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Regulation of the androgen receptor by SET9-mediated methylation.

Gaughan L, Stockley J, Wang N, McCracken SR, Treumann A, Armstrong K, Shaheen F, Watt K, McEwan IJ, Wang C, Pestell RG, Robson CN - Nucleic Acids Res. (2010)

Bottom Line: Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor.Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9.Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes.

View Article: PubMed Central - PubMed

Affiliation: Solid Tumour Target Discovery Group, Northern Institute for Cancer Research, Newcastle University, Paul O'Gorman Building, Newcastle Upon Tyne, NE2 4HH, UK. luke.gaughan@ncl.ac.uk

ABSTRACT
The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.

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AR and SET9 interact in vitro and in vivo. (A) LNCaP cells grown in steroid-depleted media and treated with and without 10 nM DHT for 6 h were subject to IP using an anti-SET9 antibody followed by western analysis using an anti-AR antibody. (B) HEK293T cells were transiently transfected with pFlag-AR and either pFlag-SET9 or pFlag-SET9H297A for 48 h prior to IP using an anti-AR antibody followed by immunoblotting with anti-AR and -Flag antibodies. (C) Diagrammatic representation of the domains of the androgen receptor showing target lysine sequences of SET9 and sequence of AR peptide used in 3H methylation assays. (D) In vitro IP between His-tagged SET9 and N- and C-terminal fragments of the AR using anti-SET9 antibody followed by western analysis using an anti-His antibody.
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Figure 1: AR and SET9 interact in vitro and in vivo. (A) LNCaP cells grown in steroid-depleted media and treated with and without 10 nM DHT for 6 h were subject to IP using an anti-SET9 antibody followed by western analysis using an anti-AR antibody. (B) HEK293T cells were transiently transfected with pFlag-AR and either pFlag-SET9 or pFlag-SET9H297A for 48 h prior to IP using an anti-AR antibody followed by immunoblotting with anti-AR and -Flag antibodies. (C) Diagrammatic representation of the domains of the androgen receptor showing target lysine sequences of SET9 and sequence of AR peptide used in 3H methylation assays. (D) In vitro IP between His-tagged SET9 and N- and C-terminal fragments of the AR using anti-SET9 antibody followed by western analysis using an anti-His antibody.

Mentions: To address a role for SET9 in the AR signalling cascade we first sought to identify an interaction between SET9 and the receptor. Androgen-dependent LNCaP prostate cancer cells were cultured in steroid-depleted media for 40-h prior to treatment with or without 10 nM dihydrotestosterone (DHT) for 8-h and subject to IP using an anti-SET9 antibody. Western analysis of immunoprecipitated material using an anti-AR antibody demonstrated an interaction between endogenous SET9 and AR that is enhanced in the presence of androgen (Figure 1A). To confirm the interaction, the reciprocal IP was performed in AR- HEK293T cells, grown in serum-containing media, ectopically expressing AR together with wild-type SET9 or the methyltransferase-inactive mutant SETH297A. As shown in Figure 1B, immunoprecipitated AR interacted equally well with both wild-type and mutant forms of SET9.Figure 1.


Regulation of the androgen receptor by SET9-mediated methylation.

Gaughan L, Stockley J, Wang N, McCracken SR, Treumann A, Armstrong K, Shaheen F, Watt K, McEwan IJ, Wang C, Pestell RG, Robson CN - Nucleic Acids Res. (2010)

AR and SET9 interact in vitro and in vivo. (A) LNCaP cells grown in steroid-depleted media and treated with and without 10 nM DHT for 6 h were subject to IP using an anti-SET9 antibody followed by western analysis using an anti-AR antibody. (B) HEK293T cells were transiently transfected with pFlag-AR and either pFlag-SET9 or pFlag-SET9H297A for 48 h prior to IP using an anti-AR antibody followed by immunoblotting with anti-AR and -Flag antibodies. (C) Diagrammatic representation of the domains of the androgen receptor showing target lysine sequences of SET9 and sequence of AR peptide used in 3H methylation assays. (D) In vitro IP between His-tagged SET9 and N- and C-terminal fragments of the AR using anti-SET9 antibody followed by western analysis using an anti-His antibody.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3045589&req=5

Figure 1: AR and SET9 interact in vitro and in vivo. (A) LNCaP cells grown in steroid-depleted media and treated with and without 10 nM DHT for 6 h were subject to IP using an anti-SET9 antibody followed by western analysis using an anti-AR antibody. (B) HEK293T cells were transiently transfected with pFlag-AR and either pFlag-SET9 or pFlag-SET9H297A for 48 h prior to IP using an anti-AR antibody followed by immunoblotting with anti-AR and -Flag antibodies. (C) Diagrammatic representation of the domains of the androgen receptor showing target lysine sequences of SET9 and sequence of AR peptide used in 3H methylation assays. (D) In vitro IP between His-tagged SET9 and N- and C-terminal fragments of the AR using anti-SET9 antibody followed by western analysis using an anti-His antibody.
Mentions: To address a role for SET9 in the AR signalling cascade we first sought to identify an interaction between SET9 and the receptor. Androgen-dependent LNCaP prostate cancer cells were cultured in steroid-depleted media for 40-h prior to treatment with or without 10 nM dihydrotestosterone (DHT) for 8-h and subject to IP using an anti-SET9 antibody. Western analysis of immunoprecipitated material using an anti-AR antibody demonstrated an interaction between endogenous SET9 and AR that is enhanced in the presence of androgen (Figure 1A). To confirm the interaction, the reciprocal IP was performed in AR- HEK293T cells, grown in serum-containing media, ectopically expressing AR together with wild-type SET9 or the methyltransferase-inactive mutant SETH297A. As shown in Figure 1B, immunoprecipitated AR interacted equally well with both wild-type and mutant forms of SET9.Figure 1.

Bottom Line: Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor.Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9.Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes.

View Article: PubMed Central - PubMed

Affiliation: Solid Tumour Target Discovery Group, Northern Institute for Cancer Research, Newcastle University, Paul O'Gorman Building, Newcastle Upon Tyne, NE2 4HH, UK. luke.gaughan@ncl.ac.uk

ABSTRACT
The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.

Show MeSH
Related in: MedlinePlus