Graduate Thesis Or Dissertation

 

Unraveling the endogenous functions of the aryl hydrocarbon receptor (AHR) and characterizing a novel long non-coding RNA (slincR) that contributes to AHR-mediated toxic responses in zebrafish Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/t435gk14c

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  • The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is a member of the basic helix-loop-helix PER-ARNT-SIM (bHLH/PAS) protein family of cellular sensors. The AHR is conserved across multiple animal phyla and is required for proper vertebrate development. A structurally diverse group of chemicals, including polycyclic aromatic hydrocarbons (PAHs), can inappropriately activate the aryl hydrocarbon receptor and lead to adverse developmental and cognitive effects in wildlife and humans. Increasing our knowledge of AHR-regulated biological processes can help foster a better understanding of the target organs and specific AHR-dependent mechanism that lead to negative health effects. In zebrafish, AHR2 and ARNT1 are the primary functional orthologs of mammalian AHR and ARNT. In this dissertation, we used the CRISPR-Cas9 genome editing method to generate and characterized an AHR2-null zebrafish line (ahr2osu1) to investigate the endogenous functional role of AHR2 in zebrafish behavioral responses, organ development, and reproduction. The ahr2osu1 line was resistant to TCDD-induced toxicity, demonstrating AHR2 is non-functional. The AHR2-null zebrafish exhibited decreased survival and fecundity compared to the wild type line. ahr2osu1 adults had malformed cranial skeletal bones and severely damaged fins. Both larval and adult ahr2osu1 zebrafish displayed abnormal behavioral responses when compared to the wild-type line. In summary, our study demonstrates that AHR2 is required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish. The AHR2-dependent signaling events that lead to toxic outcomes are poorly understood. In zebrafish, repression of sox9b has a causal role in several AHR2-mediated toxic responses, including craniofacial cartilage malformations; however, the mechanism of sox9b repression remains unknown. In this dissertation, we identified and characterized a novel long non-coding RNA called slincR that is upregulated by multiple strong AHR2 ligands (in an AHR2-dependent manner) and is located adjacent to the sox9b locus. slincR is 466 bp in length, contains 3 exons, and is localized to both the nucleus and cytoplasm. In support of the hypothesis that slincR regulates sox9b expression, we demonstrated that slincR is required for proper expression of sox9b and several of its downs stream targets. Additionally, slincR and sox9b are expressed in adjacent and overlapping tissues though out zebrafish development. We reviewed the literature and used the combination of qRT-PCR Capture Hybridization Analysis of RNA Targets, transcriptome profiling, and phenotypic analysis to develop the following hypothesis: AHR2 activation by a high affinity ligand (e.g. TCDD) results in an increase in slincR expression, slincR binds (directly or indirectly) to the 5’ untranslated region of the sox9b promoter to repress transcription of sox9b, which leads to decreases in the size and number of chondrocytes to produce malformed craniofacial cartilages. Our data also suggest slincR has a causal role in the TCDD-induced hemorrhaging phenotype and is upregulated by multiple environmentally relevant PAHs. Overall, the work presented here increases our understanding of the endogenous functions of AHR2 and enhances our mechanistic knowledge of how inappropriate AHR activation by environmental pollutants can lead to adverse health effects.
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  • Pending Publication
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  • 2017-12-12 to 2018-07-12

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