INVOLVEMENT OF AN OCTAMER-LIKE SEQUENCE WITHIN A CRUCIAL REGION OF THE ANDROGEN-DEPENDENT SLP ENHANCER

Androgen dependence of the mouse sex-limited protein (Slp) gene is con ferred by an enhancer encompassing a consensus hormone response elemen t (HRE) and sites for several nonreceptor factors, The footprint IV (F PIV) region of the enhancer plays a key role in hormone- and tissue-sp ecific response, both in vitro and in vivo. We characterized FPIV-bind ing factors by methylation interference analysis and UV cross-linking of several complexes evident in gel mobility-shift assays, The footpri nting analysis revealed that distinct base contacts within the multipl e nuclear protein-DNA complexes occurred primarily within a sequence s imilar to an octamer transcription factor (Oct-1) binding site, With a dditional data on approximate molecular weights from UV cross-linking, several plausible candidates were tested for their DNA binding and fu nctional activity at FPIV. Oct-like protein binding in gel-shift assay s with several cell and tissue extracts was evident using specific com petitors and antibodies, but was lower in affinity for FPIV than for a n Oct-1 consensus site. Site-directed mutation of the FPIV sequence to a consensus Oct-1 element within the Slp enhancer context increased O ct-1 binding in vitro, but greatly reduced hormonal induction in vivo. This suggested that Oct-1 is not directly involved in response, or al ternatively, that Oct-1 bound to the lower-affinity site interacts wit h neighboring factors significantly differently than Oct-1 bound to a consensus sequence, A sequence overlapping the Oct-like element that w as similar to a hepatic nuclear factor-4 (HNF-4) site showed no abilit y to bind HNF-4 in vitro, nor the related orphan receptor, chicken ova lbumin upstream promoter factor (COUP-TF), Intriguingly, however, expr ession of COUP-TF in transfection had a dramatic inhibitory effect on response of the androgen-specific enhancer (C'Delta 9), but did not af fect other enhancer configurations that can also be induced by glucoco rticoid (C'Delta 2). This underscores that, despite extensive sequence identity of C'Delta 9 and C'Delta 2, components of the androgen-speci fic transcription complex differ significantly from that of one that i s more generally steroid responsive.