The overall goal of this research proposal is to determine if a novel long non-coding RNA (lncRNA) serves as an upstream molecular target that connects aryl hydrocarbon receptor (AHR) signaling to tissue specific toxicity phenotypes. Using the zebrafish model, we discovered a lncRNA that is upregulated (in an AHR-dependent manner) in response to several strong AHR ligands. The lncRNA mapped directly adjacent to the Sox9b (ortholog for human Sox9) locus; hence, we named it Sox9b-lncRNA. Sox9b has been shown to be one of the most reduced transcripts in three different AHR tissue specific toxicity endpoints, and its misexpression has been correlated with multiple human diseases. Additionally, we have identified a putative lncRNA binding site in the Sox9b promoter, and the genomic spatial orientation of the lncRNA relative to Sox9b is conserved in humans and mice. Furthermore, antisense knockdown of the lncRNA in embryos exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, the classic AHR ligand) have increased expression of Sox9b when compared to controls. The preliminary and published data have led to the central hypothesis: Upon AHR activation, the conserved Sox9b-lncRNA binds to the Sox9b promoter resulting in its transcriptional repression, which leads to impaired morphogenesis and growth of the cranial cartilage. We decided to focus on the AHR-induced craniofacial malformation because Sox9b repression is both necessary and sufficient for generating this phenotype. We will test the central hypothesis of the proposal through the following aims:
Aim 1 : Determine the role of Sox9b-lncRNA in impaired morphogenesis and growth of the cranial cartilage of zebrafish embryos exposed to a strong AHR ligand Aim 2: Determine the mechanism by which Sox9b-lncRNA represses Sox9b in AHR signaling For the following high throughput assays, embryos will be exposed to TCDD or vehicle (DMSO) and cartilage cells will be isolated utilizing a transgenic reporter fish driven by a cartilage specific promoter and a MoFlo high speed cell sorter to produce a purified population of cartilage cells. Capture Hybridization Analysis of RNA Targets Sequencing (CHART-Seq) will be used to determine the Sox9b-lncRNA genome wide binding sites. The impact of Sox9b-lncRNA expression on the transcriptome will be evaluated by performing RNA-Seq from wild type and Sox9b-lncRNA-knockdown samples. The Sox9b-lncRNA regulatory network will be inferred via advanced computational data integration of the CHART-Seq and RNA-Seq data. A sub-set of Sox9b-lncRNA targets will be validated via quantitative RT-PCR. The molecular analysis will be anchored to detailed observations at the structural and functional level, and lower jaw morphometrics will be quantitatively measured. The mechanism by which Sox9b-lncRNA represses Sox9b will be investigated using spatial and quantitative reporter assays, in vitro RNA/dsDNA interaction assays, and histone/promoter co-immunoprecipitation assays.
Inappropriate activation of the aryl hydrocarbon receptor (AHR) produces a diverse number of physiological consequences that negatively impact human health; however, the downstream molecular events that link AHR signaling to tissue-specific toxicity phenotypes are not well defined. This project seeks to unravel the functional impact of AHR activation on a novel long non-coding RNA (Sox9b-lncRNA) that is elevated in multiple AHR toxicity target organs. The focus on Sox9b as the primary lncRNA target is highly relevant to toxicology and human health because this gene is known to be suppressed by AHR activation, and its misexpression has been correlated with multiple human diseases.
|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|
|Garcia, Gloria R; Noyes, Pamela D; Tanguay, Robert L (2016) Advancements in zebrafish applications for 21st century toxicology. Pharmacol Ther 161:11-21|
|Noyes, Pamela D; Garcia, Gloria R; Tanguay, Robert L (2016) ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN. Green Chem 18:6410-6430|