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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 enhances AR N- and C-terminal interaction without affecting receptor stability or nuclear shuttling. (A) HEK293T cells transiently transfected with empty vector, SET9 or SET9H297A were treated with 1 µM cycloheximide (CHX) for up to 8 h and then subject to western analysis using anti-AR and anti-α-tubulin antibodies. (B) LNCaP cells transiently transfected with non-silencing (N/S) or SET9 siRNAs were treated with 1 µM CHX and subject to western analysis as in (A). (C) LNCaP cells transiently transfected with N/S or SET9 siRNAs in steroid-depleted media were treated with and without 10 nM DHT for 6 h prior to nuclear-cytoplasmic extraction and western analysis using anti-AR, -PARP1 and -GAPDH antibodies. (D) HEK293T cells transiently transfected with AR or ARK632R were subject to the same experimental procedure as in (C) with the inclusion of an additional 10 nM DHT time-point at 2 h. (E) Mammalian two-hybrid analysis of AR-TD and AR-DBD/H/LBD fragment interaction in HEK293T cells transiently transfected with either SET9, SET9H297A or SRC-1 and Gal4-luciferase and β-galactosidase reporters. Cells were treated with and without 10 nM DHT for 24-h prior to harvesting, and luciferase and β-galactosidase analyses. Data represents the average of three independent experiments performed in quadruplicate ± standard error (asterisk represents statistical significance <0.05).
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Figure 5: SET9 enhances AR N- and C-terminal interaction without affecting receptor stability or nuclear shuttling. (A) HEK293T cells transiently transfected with empty vector, SET9 or SET9H297A were treated with 1 µM cycloheximide (CHX) for up to 8 h and then subject to western analysis using anti-AR and anti-α-tubulin antibodies. (B) LNCaP cells transiently transfected with non-silencing (N/S) or SET9 siRNAs were treated with 1 µM CHX and subject to western analysis as in (A). (C) LNCaP cells transiently transfected with N/S or SET9 siRNAs in steroid-depleted media were treated with and without 10 nM DHT for 6 h prior to nuclear-cytoplasmic extraction and western analysis using anti-AR, -PARP1 and -GAPDH antibodies. (D) HEK293T cells transiently transfected with AR or ARK632R were subject to the same experimental procedure as in (C) with the inclusion of an additional 10 nM DHT time-point at 2 h. (E) Mammalian two-hybrid analysis of AR-TD and AR-DBD/H/LBD fragment interaction in HEK293T cells transiently transfected with either SET9, SET9H297A or SRC-1 and Gal4-luciferase and β-galactosidase reporters. Cells were treated with and without 10 nM DHT for 24-h prior to harvesting, and luciferase and β-galactosidase analyses. Data represents the average of three independent experiments performed in quadruplicate ± standard error (asterisk represents statistical significance <0.05).

Mentions: Having shown a role for receptor methylation in transcriptional co-activation, we next assessed the impact of methylation on the stability of the receptor. Previous reports have shown differing effects of SET9-mediated methylation on protein stability; both p53 and ER are stabilized by SET9 (31,33) while the RelA subunit of NFκB (34) and DNMT1 (41) are both destabilized. To test our hypothesis that SET9 affects AR stability and hence up-regulates transcriptional activity of the receptor, cycloheximide (CHX) time-course experiments were undertaken in HEK293 cells over-expressing AR with and without wild-type or methylase-inactive mutant SET9. As shown in Figure 5A (left panel), in the absence of ectopic SET9, AR levels are reduced by ∼50% after 8 h CHX treatment in serum-containing media which is in line with our previous findings (42). Importantly, expression of SET9 (Figure 5A, middle panel) or SET9H297A (Figure 5A, right panel), had no additional effect on AR protein levels indicating that SET9 does not impact on receptor stability. To test this further, LNCaP cells transiently transfected with SET9 siRNA or control were subject to CHX time-course experiments as above. Endogenous AR destabilization rates in the SET9-depleted cells were equivalent to control cells (Figure 5B). Moreover, overexpression of SET9 or SET9H297A did not affect AR or ARK632R protein levels (Figures 2D and 3C) confirming that SET9 does not regulate AR turnover.Figure 5.


