Coactivator Interactions at the Transcription Activation Units of the Androgen Receptor

Abstract

The androgen receptor (AR) belongs to the family of nuclear receptors (NRs). These NRs are ligand-inducible transcription factors that are directed to their target genes through an interaction of their DNA-binding domain with specific sequences in the DNA. A large number of coactivators is sequentiallyrecruited by the NRs via a coactivator-binding groove at the surface of their ligand-binding domain (LBD). Although the LBD of the AR is structurally very similar to that of other NRs, its activation function (called AF-2) is very weak, a characteristic that correlates with a strong interaction between theamino-terminal domain and the coactivator-binding groove. While the DNA-binding and the ligand-binding domains are strongly conserved within the complete family of nuclear receptors, the amino-terminal domain (NTD) is very divergent both in length and in sequence. The amino-terminal domains of the steroid receptors in particular are known to cover strong activation functions, but the mechanisms behind the activation remain much less clear compared to that of the ligand-binding domain. Indeed,the coactivator mechanisms of this domain do not seem to be conserved even between different steroid receptors. For the AR, the NTD is about 550 amino acids long and contains two transcription activation units, Tau-1 and Tau-5, which can work independently of each other as activation domains. For Tau-5, we and others described an interaction with p160 coactivators, not via their LxxLL motifs, but via aglutamine rich (Qr) region (Callewaert et al. 2006). In the first part of this study we analyzed the interactions between Tau-5 and the p160s. The boundaries and key characteristics of both the Tau-5 and the p160 Qr fragment were determined to delineate the specific interaction surface. Since both domains separately were difficult to express inprokaryotes, we aimed to purify the complex of Tau-5 with SRC1-Qr. Furtheroptimization is needed before the protein quantities will be sufficient forfurther structural analyses. In the second part of this work, we studied Tau-1, and focused on identifying its coactivator(s). The copurification of Tau-1 interacting proteins revealed interactions of HSP70-1a. We are convinced that this reflects the hydrophobic nature of the isolated AR-fragment that was used in the copurification assay, although a role of HSP70 in the action mechanisms of Tau-1 cannot be excluded.We also present the Transcription Intermediary Factor 1 ß (TIF1ß) as a Tau-1 coactivator. This factor can act both as a positive and a negative regulator for different transcription factors. We can show an interaction between TIF1ß and the AR and demonstrate that its coactivation depends on thepresence of Tau-1. Strong physiological evidence comes from the observation that a mutation in the Tau-1 domain found in an AIS patient destroys the coactivation by TIF1ß almost completely, indicating that the interaction also plays an important role in vivo. Compared to Tau-5, Tau-1 is less conserved, especially in Danio rerio, but we think this is linked with the fact that TIF1ß is lost in the Clupeocephala, where Danio rerio belongs to. A structure-function analysis of TIF1ß shows that the domains involved in its action as a corepressor are different from those involved in coactivation of the AR. In conclusion, we describe TIF1ß as a new coactivator for Tau-1. Since we demonstrate its expression in prostate cells, this AR-TIF1ß axis could become a a new target for prostate cancer therapy.status: publishe