Leen Callewaert

The Hinge Region Regulates DNA Binding, Nuclear Translocation, and Transactivation of the Androgen Receptor

The androgen receptor (AR) encoding gene can undergo mutations during the development and treatment of prostate cancer. Even in hormone-independent stages, mutations in the receptor paradoxically seem to result in an increased AR function. Two such point mutations have been described in the part of the AR involved in DNA binding and nuclear translocation, namely the hinge region. Despite a decreased nuclear translocation, these mutant ARs display increased transactivating potencies. Through detailed analysis of the hinge region, we found that deletion of residues 629 to 636 resulted in a stronger androgen response on different reporters, although this mutant displays an extremely low in vitro affinity for androgen response elements. This superactivity is independent of nuclear localization and can be inhibited by antiandrogens. Surprisingly, the AR activation functions, AF1 and AF2, are not dramatically affected when the inhibitory region (629-RKLKKLGN-636) is deleted, although cotransfected p160 coactivator TIF2 had a stronger potentiating effect in the absence of this motif. The ligand-dependent interaction between the amino-terminal domain and the ligand-binding domain (N/C interaction) plays an important role in transactivation by the AR. We found that this interaction is strongly enhanced by deletion of the inhibitory region. In conclusion, the description of prostate cancer mutations has led to the discovery of a complex role of the hinge region in nuclear localization, DNA binding, coactivator recruitment, and N/C interaction of the AR.

Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model

Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction.

Interplay between two hormone-independent activation domains in the androgen receptor.

The androgen receptor (AR) plays a key role in prostate cancer development, as well as its treatments, even for the hormone-refractory state. Here, we report that an earlier described lysine-to-arginine mutation at position 179 in AR leads to a more potent AR. We show that two activation domains (Tau-1 and Tau-5) are necessary and sufficient for the full activity of AR and the intrinsic activity of the AR-NTD. Two alpha-helices surrounding the Lys179 define the core of Tau-1, which can act as an autonomous activation function, independent of p160 coactivators. Furthermore, we show that although the recruitment of p160 coactivators is mediated through Tau-5, this event is attenuated by core Tau-1. This better definition of the mechanisms of action of both Tau-1 and Tau-5 is instrumental for the design of alternative therapeutic strategies against prostate cancer.

Implications of a polyglutamine tract in the function of the human androgen receptor.

The androgen receptor (AR) is a ligand-dependent transcription factor and belongs to the nuclear receptor family. The AR gene contains a long polymorphic CAG repeat, coding for a polyglutamine tract. In the full size AR, the deletion of the polyglutamine tract results in an increase in the transactivation through canonical AREs. However, this effect is clearly dependent on the response elements, since it is not observed on selective elements. In our assays, a deletion of the repeat positively affected the interactions of the ligand-binding domain with the amino-terminal domain as well as the recruitment of the p160 coactivator SRC-1e to the amino-terminal domain of the AR. This is reflected by an enhanced coactivation of the AR by SRC-1e.

Characterization of the Two Coactivator-interacting Surfaces of the Androgen Receptor and Their Relative Role in Transcriptional Control*

The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain. The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s. We here describe the implication of two conserved motifs in the latter interaction. The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain. Both domains are, however, implicated in the recruitment of the p160s. Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested. We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer.

Detailed functional studies on androgen receptor mild mutations demonstrate their association with male infertility.

Context   Mutations in the androgen receptor (AR) gene can cause the androgen insensitivity syndrome (AIS). For complete and severe partial AIS, well‐characterized in vitro functional assays can be used for genotype–phenotype correlation; however, for mild forms of AIS, as associated with male infertility, experimental evidence is scarce or lacking. In particular, optimal in vitro functional tests informative about the genotype–phenotype relation have not been described.

