DNA dependent recruitment of DDX17 and other interacting proteins by the human androgen receptor.
An oligonucleotide-based assay (OBA) was used to identify novel co-factors that can be recruited by the deoxyribonucleic acid (DNA)-bound androgen receptor (AR). Nuclear extracts obtained from LNCaP cells, after incubation with R1881, were incubated with biotinylated oligonucleotides bound to streptavidin coated beads. The oligonucleotides contain 3 copies in tandem of the androgen responsive element ARE1 from the prostate specific antigen (PSA) gene promoter. As control incubation, a scrambled version of the tandem ARE1 was used. Immunoblots of the eluents revealed that the AR was bound to the ARE1 oligonucleotide and to a much lesser extent to the scrambled oligonucleotide. Proteins eluted from the oligonucleotides, were separated on a 5-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel, followed by identification using mass spectrometry. Identified proteins were scored for having one or more of the following known properties: nuclear localization, involved in transcription regulation, involvement in steroid hormone receptor (SHR) function, or specifical involvement in AR function. A total number of 85 nuclear proteins were found in two separate OBAs. Based on peptide counting, we found enrichment of 7 proteins eluted from the ARE1 oligonucleotide, compared to the scrambled oligonucleotide. Taken together with the obtained scores, these proteins are considered putative AR co-factors. One of these proteins, DDX17, is known to be a co-factor for estrogen receptor alpha (ERalpha), but has never been associated with AR function. The results indicate that the ARE oligonucleotide-based assay may allow enrichment of new candidate DNA-bound AR interacting proteins.
Wong
Hao Yun
HY
Department of Reproduction and Development, Erasmus MC, 3000 CA Rotterdam, The Netherlands. h.wong@erasmusmc.nl
Demmers
Jeroen A A
JA
Bezstarosti
Karel
K
Grootegoed
J Anton
JA
Brinkmann
Albert O
AO
D001710:Biotin; D045744:Cell Line, Tumor; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008297:Male; D015741:Metribolone / Q000494:pharmacology; D009841:Oligonucleotides; D011401:Promoter Regions, Genetic; D017430:Prostate-Specific Antigen / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011944:Receptors, Androgen / Q000235:genetics / Q000502:physiology*; D020218:Response Elements; D019809:Streptavidin
D009841:Oligonucleotides; D011944:Receptors, Androgen; D015741:Metribolone; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D017430:Prostate-Specific Antigen; C102004:DDX17 protein, human; D053487:DEAD-box RNA Helicases