Abstract

Transcriptional regulation by the androgen receptor (AR) requires its binding to hormone response element nucleotide sequences in DNA. A consensus glucocorticoid response element (GRE) can mediate transactivation by AR and other members of the AR/glucocorticoid (GR)/progesterone (PR)/mineralocorticoid (MR) receptor subfamily. We identified putative androgen response element (ARE) sequences by binding of a human AR DNA-binding domain fusion protein to DNA in a random sequence selection assay. A 17-base pair consensus nucleotide sequence, termed IDR17, containing three potential GRE-like core binding sites organized as both inverted and direct repeats, was determined from a pool of degenerate oligonucleotides. IDR17 was active in mediating androgen-dependent induction of reporter gene expression in transient transfection assays. Dissection of the IDR17 sequence revealed an 11-base pair sequence (DR-1), consisting of two potential core binding sites oriented as an overlapping direct repeat, as the most potent ARE. DR-1 demonstrated a strong preference for AR binding and transactivation when compared with GR. To our knowledge, this is the first observation that a direct repeat of GRE-like core motifs functions as a preferred hormone response element within the AR/GR/PR/MR subfamily of nuclear receptors.

Highlights

  • Transcriptional regulation by the androgen receptor (AR) requires its binding to hormone response element nucleotide sequences in DNA

  • The AR protein was a recombinant protein composed of amino acids 559 – 644 of the human AR-DBD fused to glutathione S-transferase (GST)

  • The AR-DBD fusion protein was incubated with an oligonucleotide containing a consensus glucocorticoid response element (GRE)/androgen response element (ARE) nucleotide sequence and was shown to form a protein-DNA complex by Electrophoretic Mobility Shift Assays (EMSAs)

Read more

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The following plasmid DNAs were generous gifts: OB7 (hGR) from Dr R. The 5Ј and 3Ј termini contained restriction endonuclease sites for BamHI and EcoR I, respectively, for subcloning purposes and were utilized for primer annealing to convert oligonucleotides from single- to double-stranded DNA and for subsequent PCR amplification. A 90-bp DNA fragment was isolated and radiolabeled by Klenow fragment in the presence of [␣-32P]dCTP For these experiments, 50 –500 ng of either AR-DBD or GR-DBD fusion proteins were incubated with 32P-labeled DNAs in binding reactions. The proteins were first incubated with 10 –1000-fold excess of unlabeled double-stranded oligonucleotide competitor at 25 °C for 10 min before the addition of the radiolabeled DNAs. PCR Amplification of Selected DNA—Oligonucleotide DNAs recovered from EMSA were dissolved in 10 ␮l of 0.1 ϫ TE buffer. Dimers of IDR17, IR0, IR5, and DR-1 were excised from the gel, eluted, recovered by ethanol precipitation, and ligated into the XbaI site of the pBLCAT2 plasmid. CAT activities were normalized for transfection efficiency based upon their corresponding ␤-galactosidase activities

RESULTS
Frequency Consensus
DISCUSSION
Full Text
Paper version not known

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call