Negative regulation of the androgen receptor gene promoter by NFI and an adjacently located multiprotein-binding site

The upstream promoter of the rat androgen receptor (AR) gene contains a str ong negative regulatory region located at the -388 to -340 nucleotide posit ion. The distal part (-388/-373) of this regulatory region binds NFI, a ubi quitous transcription factor, while the proximal portion (-372/-340) contai ns an overlapping binding site for two nuclear proteins. This composite reg ulatory region (-388/-340) was initially defined by deoxyribonuclease I foo tprinting as the continuous stretch of a nuclease-protected site. NFI speci ficity of the distal portion (-388/-373) of the footprint was established t hrough cross-competition in electrophoretic mobility shift assay (EMSA) usi ng the well characterized NFI element of the adenovirus major late promoter and by immunoreactivity to the NFI antibody. EMSA with oligonucleotide dup lexes corresponding to the proximal domain (-372/-340) indicated multiple r etarded bands with at least two major DNA-protein complexes. Further analys is with truncated oligonucleotide duplexes showed that these two major prot eins bind to this domain in an overlapping manner. Within this overlapping area, the position spanning -359 to -347 is essential for the formation of either of these two complexes. Substitution of four G with T residues in th e overlapping area totally abolished all protein binding at the downstream -372/-340 site. Point mutations that abolish specific binding at either the NFI or immediately downstream multiprotein-binding site caused about a 10- fold increase in AR promoter activity in transfected HepG2 cells. Double mu tation involving both the NFI and proximal overlapping protein-binding site s failed to cause any additional increase in promoter function. From these results we conclude that the AR promoter contains a composite negative regu latory region at -388/-340, and the repressor function may involve a coordi nate interaction between NFI and at least two other nuclear factors.