Identification of novel androgen receptor (AR) coactivator complexes and a molecular examination of N-terminal glutamine repeat polymorphisms on AR function
|University of Maryland, Baltimore. Molecular Medicine. Ph.D. 2002
|The androgen receptor (AR) is a ligand-activated transcription factor that regulates the expression of genes involved in differentiation, development and maintenance of male reproductive functions. A major obstacle for studies addressing AR function and identifying physiologically relevant AR coregulatory factors is the lack of an appropriate cell model system. In this study, we generated a human cell line (termed E19) that conditionally expresses a tetracycline-regulated FLAG epitope-tagged human AR that is transcriptionally active in vivo . Using the FLAG-tag as a means of immunopurification, we show that AR can be isolated in association with coactivators containing histone acetyltranferase (HAT) activity (RAC 3 and CBP), and with a large multisubunit coactivator complex that directly interacts with the basal transcriptional apparatus (i.e., the TRAP/Mediator complex). Consistent with a TRAP/Mediator coactivator role in AR-mediated transcription, overexpression of several TRAP subunits enhances ligand-dependent transcription by AR in transient transfection experiments. We further show that AR can directly interact with one of the TRAP/mediator subunits (TRAP220) possibly implicating this subunit as targeting the holocomplex to AR. Taken together, these data suggest a multistep pathway of AR-regulated gene expression involving the recruitment of both chromatin modifying enzymes and cofactors that directly interact with the basal apparatus. Finally, we examined the molecular consequences of a pathologically-associated polymorphic glutamine (Gln) repeat region found at the N-terminal end of the human AR gene. Using prostate cancer cell lines transiently and/or stably transfected with AR Gln-repeat variants, we found that AR transcriptional activity increases as the number of Gln repeats decreases. Whereas DNA binding activity appears to be unaffected by variation in the Gln repeats region, we found AR containing short Gln repeats has a higher ligand binding affinity than wild-type AR. More importantly, we found that a specific ligand-induced intramolecular interaction between AR's N-terminal and C-terminal domains is enhanced in AR proteins containing shorter Gln repeats. Given that this AR-intramolecular interaction is believed to generate a binding surface for AR-coregulatory factors, our results suggest that decreasing length of the Gln repeats may play a molecular role in promoting cofactor binding to AR.
|Identification of novel androgen receptor (AR) coactivator complexes and a molecular examination of N-terminal glutamine repeat polymorphisms on AR function
|Fondell, Joseph D.