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 enhances AR N- and C-terminal interaction without affecting receptor stability or nuclear shuttling. (A) HEK293T cells transiently transfected with empty vector, SET9 or SET9H297A were treated with 1 µM cycloheximide (CHX) for up to 8 h and then subject to western analysis using anti-AR and anti-α-tubulin antibodies. (B) LNCaP cells transiently transfected with non-silencing (N/S) or SET9 siRNAs were treated with 1 µM CHX and subject to western analysis as in (A). (C) LNCaP cells transiently transfected with N/S or SET9 siRNAs in steroid-depleted media were treated with and without 10 nM DHT for 6 h prior to nuclear-cytoplasmic extraction and western analysis using anti-AR, -PARP1 and -GAPDH antibodies. (D) HEK293T cells transiently transfected with AR or ARK632R were subject to the same experimental procedure as in (C) with the inclusion of an additional 10 nM DHT time-point at 2 h. (E) Mammalian two-hybrid analysis of AR-TD and AR-DBD/H/LBD fragment interaction in HEK293T cells transiently transfected with either SET9, SET9H297A or SRC-1 and Gal4-luciferase and β-galactosidase reporters. Cells were treated with and without 10 nM DHT for 24-h prior to harvesting, and luciferase and β-galactosidase analyses. Data represents the average of three independent experiments performed in quadruplicate ± standard error (asterisk represents statistical significance <0.05).
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Figure 5: SET9 enhances AR N- and C-terminal interaction without affecting receptor stability or nuclear shuttling. (A) HEK293T cells transiently transfected with empty vector, SET9 or SET9H297A were treated with 1 µM cycloheximide (CHX) for up to 8 h and then subject to western analysis using anti-AR and anti-α-tubulin antibodies. (B) LNCaP cells transiently transfected with non-silencing (N/S) or SET9 siRNAs were treated with 1 µM CHX and subject to western analysis as in (A). (C) LNCaP cells transiently transfected with N/S or SET9 siRNAs in steroid-depleted media were treated with and without 10 nM DHT for 6 h prior to nuclear-cytoplasmic extraction and western analysis using anti-AR, -PARP1 and -GAPDH antibodies. (D) HEK293T cells transiently transfected with AR or ARK632R were subject to the same experimental procedure as in (C) with the inclusion of an additional 10 nM DHT time-point at 2 h. (E) Mammalian two-hybrid analysis of AR-TD and AR-DBD/H/LBD fragment interaction in HEK293T cells transiently transfected with either SET9, SET9H297A or SRC-1 and Gal4-luciferase and β-galactosidase reporters. Cells were treated with and without 10 nM DHT for 24-h prior to harvesting, and luciferase and β-galactosidase analyses. Data represents the average of three independent experiments performed in quadruplicate ± standard error (asterisk represents statistical significance <0.05).
Mentions: Having shown a role for receptor methylation in transcriptional co-activation, we next assessed the impact of methylation on the stability of the receptor. Previous reports have shown differing effects of SET9-mediated methylation on protein stability; both p53 and ER are stabilized by SET9 (31,33) while the RelA subunit of NFκB (34) and DNMT1 (41) are both destabilized. To test our hypothesis that SET9 affects AR stability and hence up-regulates transcriptional activity of the receptor, cycloheximide (CHX) time-course experiments were undertaken in HEK293 cells over-expressing AR with and without wild-type or methylase-inactive mutant SET9. As shown in Figure 5A (left panel), in the absence of ectopic SET9, AR levels are reduced by ∼50% after 8 h CHX treatment in serum-containing media which is in line with our previous findings (42). Importantly, expression of SET9 (Figure 5A, middle panel) or SET9H297A (Figure 5A, right panel), had no additional effect on AR protein levels indicating that SET9 does not impact on receptor stability. To test this further, LNCaP cells transiently transfected with SET9 siRNA or control were subject to CHX time-course experiments as above. Endogenous AR destabilization rates in the SET9-depleted cells were equivalent to control cells (Figure 5B). Moreover, overexpression of SET9 or SET9H297A did not affect AR or ARK632R protein levels (Figures 2D and 3C) confirming that SET9 does not regulate AR turnover.Figure 5.

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