Determinants of Gli2 co-activation of wildtype and naturally truncated androgen receptors

Na Li, Mengqian Chen, Sarah Truong, Chunhong Yan, Ralph Buttyan

Research output: Contribution to journalArticle

8 Citations (Scopus)

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. Result 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. CONCLUSION 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. Prostate 74:1400-1410, 2014.

Original languageEnglish (US)
Pages (from-to)1400-1410
Number of pages11
JournalProstate
Volume74
Issue number14
DOIs
StatePublished - Jan 1 2014

Fingerprint

Androgen Receptors
Prostatic Neoplasms
Androgens
Chromatin Immunoprecipitation
Response Elements
Chromatin
Protein Interaction Domains and Motifs
Castration
Growth
Immunoprecipitation
Transcriptional Activation
Genes
Prostate

Keywords

  • Gli2
  • androgen receptor
  • prostate cancer
  • transcriptional co-activation

ASJC Scopus subject areas

  • Oncology
  • Urology

Cite this

Determinants of Gli2 co-activation of wildtype and naturally truncated androgen receptors. / Li, Na; Chen, Mengqian; Truong, Sarah; Yan, Chunhong; Buttyan, Ralph.

In: Prostate, Vol. 74, No. 14, 01.01.2014, p. 1400-1410.

Research output: Contribution to journalArticle

Li, Na ; Chen, Mengqian ; Truong, Sarah ; Yan, Chunhong ; Buttyan, Ralph. / Determinants of Gli2 co-activation of wildtype and naturally truncated androgen receptors. In: Prostate. 2014 ; Vol. 74, No. 14. pp. 1400-1410.
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N2 - 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. Result 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. CONCLUSION 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. Prostate 74:1400-1410, 2014.

AB - 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. Result 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. CONCLUSION 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. Prostate 74:1400-1410, 2014.

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