Aryl hydrocarbon Receptor (AhR), a mediator of xenobiotic toxicity, is known to function as a ligand-activated transcription factor that binds to a xenobiotic response element (XRE, GCGTG motif) in association with its heterodimerization partner, the AhR nuclear translocator (Arnt) protein. This proposal is built on our observation that cinnabarinic acid (CA) mediated AhR-dependent induction of stanniocalcin 2 (stc2) attenuates stress induced apoptosis and protects against liver injury both in vitro and in vivo. stc2 induction was achieved in response to endogenous AhR agonist CA but not the classic exogenous AhR ligand 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Moreover, CA in contrast to TCDD, was unable to trigger prototypical AhR target gene, cyp1a1 in hepatocytes and did not regulate xenobiotic metabolism. Therefore, stc2 and cyp1a1 genes exhibit mutually exclusive agonist-specific AhR-mediated transcriptional responsiveness with distinct physiological consequences. As a part of my K01 research, we observed interaction of Metastasis-associated protein 2 (MTA2), a known chromatin remodeling protein, with AhR exclusively upon CA treatment. AhR-MTA2 complex was recruited only to stc2, but not cyp1a1 promoter. In addition, CA treatment was able to exhibit histone H4 lysine 5 acetylation specifically at stc2 promoter concomitant with AhR-MTA2 complex recruitment. Our preliminary studies identified that chromatin architecture in addition to the primary DNA sequence significantly contributes to the agonist-specific differential gene expression. Building on our preliminary studies this proposal tests the hypothesis that CA-specific AhR-dependent epigenetic modifications and chromatin restructuring dictates transcription regulation of stc2, which in turn is responsible for cytoprotection following liver injury.
Specific Aim 1 of this application will profile CA and TCDD specific epigenetic modifications as well as will identify the readers-writers-erasers of the histone modifications recruited to the XRE bounds AhR-MTA2 complex.
Specific Aim 2 will determine role of chromatin architecture in agonist specific stc2 regulation by using CRISPR/Cas9 gene editing.
Specific Aim 3 will interrogate mechanism by which CA-triggered stc2 protects against ethanol-induced apoptosis by identifying downstream cytoprotective pathway components activated by stc2. Therefore, the objective of this application is to decode CA-specific AhR-mediated transcription regulation of stc2 and decipher its cytoprotective function. Given stc2?s involvement in cytoprotection against liver injury, understanding its CA-specific regulation may serve as a key to developing future molecular therapeutics targeting hepatic diseases.
Endogenous and exogenous Aryl hydrocarbon Receptor agonists, cinnabarinic acid and dioxin exhibit mutually exclusive transcription regulation of target genes, stanniocalcin 2 and cyp1a1 respectively with distinct physiological consequences. Cinnabarinic acid induced Aryl hydrocarbon Receptor-dependent stanniocalcin 2 activation can protect against ER/oxidative stress induced hepatocyte apoptosis, steatosis and liver injury. The experiments outlined in this proposal are focused on deciphering Aryl hydrocarbon Receptor-mediated epigenetic regulation of stanniocalcin 2 by cinnabarinic acid as well as will interrogate downstream cytoprotective pathways regulated by stc2, which may prove instrumental in developing therapeutic strategies targeting hepatic diseases.
