Characterization of Aryl Hydrocarbon receptor and Kruppel-Like-Factor-6 interaction
Pereira De Castro, Karen Larissa
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The aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor which regulates toxic response to environmental pollutants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Upon ligand activation, the AhR translocates to the nucleus, dissociates from its chaperone complex, and dimerizes with the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT). The canonical AhR-ARNT complex binds to DNA regions known as Xenobiotic Response Elements (XRE), inducing the transcription of target genes. Our previous results demonstrated that the AhR is also involved in a novel protein-DNA complex with the Kruppel Like Factor 6 (KLF6), a zinc finger that is involved in processes such as angiogenesis, cell proliferation, and apoptosis. The AhR-KLF6 heterodimer functions differently from the ARNT-AhR complex by binding to a non-consensus XRE (NC-XRE) in a ligand-dependent manner. Using full length proteins as well as a series of point mutation and deletion proteins produced in vitro, we demonstrated by electrophoretic mobility shift assays (EMSAs) that the glutamine rich region (Q-rich) of AhR is crucial for protein-DNA binding. We identified the specific residues in the AhR Q-rich domain responsible for protein DNA-binding. We also identified the key amino acids of KLF6 and the regions of AhR that are responsible for protein-protein interaction. We have applied ChIP-seq (Chromatin Immunoprecipitation–Sequencing) and RNA-sequencing to correlate DNA binding events with changes in transcriptomics in the liver of mice exposed to TCDD, linking AhR activation by TCDD with biological functions such as regulation of cell death, apoptotic process, cellular proliferation and response to xenobiotic stimulus.