Multiple Components of a Complex Androgen-Dependent Enhancer

Abstract

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.

The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences.

“…We demonstrated previously that the 120-bp Slp enhancer fragment, C'A9, is hormonally responsive in CV-1 cells with cotransfected AR, but not GR, expression plasmids (1). This fragment contains several sequences similar to HREs, but only the 3'-most element, HRE-3, matches a full consensus, confers hormonal response on a reporter gene independent of additional elements, and binds full-length AR protein in vitro (2). HRE-3, however, did not account for the specificity of androgen activation, as multiple copies of this sequence inserted before a tkCAT reporter gene conferred even greater response to GR than to AR, suggesting that other elements in C'A9 must be required to delimit induction (1).…”

“…In an earlier study of the Slp enhancer, we showed that multiple elements interact to augment, or diminish, androgen response (2). To examine the role of these elements in specific hormonal response, we used PCR to introduce clustered base changes throughout C'A9.…”

“…LS 147-154, which alters HRE-3, the proven AR binding site, and LS 133-140, which alters a sequence (HRE-2) similar to the consensus but with no inducibility on its own (2) HRE-3 [2]); this mutant retained only 14% of the response of C'A9. Mutation of the nonconsensus half of HRE-1 (LS 110-117), coinciding with the FPIV region protected by various nuclear extracts (21) and similar in sequence to an octamer binding site, impaired response by about 50%.…”

“…C'A9tkCAT has been described previously; this reporter contains a 120-bp DNA fragment from 2 kb upstream of the Slp gene inserted before the 105-bp herpes simplex virus thymidine kinase promoter driving chloramphenicol acetyltransferase (CAT) (2). Clustered base changes to a restriction site (linker scanning [LS] mutations) were introduced by overlap extension polymerase chain reaction (PCR)-mediated site-directed mutagenesis (16).…”

“…Slp arose from a duplicated fourth component of complement gene that acquired andro-ANDROGEN-SPECIFIC GENE ACTIVATION 6327 gen dependence from a proviral insertion (37). The Slp enhancer, located within the proviral long terminal repeat, contains a near-perfect consensus HRE that is necessary but not sufficient for hormonal induction (2). Although multimers of this HRE can be activated by GR and PR as well as by AR, hormonal specificity is enforced by interactions with nonreceptor elements within the enhancer (1).…”