Anna Haelens

Androgen receptor knockout and knock-in mouse models

Androgens play an important role in male reproductive development and function. These steroid hormones mediate their actions by binding to the androgen receptor (AR). Diseases such as androgen insensitivity syndrome, prostate cancer, Kennedys disease, and infertility can be caused by mutations in the AR. To get a better insight into the molecular working mechanisms of the AR, several knockout and knock-in mouse models have been developed. These models are reviewed here and are compared with human diseases.

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.

The first exon of the human sc gene contains an androgen responsive unit and an interferon regulatory factor element.

Secretory component (SC) plays a key role in the transport of IgA and IgM to the lumina of many glands. The gene is constitutively expressed, but can be modulated by hormonal and immunological stimuli. Recently, the promoter and the first exon of the human sc gene have been cloned. The first exon contains a putative androgen/glucocorticoid response element (ARE/GRE) and an Interferon Regulatory Factor Element (IRF-E). Here we show that the ARE/GRE can bind the DNA-binding domain (DBD) of both the androgen (AR) and glucocorticoid receptor (GR) with a preference for the AR-DBD. In transient transfection experiments, this element confers higher responsiveness to androgens than to glucocorticoids. The IRF-E can function as an IRF-2, but surprisingly not as an IRF-I responsive element. We postulate that these two regulatory elements play a key role in the complex regulation of the sc gene in vivo.

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

Androgen-regulated transcription in the epithelium of the rat lacrimal gland

Androgens are male sex hormones produced by the testes and, to a lesser extent, by the adrenals and ovaries. The responses evoked by androgens can be very diverse depending on the tissue under investigation: sexual accessory glands depend on androgens for organogenesis, maintenance, and cellular differentiation, whereas in other organs such as kidney, liver, salivary gland, and lacrimal gland, a more limited number of genes are influenced.1

Multitasking and Interplay Between the Androgen Receptor Domains

The androgen receptor, like the other nuclear receptors, consists of three canonical domains: the aminoterminal domain (NTD), the DNA-binding domain (DBD) and the ligand-binding domain (LBD). The flexible hinge between the DBD and LBD can also be considered as a separate entity. Each of these domains has multiple functions. The NTD harbors two interdependent transactivation functions Tau-1 and Tau-5, two SUMOylation sites that seem to control cooperativity of the AR, and an 23FQNLF27 motif that interacts with high affinity with the ligand-binding domain. The DBD is involved in the correct interactions of the AR with its response elements, but it also contains a nuclear export signal as well as a nuclear translocation signal. The hinge region controls the interactions of the AR with selective AREs. It harbors an acetylation and a phosphorylation acceptor site, overlaps with the nuclear translocation signal, and seems involved in the control of the steady state of the AR. The LBD binds its natural agonists with high affinity; it interacts with heat-shock protein complexes when unbound and with a series of coregulators when bound by agonists. Many of these coregulators harbor motifs that resemble the 23FQNLF27-motif of the NTD.