Guy Verrijdt

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.

Comparative analysis of the influence of the high-mobility group box 1 protein on DNA binding and transcriptional activation by the androgen, glucocorticoid, progesterone and mineralocorticoid receptors

We performed a comparative analysis of the effect of high-mobility group box protein 1 (HMGB1) on DNA binding by the DNA-binding domains (DBDs) of the androgen, glucocorticoid, progesterone and mineralocorticoid receptors. The affinity of the DBDs of the different receptors for the tyrosine aminotransferase glucocorticoid response element, a classical high-affinity binding element, was augmented up to 7-fold by HMGB1. We found no major differences in the effects of HMGB1 on DNA binding between the different steroid hormone receptors. In transient transfection assays, however, HMGB1 significantly enhances the activity of the glucocorticoid and progesterone receptors but not the androgen or mineralocorticoid receptor. We also investigated the effect of HMGB1 on the binding of the androgen receptor DBD to a subclass of directly repeated response elements that is recognized exclusively by the androgen receptor and not by the glucocorticoid, progesterone or mineralocorticoid receptor. Surprisingly, a deletion of 26 amino acid residues from the C-terminal extension of the androgen receptor DBD does not influence DNA binding but destroys its sensitivity to HMGB1. Deletion of the corresponding fragment in the DBDs of the glucocorticoid, progesterone and mineralocorticoid receptor destroyed their DNA binding. This 26-residue fragment is therefore essential for the influence of HMGB1 on DNA recognition by all steroid hormone receptors that were tested. However, it is dispensable for DNA binding by the androgen receptor.

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.

Influence of nucleophosmin/B23 on DNA binding and transcriptional activity of the androgen receptor in prostate cancer cell.

The promotion and progression of prostate cancer (PCa) are associated with androgen receptor (AR) signalling. AR functions are modulated by a variety of co-factors amongst which we identified the nucleophosmin (NPM/B23), a member of the histone chaperone family. Here, we show that NPM is overexpressed in PCa compared to normal adjacent tissues. AR and NPM interact in vitro and in vivo, and NPM is critical for androgen-dependent transcriptional activation in LNCaP cells as an anti-NPM siRNA downregulates transcription of a transfected androgen response element (ARE)-containing reporter promoter as well as expression of the endogenous androgen responsive prostate-specific antigen (PSA) gene. By investigating the effect of NPM on AR, we have also observed that NPM enhances AR binding to an ARE in vitro in electrophoretic gel mobility-shift assay experiments. Chromatin immunoprecipitation studies further demonstrated that both AR and NPM associate with AREs of the PSA gene in vivo. Altogether, our data suggest that the molecular histone chaperone NPM could regulate AR functions by promoting assembly of AR-containing regulatory complexes and that high levels of NPM might alter AR functions in PCa.

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.

Functional Interplay between Two Response Elements with Distinct Binding Characteristics Dictates Androgen Specificity of the Mouse Sex-limited Protein Enhancer

Many of the aspects involved in steroid-specific transcriptional regulation are still unsolved to date. We describe here the detailed characterization of the mouse sex-limited protein enhancer as a paradigm for androgen-specific control of gene expression. By deletion analysis, we delineate the minimal enhancer region displaying androgen sensitivity and specificity. We also show that each of the three hormone response elements (HRE), which constitute this minimal enhancer region, is essential but not sufficient for its functionality. When investigated as isolated elements, HRE1 is inactive and HRE3 is a potent androgen response element as well as GRE. Only the non-canonical HRE2 (5-TGGTCAgccAGTTCT-3′) is capable of conferring an androgen-specific transcriptional response to a heterologous promoter. This finding is correlated with the fact that HRE2 is recognized in binding assaysin vitro by the DNA-binding domain (DBD) of the androgen but not the glucocorticoid receptor, while HRE3 is recognized by both DBDs. Differential binding of the androgen receptor to HRE2 in the context of the enhancer was analyzed in more detail in footprinting assays in vitro. In transient transfection experiments using chimeric receptors, the inability of the glucocorticoid receptor to transactivate via the slp-ARU as well as the isolatedslp-HRE2 was rescued by the replacement of its DNA-binding domain with that of the androgen receptor. Our data suggest that the functional interplay between the weak, but highly androgen-specific HRE2 and the adjacent strong, but non-selective HRE3 is the major determinant in the generation of androgen specificity of transcriptional response via the sex-limited protein enhancer.

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.

Anti-androgenic properties of Compound A, an analog of a non-steroidal plant compound

We investigated the interactions between Compound A (CpdA), an analog of a hydroxyphenyl aziridine precursor found in an African shrub, and the androgen receptor (AR). CpdA represses androgen-induced activation of both specific and non-specific androgen DNA response elements. While a similar effect was obtained for the progesterone receptor (PR) via a non-specific hormone response element, CpdA had no effect on the actions of the glucocorticoid and mineralocorticoid receptors. CpdA represses the ligand-dependent interaction between the NH(2)- and COOH-terminal domains of the AR, similar to well-characterised anti-androgens. CpdA also interferes with the interaction of steroid receptor co-activator 1 (SRC1) with the activation domain AF2 but not with AF1. However, CpdA does not compete with androgen for binding to the AR. These results demonstrate that CpdA elicits anti-androgenic actions by a mechanism other than competitive binding for the AR.

Transfection with steroid-responsive reporter constructs shows glucocorticoid rather than androgen responsiveness in cultured Sertoli cells.

It remains unclear why it has proven so difficult to identify androgen target genes in cultured Sertoli cells. Given the lack of useful endogenous reporter genes, we studied the androgen and glucocorticoid responsiveness of these cells by transfection with three different steroid-responsive reporter constructs. The constructs were driven by the tyrosine aminotransferase steroid-responsive region (TAT-GRE4x-Luc), the mouse mammary tumor virus promoter (MMTV-Luc) and the Pem homeobox gene proximal promoter respectively (Pem-Luc). These constructs can be activated either by both the glucocorticoid receptor (GR) and the androgen receptor (AR) (TAT-GRE4x-Luc and MMTV-Luc) or selectively by the AR (Pem-Luc). Despite high transfection efficiency (30-40%) none of the constructs could be activated by treatment of the Sertoli cells with testosterone, 5alpha-dihydrotestosterone or synthetic androgens. Even pretreatment with follicle-stimulating hormone to raise AR levels (from 31 up to 82fmol/mg protein) did not result in androgen responsiveness. In contrast, treatment with dexamethasone markedly stimulated TAT-GRE4x-Luc and MMTV-Luc activity. GR levels reached a value of 172fmol/mg protein in the cultured cells and both AR and GR displayed homogeneous distribution by immunocytochemical evaluation. Androgen responsiveness was restored and glucocorticoid responsiveness was increased by cotransfection with AR or GR expression constructs. Under cotransfection conditions, 1nM of testosterone (a concentration that is some 100 times lower than that estimated to be present in the testis) was sufficient to stimulate the TAT-GRE4x-Luc maximally. Our data indicate that cultured Sertoli cells respond better to glucocorticoids than to androgens and that one of the factors limiting androgen responsiveness is the availability of AR. Other factors limiting the transactivation capacity of the (endogenous) AR, however, cannot be excluded.