new ahr2 osu1 line [ 40 , 41 ]. The expression of cytochrome p450s ( cyp1a , cyp1b , cyp1c1 ), ahhra , and ahrrb transcripts increased in both the TCDD-exposed wild type and mutant lines at 5 dpf when compared to the vehicle control; however, the wild type line had a significantly greater increase in expression compared to the AHR2-null line.…”

“…In response to TCDD, both the ahr2 + and ahr2 osu1 larval fish had a significant increase in the relative expression levels of cyp1a , cyp1b1 , cyp1c1 , ahrra , and ahrrb , in comparison to the vehicle-treated wild type; however, the wild type fish exposed to TCDD had a greater log 2 fold increase in expression of known AHR2 target genes ( Fig 3B ). In zebrafish, developmental exposure to TCDD causes an AHR2-dependent increase in cyp3a65 and slincR transcript levels and a decrease in sox9b mRNA expression [ 40 , 41 ]. Only the wild-type strain resulted in a significant increase in cyp3a65 and slincR expression and decrease in sox9b expression in response to TCDD exposure.…”

“…In zebrafish, repression of the transcription factor sox9b is hypothesized to have a causal role in TCDD-induced craniofacial cartilage malformations [ 28 ]. At 48 hpf, the repression of sox9b is mediated by the AHR2-dependent increase in expression of a long non-coding RNA called slincR that binds to the 5’ untranslated region of the sox9b promoter to repress transcription ([ 40 ] and Garcia and Tanguay (manuscript in preparation). To determine if AHR2 has a functional role in normal cartilage development, we stained and measured the cartilage of 5 dpf zebrafish.…”

Role of DNA methylation in altered gene expression patterns in adult zebrafish (Danio rerio) exposed to 3, 3’, 4, 4’, 5-pentachlorobiphenyl (PCB 126)

“…One such mechanism of sox9b regulation is by non-coding RNA. A recent study has demonstrated that a long intervening non-coding RNA (lincRNA; si: ch1073–384e4.1) regulates the expression of sox9b in zebrafish embryos [ 53 ]. We also observed significant upregulation of this lincRNA in response to PCB126 exposure (4.5 log FC; FDR 6.03E–12), suggesting multiple modes of action of AHR agonists in altering sox9b expression in zebrafish.…”

lncRNAs: function and mechanism in cartilage development, degeneration, and regeneration

“…Aryl hydrocarbon receptor (AHR) is a conserved receptor from invertebrates to vertebrates, loss of which will protect against toxicity phenotypes, including cardiac malformation, cartilage malformation, and reduced peripheral blood flow [42,43]. Garcia et al [44] found that a novel lncRNA named slincR is associated with AHR2 and sox9b expression during normal development. LncRNA slincR acts as an intermediate between AHR2 and sox9b mRNA.…”