Transcriptional regulation of the human aryl hydrocarbon receptor: Identification of multiple transactivation sub-domains and role of coactivators in modulating transactivation potential.

摘要

The Aryl hydrocarbon Receptor (AhR) is a ligand activated transcription factor which belongs to the bHLH-PAS protein superfamily and mediates many, if not all, of the toxic and biological effects of aromatic hydrocarbons which include the environmental contaminant, TCDD. The binding of AhR/ARNT to dioxin responsive elements leads to graded, localized chromatin remodeling of the CYP1A1 gene and binding of proteins to elements at the promoter and promoter proximal regions. Although, some aspects of the process of transcriptional activation are known, the details including recruitment of coactivators, and the subsequent or concomitant assembly of a pre-initiation complex are not elucidated.; This thesis describes the identification of potential coactivators of the AhR using a DRE-driven reporter assay. Coactivators, SRC-1 and RIP140 were found to interact with the hAhR via its transactivation domain. The mechanisms of interaction of the two co-factors appear to be distinct. Mapping of the site of interaction with AhR indicated that RIP140 interacts with AhR via regions distinct from those involved in nuclear receptor interactions. The LXXLL motifs in SRC-1 were necessary for both, physical interaction with the AhR and to mediate transcriptional activation of a DRE-driven reporter gene. This suggests a possible diversity in the mechanism of recruitment of coactivators to AhR. However, both coactivators interact with the AhR via the Q-rich sub-domain in vitro. Reporter gene assays using mutant Hepa-1 cells which lack ARNT, revealed that the ARNT transactivation domain is not required for SRC-1 coactivation.; Extensive mapping of the hAhR transactivation domain revealed that the Q-rich subdomain is critical for transactivation potential. In addition, the acidic sub-domain by itself did not appear to contribute to the hAhR transactivation potential. Deletion of the P/S/T-rich sub-domain appeared to have a stimulatory effect. Further characterization of the Q-rich sub-domain identified a 23 AA long stretch of amino acids in the Q-rich sub-domain between AA residues 666-688, which is required for optimal transactivation potential. Alanine scanning mutagenesis of this region revealed that a leucine residue (L678) is critical for hAhR transactivation potential.