Since the discovery of the Aryl Hydrocarbon Receptor (AHR) 20 years ago, it is clear that this ligand activated transcription factor is intimately integrated with numerous signaling pathways that modulate biological processes. Exposure to AHR ligands profoundly impacts embryonic development, immune function, and promote tumor development. It has been implicated in multiple human diseases. Furthermore, selective AHR modulators are now being developed for therapeutic uses to suppress inflammation. Elucidating the complex events immediately downstream of AHR activation is critical to parse toxicity and improve therapeutic efficacy. The bulk of research into the downstream AHR targets has focused on mRNA induction, despite ample evidence that numerous transcripts are repressed by AHR activation. One key down-regulated target is sox9, a transcription factor that controls specification and differentiation of numerous progenitor and differentiated cell types. Sox9 haploinsufficiency causes a multitude of severe developmental problems in humans, but the mechanisms governing sox9 transcription remain unsolved. We and others have reported that TCDD leads to a rapid down regulation of sox9 transcription in an AHR dependent mechanism in developing mammals and fish, which is manifest through craniofacial and reproductive effects. Understanding the mechanisms of AHR- dependent transcriptional repression of sox9b has stymied us for six years, until we completed an unbiased deep RNA-seq study that identified a novel long non-coding RNA (lncRNA) transcript induced by developmental exposure to AHR ligands. This lncRNA is directly adjacent to the sox9b gene. Importantly the lncRNA-sox9b genomic relationship is conserved in mice and humans. We hypothesize a new adverse outcome pathway whereby AHR activation induces sox9b-lncRNA which in turn down-regulates sox9b transcription. Using AHR2-null fish, we confirmed that the sox9b-lncRNA induction is absolutely AHR2-dependent. We also identified AHR regulatory elements in the promoters of both zebrafish and mammalian lncRNA-sox9b genes. To test the hypothesis that the conserved sox9-lncRNA is a direct AHR target gene that upon AHR activation represses sox9b leading to target organ-specific toxicity, we will complete two Specific Aims: 1) We will determine the temporal and spatial expression of zebrafish sox9b-lncRNA relative to the sox9b mRNA, determine its binding to the sox9b promoter in vivo, and identify other potential targets of the sox9b-lncRNA by genome-wide analysis; 2) Using sox9b-lncRNA gain and loss-of-function studies, we will also define the role of sox9b-lncRNA in AHR-dependent developmental toxicity. Collectively, these studies are ideal for the R21 mechanism to build the new tools need to probe the role of lncRNAs in toxicity and develop a new approach for unraveling an unexpected layer of complexity affecting adverse outcome pathways.
There is increased evidence that some environmental contaminants that bind the aryl hydrocarbon receptor (AHR) produce birth defects in humans. This project explores a novel mechanism to explain how AHR receptor activation leads to changes in the expression of genes important for normal human development.
| Garcia, Gloria R; Bugel, Sean M; Truong, Lisa et al. (2018) AHR2 required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish. PLoS One 13:e0193484 |
| Garcia, Gloria R; Goodale, Britton C; Wiley, Michelle W et al. (2017) In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression. Mol Pharmacol 91:609-619 |
