title: Determinants of Gli2 co-activation of wildtype and naturally truncated androgen receptors.
abstract: BACKGROUND
  Gli2, a transcription factor in the Hedgehog pathway, is overexpressed in castrate-resistant prostate cancer (PCa). Previously we showed that Gli2 overexpression increased transcriptional activity of androgen receptor (AR) and conferred androgen growth-independence to normally growth-dependent PCa cells. Here we localized the regions of AR-Gli2 protein interaction and determined the domains within Gli2 needed for AR co-activation.
  
  
  METHODS
  Co-immunoprecipitation and GST-pulldown assays were used to define AR-Gli binding domains. Co-activation assays using androgen-responsive promoter reporters were used to define Gli2 regions needed for AR co-activation. Chromatin immunoprecipitation (ChIP) assays were used to confirm nuclear interactions of Gli2 with AR in PCa cells.
  
  
  RESULTS
  The Gli2 C-terminal domain (CTD) is sufficient for AR co-activation. Two elements within the CTD were required: (1) an AR binding domain within aa628-897; and (2) at least part of the Gli2 transactivation domain within aa1252-1586. In turn, Gli2 binds the tau5/AF5 ligand-independent activation domain in the AR N-terminus. Mutations in the WxxLF motif in tau5/AF5 greatly diminished binding to Gli2-CTD. Gli2 interaction with AR tau5/AF5 was further substantiated by the ability of Gli2/Gli2-CTD to co-activate truncated AR splice variants (AR-V7/ARV567es). ChIP assays confirmed that Gli2 associates with chromatin at androgen response elements found near androgen-responsive genes in LNCaP cells. These assays also showed that AR associates with chromatin containing a Gli-response element near a Gli-responsive gene.
  
  
  CONCLUSIONS
  Our findings indicate that Gli2 overexpression in PCa cells might support development of castration resistant PCa through AR co-activation and suggests that AR might modulate transcription from Gli2.
authors.affiliation: The Vancouver Prostate Centre, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.forename: Mengqian
authors.initials: M
authors.lastname: Chen
authors.forename: Sarah
authors.initials: S
authors.lastname: Truong
authors.forename: Chunhong
authors.initials: C
authors.lastname: Yan
authors.forename: Ralph
authors.initials: R
authors.lastname: Buttyan
mesh_terms: D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D064129:Prostatic Neoplasms, Castration-Resistant / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2
chemical_list: D004273:DNA, Neoplasm; C400551:GLI1 protein, human; C082593:GLI2 protein, human; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D011944:Receptors, Androgen; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2
keywords: Gli2; androgen receptor; prostate cancer; transcriptional co-activation