Inhibition of Androgen Receptor-Mediated Transcription by Amino-Terminal Enhancer of split

A yeast two-hybrid assay has identified an androgen-dependent interaction of androgen receptor (AR) with amino-terminal enhancer of split (AES), a member of the highly conserved Groucho/TLE family of corepressors. Full-length AR, as well as the N-terminal fragment of AR, showed direct interactions with AES in in vitro protein-protein interaction assays. AES specifically inhibited AR-mediated transcription in a well-defined cell-free transcription system and interacted specifically with the basal transcription factor (TFIIE) in HeLa nuclear extract. These observations implicate AES as a selective repressor of ligand-dependent AR-mediated transcription that acts by directly interacting with AR and by targeting the basal transcription machinery. Androgen receptor (AR) is a member of the superfamily of ligand-inducible transcription factors and mediates the biological actions of androgens (19). Like other superfamily members, AR contains a central DNA-binding domain, a C-terminal ligand-binding domain with an associated AF-2 activation domain, and a large N-terminal region containing the AF-1 activation domain (4, 26). Nuclear receptors regulate the transcription of their target genes through the agency of various coactivators and corepressors that are recruited to target genes through interactions with promoter-bound receptors (56). Many of the known coactivators for nuclear receptors contain histone acetyltransferase activities and are thought to act mainly through targeted chromatin structural perturbations that facilitate the subsequent recruitment (to the promoter) and function of other transcriptional coactivators and basal transcriptional factors (3). Transcriptional corepressors, by contrast, mediate repression by various nuclear receptors. Some nuclear receptors (including retinoid receptor, thyroid hormone receptor, vitamin D receptor, and certain orphan receptors) that are not associated with heat shock proteins in their unliganded state repress transcription by recruitment of corepressor complexes (15, 35). Corepressor complexes contain histone deacetylase (HDAC) activities that maintain chromatin in a configuration that excludes functional interactions of the general transcriptional machinery with the promoter. In contrast, unliganded steroid receptors (including AR) generally associate with heat shock proteins and, upon ligand binding, dissociate from the heat shock proteins, translocate to the nucleus, and associate with coactivators to activate or repress target genes (30). Another type of corepressor, implicated in the function of other types of repressors, is the Groucho/TLE family (see Fig. 1D) (5, 11). The larger family members such as Drosophila Groucho and its mammalian homologues, the TLE proteins (transducin-like enhancer of split [TLE1-3]), share five domains. A carboxyl-terminal WD-40 repeat domain (WD-40) and an amino-terminal glutamine-rich domain (Q) are highly conserved. In the much less well-conserved central region, there is a loosely conserved CcN motif (CcN), consisting of putative cdc2 kinase and casein kinase II phosphorylation sites, and two poorly conserved regions (GP and SP) that are characteristically rich in either glycine and proline (GP) or serine and proline (SP) residues. A shorter family member, human TLE4, is similar except for the absence of the amino-terminal Q and GP domains. The shortest family member, amino-terminal enhancer of split (AES), shares only the first two regions of the amino terminus. The Q domain mediates both homo- and hetero-oligomerization between Groucho/TLE family proteins, whereas the WD-40 repeats appear to mediate protein-protein interactions with relevant DNA-binding activators and repressors. Groucho/TLE proteins do not have recognizable DNA-binding domains but can repress transcription directly if tethered to DNA through a Gal4 DNA-binding domain or if recruited to DNA through interactions with other DNA-binding activators and repressors. The function of AES remains controversial. It was suggested that AES might act as an inhibitor of Groucho/TLE corepressors by dominant negative mechanisms (28, 45). On the other hand, AES has been shown to mediate Blimp-1dependent repression of the beta interferon gene (41) and to repress NF-kB-driven gene expression (51) in vivo. Here we demonstrate that AES physically interacts with human AR both in vivo and in vitro and that it represses AR-dependent transcription both in transient-transfection assays and in a purified cell-free transcription system. In addition, we find that AES interacts selectively with the basal transcription factor TFIIE. These observations indicate that AES represses AR-driven transcription by directly targeting the basal transcription machinery.