Functional role of a conformationally flexible homopurine/homopyrimidine domain of the androgen receptor gene promoter interacting with Sp1 and a pyrimidine single strand DNA-binding protein.
The androgen receptor (AR) gene promoter does not contain the TATA or CAAT box, but it contains a long (approximately 90-bp) homopurine/homopyrimidine (pur/ pyr) stretch immediately upstream of the Sp1-binding GC box site. This pur-pyr stretch is conserved at the same proximal position in the rat, mouse, and human AR gene promoters. Mutation of this region results in a 3-fold decline in promoter activity, indicating an important regulatory function. Examination of the conformational state of the AR pur/pyr region with the single-strand-specific S1 nuclease showed that it is capable of forming a non-B DNA structure involving unpaired single strands. Fine mapping of the S1-sensitive site revealed an unsymmetric cleavage pattern indicative of an intramolecular triple helical H-form DNA conformation. Electrophoretic mobility shift analyses showed that the pur/pyr region of the AR promoter can bind a novel pyrimidine single-strand-specific protein (ssPyrBF) and also a double-strand DNA-binding protein. Both oligonucleotide cross-competition and antibody supershift experiments established that the double-strand binding protein is equivalent to Sp1. Deoxyribonuclease I (DNase I) footprinting analysis showed multiple Sp1-binding to the pur/pyr site and a weaker Sp1 interaction to this region compared with the adjacently located GC box, where Sp1 functions to recruit the TFIID complex. These results suggest that the pur/pyr domain of the AR gene can serve to attract additional Sp1 molecules when it exists in the double-stranded B-DNA conformation. However, binding of ssPyrBF and the resultant stabilization of the non-B DNA structure is expected to prevent its interaction with Sp1. We speculate that in the TATA-less AR gene promoter, multiple weak Sp1 sites at the pur/pyr region adjacent to the GC box can provide a readily available source of this transcription factor to the functional GC box, thereby facilitating the assembly of the initiation complex.
Chen
S
S
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio 78284-7762, USA.
Supakar
P C
PC
Vellanoweth
R L
RL
Song
C S
CS
Chatterjee
B
B
Roy
A K
AK
D000818:Animals; D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008099:Liver; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D011944:Receptors, Androgen / Q000737:chemistry*; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013045:Species Specificity; D014162:Transfection
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011944:Receptors, Androgen; D016329:Sp1 Transcription Factor; C051802:triplex DNA; D004247:DNA