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 strong negative regulatory region located at the -388 to -340 nucleotide position. The distal part (-388/-373) of this regulatory region binds NFI, a ubiquitous transcription factor, while the proximal portion (-372/-340) contains an overlapping binding site for two nuclear proteins. This composite regulatory region (-388/-340) was initially defined by deoxyribonuclease I footprinting as the continuous stretch of a nuclease-protected site. NFI specificity of the distal portion (-388/-373) of the footprint was established through cross-competition in electrophoretic mobility shift assay (EMSA) using the well characterized NFI element of the adenovirus major late promoter and by immunoreactivity to the NFI antibody. EMSA with oligonucleotide duplexes corresponding to the proximal domain (-372/-340) indicated multiple retarded bands with at least two major DNA-protein complexes. Further analysis with truncated oligonucleotide duplexes showed that these two major proteins 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 the 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 mutation involving both the NFI and proximal overlapping protein-binding sites failed to cause any additional increase in promoter function. From these results we conclude that the AR promoter contains a composite negative regulatory region at -388/-340, and the repressor function may involve a coordinate interaction between NFI and at least two other nuclear factors.
Song
C S
CS
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 78284-7762, USA.
Jung
M H
MH
Supakar
P C
PC
Chatterjee
B
B
Roy
A K
AK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D009154:Mutation; D051839:NFI Transcription Factors; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D011916:Rats, Inbred F344; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics / Q000502:physiology; D014018:Tissue Distribution; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C080333:transcription factor nuclear factor 1; D004247:DNA