DNA recognition by the androgen receptor: evidence for an alternative DNA-dependent dimerization, and an active role of sequences flanking the response element on transactivation

Abstract

The androgen receptor has a subset of target DNA sequences, which are not recognized by any other steroid receptors. The androgen selectivity of these sequences was proposed to be the consequence of the ability of the androgen receptor to dimerize on direct repeats of 5'-TGTTCT-3'-like sequences. This is in contrast with the classical non-selective elements consisting of inverted repeats of the 5'-TGTTCT-3' elements separated by three nucleotides and which are recognized by other steroid receptors in addition to the androgen receptor. We demonstrate that while the DNA-binding domain of the oestrogen receptor is unable to dimerize on direct repeats, dimeric binding can be rescued by replacing the second Zn finger and part of the hinge region by the corresponding fragment of the androgen receptor, but not the glucocorticoid receptor. In this study, we investigate the androgen receptor binding to all natural androgen-selective response elements described so far. We show that a 12-amino acid C-terminal extension of the DNA-binding domain is required for high-affinity binding of the androgen receptor to all these elements. For one androgen-specific low-affinity binding site, the flanking sequences do not contribute to the in vitro affinity of the androgen receptor DNA-binding domain. Surprisingly, however, they control the transcriptional activity of the androgen receptor in transient transfection experiments. In conclusion, we give evidence that the alternative DNA-dependent dimerization of the androgen receptor on direct repeats is a general mechanism for androgen specificity in which the second Zn finger and hinge region are involved. In addition, the sequences flanking an androgen-response element can control the activity of the androgen receptor.

Stepwise androgen receptor dimerization

“…Although amino acid residues involved in glucocorticoid receptor (GR) LBD-LBD interactions are conserved in AR (Centenera et al, 2008), in crystallographic studies, the isolated AR LBD is present as a monomer in solution, in contrast to GR, progesterone receptor (PR) and estrogen receptor (ER) LBDs (Bledsoe et al, 2002;Matias et al, 2000;Sack et al, 2001;Tanenbaum et al, 1998;Williams and Sigler, 1998). However, a (weak) dimerization function in the hinge region as suggested for GR, or in the C-terminal extension of the AR DBD cannot be completely excluded (Centenera et al, 2008;Haelens et al, 2003;Savory et al, 2001).…”

c-Jun enhancement of androgen receptor transactivation is associated with prostate cancer cell proliferation

“…This interaction, which may be intermolecular or intramolecular, is thought responsible for forming a transcriptional competent AR dimer (Langley et al, 1998). Interestingly, the dimerization of AR, like other nuclear receptors, is driven by the D box in the second zincfinger of the DNA-binding domain (Haelens et al, 2003). Other AR repressors include histone deacetylase 1 (HDAC1) (Korkmaz et al, 2004), Hey1 (Belandia et al, 2005), PIASg (Gross et al, 2004), silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) (Dotzlaw et al, 2002), and HBO1 (Sharma et al, 2000).…”

Dual Function of an Amino-terminal Amphipatic Helix in Androgen Receptor-mediated Transactivation through Specific and Nonspecific Response Elements

“…Plasmid Constructs-The expression vectors pSG5AR 538 -919 (encoding the hAR-DBD-LBD), pSG5AR (expressing FLAG-tagged fulllength hAR), and pSG5SRC-1e and the vector Qr SRC-1 (expressing the Qr domain of SRC-1 fused to the Gal4-DBD) are described elsewhere (14,18,39). The Gal4-DBD and the VP16 fusion constructs hAR , hAR 1-529 ⌬FQNLF, and hAR 1-529 L435P were made by a PCRbased method.…”

“…Gel Shift Assays and Western Blots-Synthetic complementary oligonucleotides were hybridized, radioactively labeled, and used in band shift assays as described previously (14). In brief, 5 l of total cell extract was preincubated with 1 l of poly(dI-dC) (1 g/l), 10 l of D100 (20 mM Hepes, 5 mM MgCl 2, 0.1 mM EDTA, 17% glycerol, 100 mM NaCl), 1 l dithiothreitol (20 mM), 1 l Triton X-100 (1%), and 1 l of water.…”

“…Selective DNA binding by the AR could be one possible mechanism for hormone-specific gene regulation. It has been reported that the AR-DBD does not only recognize response elements organized as inverted repeats of 5Ј-TGTTCT-3Ј sequences with a threenucleotide spacer but can also bind to direct repeats of 5Ј-TGTTCT-3Ј-like sequences, which is proposed to contribute to the AR specificity of transcriptional responses (7)(8)(9)(10)(11)(12)(13)(14). The NTD of the AR is indispensable for AR transactivation and contains a strong transcription activation function (AF-1).…”