The mechanism by which specific hormonal regulation of gene expression is attained in vivo is a paradox in that several of the steroid receptors recognize the same DNA element in vitro. We have characterized a complex enhancer of the mouse sex-limited protein (Slp) gene that is activated exclusively by androgens but not by glucocorticoids in transfection. Potent androgen induction requires both the consensus hormone response element (HRE) and auxiliary elements residing within the 120-bp DNA fragment C'A9. Multiple nonreceptor factors are involved in androgen specificity, with respect to both the elevation of androgen receptor activity and the inactivity of glucocorticoid receptor (GR), since clustered base changes at any of several sites reduce or abolish androgen induction and do not increase glucocorticoid response. However, moving the HRE as little as 10 bases away from the rest of the enhancer allows GR to function, suggesting that GR is repressed by juxtaposition to particular factors within the androgen-specific complex. Surprisingly, some sequence variations of the HRE itself, within the context of C'A9, alter the stringency of specificity, as well as the magnitude, of hormonal response. These HRE sequence effects on expression correspond in a qualitative manner with receptor binding, i.e., GR shows a threefold difference in affinities for HREs amongst which androgen receptor does not discriminate. Altering the HRE orientation within the enhancer also affects hormonal stringency, increasing glucocorticoid but not androgen response. The effect of these subtle variations suggests that they alter receptor position with respect to other factors. Thus, protein-protein interactions that elicit specific gene regulation are established by the array of DNA elements in a complex enhancer and can be modulated by sequence variations within these elements that may influence selection of precise protein contacts.

Polymorphism in the length of the N-terminal glutamine (Q) tract in the human androgen receptor (AR) has been implicated in affecting aspects of male health ranging from fertility to cancer. Extreme expansion of the tract underlies Kennedy disease, and in vitro the AR Q tract length correlates inversely with transactivation capacity. However, whether normal variation influences physiology or the etiology of disease has been controversial. To assess directly the functional significance of Q tract variation, we converted the mouse AR to the human sequence by germline gene targeting, introducing alleles with 12, 21, or 48 glutamines. These three "humanized" AR (h/mAR) mouse lines were grossly normal in growth, behavior, fertility, and reproductive tract morphology. Phenotypic analysis revealed traits that varied subtly with Q tract length, including body fat amount and, more notably, seminal vesicle weight. Upon molecular analysis, tissue-specific differences in AR levels and target gene expression were detected between mouse lines. In the prostate, probasin, Nkx3.1, and clusterin mRNAs trended in directions predicted for inverse correlation of Q tract length with AR activation. Remarkably, when crossed with transgenic adenocarcinoma of mouse prostate (TRAMP) mice, striking genotype-dependent differences in prostate cancer initiation and progression were revealed. This link between Q tract length and prostate cancer, likely due to differential activation of AR targets, corroborates human epidemiological studies. This h/mAR allelic series in a homogeneous mouse genetic background allows examination of numerous physiological traits for Q tract influences and provides an animal model to test novel drugs targeted specifically to human AR.

An unresolved question in the study of the polyglutamine neurodegenerative disorders is the extent to which partial loss of normal function of the mutant protein contributes to the disease phenotype. To address this , we studied Kennedy disease , a degenerative disorder of lower motor neurons caused by a CAG/glutamine expansion in the androgen receptor (Ar) gene. Signs of partial androgen insensitivity , including testicular atrophy and decreased fertility , are common in affected males , although the underlying mechanisms are not well understood. Here , we describe a knock-in mouse model that reproduces the testicular atrophy , diminished fertility , and systemic signs of partial androgen insensitivity that occur in Kennedy disease patients. Using this model , we demonstrate that the testicular pathology in this disorder is distinct from that mediated by loss of AR function. Testes pathology in 113 CAG knock-in mice was characterized by morphological abnormalities of germ cell maturation , decreased solubility of the mutant AR protein , and alterations of the Sertoli cell cytoskeleton , changes that are distinct from those produced by AR loss-offunction mutation in testicular feminization mutant mice. Our data demonstrate that toxic effects of the mutant protein mediate aspects of the Kennedy disease phenotype previously attributed to a loss

Sex-limited protein (Slp) is expressed in adult male mice. A 160-basepair fragment 2 kilobases upstream of the gene serves as an androgen-dependent enhancer of chloramphenicol acetyltransferase expression in transient transfection assays in cells with endogenous or cotransfected androgen receptor. One element that is necessary, but not sufficient, for induction is a consensus glucocorticoid (or hormone) response element (HRE). This element binds to the mouse androgen receptor in vitro, but with apparent weak affinity. Induction by the HRE is greatly augmented by an accessory sequence within the 160 basepairs, suggesting that cooperative interactions confer strong response to androgen. Additional elements within the enhancer modulate induction, positively or negatively, and exhibit cell-specific behavior. Of particular interest are two degenerate HREs that are adjacent to the consensus sequence; they show no independent activity, but are functionally significant in conjunction with other elements. The complexity of this enhancer may reflect biological mechanisms that ensure specificity of hormonal response and allow gene expression to respond to changes in hormone concentration.

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