The androgen receptor DNA-binding domain determines androgen selectivity of transcriptional response

The AR (androgen receptor) is a hormone-dependent transcription factor that translates circulating androgen hormone levels into a physiological cellular response by directly regulating the expression of its target genes. It is the key molecule in e.g. the development and maintenance of the male sexual characteristics, spermatocyte production and prostate gland development and growth. It is also a major factor in the onset and maintenance of prostate cancer and a first target for pharmaceutical action against the further proliferation of prostate cancer cells. The AR is a member of the steroid hormone receptors, a group of steroid-inducible transcription factors sharing an identical consensus DNA-binding motif. The problem of how specificity in gene activation is achieved among the different members of this nuclear receptor subfamily is still unclear. In this report, we describe our investigations on how the AR can specifically activate its target genes, while the other steroid hormone receptors do not, despite having the same consensus monomeric DNA-binding motif. In this respect, we describe how the AR interacts with a newly identified class of steroid-response elements to which only the AR and not, for example, the glucocorticoid receptor can bind.

Molecular biology of the androgen responses

The androgen receptor is a ligand-inducible transcription factor with very specific target genes. This definition implies the activation by the cognate ligand through the ligand-binding domain, the recognition of the target genes by means of the DNA-binding domain and the transcriptional activation through different activation functions. When the first androgen-responsive genes were cloned, we identified receptor-binding sites by means of a DNAcellulose competition assay with partially purified androgen receptor from rat prostate (Claessens et al., 1990). Once the receptor cDNA was cloned, the separate DNAbinding domain was expressed and shown to have similar, if not identical DNA recognition properties as the full size receptor. The binding sites were proven functional in transient transfection experiments with reporter genes cloned downstream of these sites (Claessens et al., 1993). The motifs which are recognized by the receptor are called androgen response elements (ARE), and a consensus of the first identified AREs pointed out that it is very similar to the glucocorticoid/progesterone response element (GRE/PRE) consensus 5¢-GGTACAnnnTGTTCT-3¢. Not surprisingly, these AREs also act as GRE/PRE in transient transfections. The probasin promoter region also contains two AR-binding sites, but in contrast to what was observed for the earlier AREs, these are not recognized by the glucocorticoid receptor. Later on, several other selective AREs were characterized in the slp and sc enhancers (Verrijdt et al., 2000). A comparison of the DNA-binding domains of the androgen and glucocorticoid receptors revealed specific residues which are involved in the recognition of these selective AREs, but not in the recognition of the classical AREs. These residues are not situated within the first zinc-coordinated module or zinc finger, but rather in the second one, as well as in a carboxy-terminal extension of the DNA-binding motif (Schoenmakers et al., 2000). This hinted to us that the recognition of the selective AREs occurs through an alternative dimerization of the DNA-binding domain that would be specific for the androgen receptor. Indeed, when the direct repeat nature of the selective AREs was changed into inverted repeat nature, the selectivity of the AREs and of the enhancers, of which they form part, was lost (Verrijdt et al., 2000). The silico screening of human genome has led to the definition of several additional selective AREs. In collaboration with the group of Daniel Gewirth, we were able to solve a crystal structure of the DNA-binding domain of the androgen receptor complexed to a perfect direct repeat of the 5¢TGTTCT-3¢ hexamer (Shaffer et al., 2004). This revealed that the domain is folded into two zinc-coordinated modules very similar to what has been described for other nuclear receptors. The two monomers are organized in a head-to-head configuration. Specific for the androgen receptor is the increased strength of the dimerisation interface due to an enlarged contact surface as well as to three additional hydrogen bonds. A functional analysis of the carboxyterminal extension of the DBD, which is part of the hinge region, revealed that it has more functions besides contributing to selective DNA binding. It overlaps with part of a nuclear localization signal and it is involved in the control of transactivation. Indeed, opposite to what is expected, deletions within this region result in a superactive androgen receptor, even when DNA binding in band shifts becomes difficult to demonstrate. The transcription activation by the androgen receptor is complex in the sense that different domains are contributing to it. For all steroid receptors, two activation functions have been described: the activation function 1 (AF1) in the amino-terminal domain and activation function 2 (AF2) in the ligand-binding domain. The androgen receptor is an exception since the AF2 is weak and in most experiments difficult to demonstrate. A possible explanation for this was found in a strong interaction between the ligand-binding domain and the amino-terminal domain of the androgen receptor. This occurs through a motif at the amino-terminal end of the receptor that interacts with AF2, described as a hydrophobic cleft on the surface of the ligand-binding domain. This interaction seems to prevent recruitment of the known p160 co-activators to