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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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SET9 is an AR co-activator. (A) LNCaP cells grown in steroid-depleted media were transiently transfected with non-silencing (N/S) or SET9 siRNAs for 66 h prior to treatment with and without 10 nM DHT for 6 h followed by RNA extraction and real-time PCR analysis of PSA and TMPRSS2 mRNA expression. (B) HEK293T and LNCaP cells were transiently transfected for 48 h with AR (HEK293T cells only) and either SET9 or SET9H297A together with both MMTV-luciferase and β-galactosidase reporters prior to luciferase and β-galactosidase expression analyses. (C) HEK293T cells were transiently transfected and analysed as in (B) with the inclusion of ARK632R. Data represent average of three independent repeats ± standard error (A–C).
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Figure 4: SET9 is an AR co-activator. (A) LNCaP cells grown in steroid-depleted media were transiently transfected with non-silencing (N/S) or SET9 siRNAs for 66 h prior to treatment with and without 10 nM DHT for 6 h followed by RNA extraction and real-time PCR analysis of PSA and TMPRSS2 mRNA expression. (B) HEK293T and LNCaP cells were transiently transfected for 48 h with AR (HEK293T cells only) and either SET9 or SET9H297A together with both MMTV-luciferase and β-galactosidase reporters prior to luciferase and β-galactosidase expression analyses. (C) HEK293T cells were transiently transfected and analysed as in (B) with the inclusion of ARK632R. Data represent average of three independent repeats ± standard error (A–C).

Mentions: Given that SET9-mediated methylation up-regulates p53 and ER transcriptional activity (31,33), we hypothesised that SET9 would also enhance transcription of androgen-regulated genes. To this end, we depleted endogenous SET9 levels in LNCaP cells by siRNA and assessed hormone-dependent expression of the AR-target genes PSA and TMPRSS2 by quantitative PCR. LNCaP cells grown in steroid-depleted media supplemented with or without 10 nM DHT showed robust reductions in both androgen-dependent PSA (Figure 4A, upper panel) and TMPRSS2 (Figure 4A, lower panel) expression compared to the non-silencing control indicating a role for SET9 in co-activation of the AR.Figure 4.


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)

SET9 is an AR co-activator. (A) LNCaP cells grown in steroid-depleted media were transiently transfected with non-silencing (N/S) or SET9 siRNAs for 66 h prior to treatment with and without 10 nM DHT for 6 h followed by RNA extraction and real-time PCR analysis of PSA and TMPRSS2 mRNA expression. (B) HEK293T and LNCaP cells were transiently transfected for 48 h with AR (HEK293T cells only) and either SET9 or SET9H297A together with both MMTV-luciferase and β-galactosidase reporters prior to luciferase and β-galactosidase expression analyses. (C) HEK293T cells were transiently transfected and analysed as in (B) with the inclusion of ARK632R. Data represent average of three independent repeats ± standard error (A–C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045589&req=5

Figure 4: SET9 is an AR co-activator. (A) LNCaP cells grown in steroid-depleted media were transiently transfected with non-silencing (N/S) or SET9 siRNAs for 66 h prior to treatment with and without 10 nM DHT for 6 h followed by RNA extraction and real-time PCR analysis of PSA and TMPRSS2 mRNA expression. (B) HEK293T and LNCaP cells were transiently transfected for 48 h with AR (HEK293T cells only) and either SET9 or SET9H297A together with both MMTV-luciferase and β-galactosidase reporters prior to luciferase and β-galactosidase expression analyses. (C) HEK293T cells were transiently transfected and analysed as in (B) with the inclusion of ARK632R. Data represent average of three independent repeats ± standard error (A–C).
Mentions: Given that SET9-mediated methylation up-regulates p53 and ER transcriptional activity (31,33), we hypothesised that SET9 would also enhance transcription of androgen-regulated genes. To this end, we depleted endogenous SET9 levels in LNCaP cells by siRNA and assessed hormone-dependent expression of the AR-target genes PSA and TMPRSS2 by quantitative PCR. LNCaP cells grown in steroid-depleted media supplemented with or without 10 nM DHT showed robust reductions in both androgen-dependent PSA (Figure 4A, upper panel) and TMPRSS2 (Figure 4A, lower panel) expression compared to the non-silencing control indicating a role for SET9 in co-activation of the AR.Figure 4.

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