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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 recruited to the AR-regulated PSA promoter to methylate histone H3K4 and facilitate AR recruitment. (A) LNCaP cells grown in steroid-depleted media for 72-h were treated with 10 nM DHT over a time-course of 0–120 min and then subject to ChIP analysis using an anti-SET9 antibody, or non-specific isotype control, followed by quantitative PCR analysis using primers specific to the proximal (ARE I) and distal (ARE III) regions of the PSA promoter. (B) Representative SET9 knockdown and AR levels from the stable doxycycline-inducible SET9 knockdown LNCaP cell line (LNCaP-SET9K/D) compared to the equivalent non-silencing (N/S) cell line (LNCaP-N/SK/D). (C) ChIP analysis of histone H3K4 mono-methylation (H3K4me1) at ARE III in LNCaP-SET9K/D cells treated with and without doxycycline for 48 h and 10 nM DHT for 0 or 120 min. (D) Representative western analysis of global histone H3K4me1 and H3K9me2 modifications in LNCaP cells transiently transfected with 25 and 50 nM N/S or SET9 siRNAs. (E) An anti-AR antibody was used in ChIP analysis as in (B). Data represents three independent repeats ± standard error (A and E).
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Figure 6: SET9 is recruited to the AR-regulated PSA promoter to methylate histone H3K4 and facilitate AR recruitment. (A) LNCaP cells grown in steroid-depleted media for 72-h were treated with 10 nM DHT over a time-course of 0–120 min and then subject to ChIP analysis using an anti-SET9 antibody, or non-specific isotype control, followed by quantitative PCR analysis using primers specific to the proximal (ARE I) and distal (ARE III) regions of the PSA promoter. (B) Representative SET9 knockdown and AR levels from the stable doxycycline-inducible SET9 knockdown LNCaP cell line (LNCaP-SET9K/D) compared to the equivalent non-silencing (N/S) cell line (LNCaP-N/SK/D). (C) ChIP analysis of histone H3K4 mono-methylation (H3K4me1) at ARE III in LNCaP-SET9K/D cells treated with and without doxycycline for 48 h and 10 nM DHT for 0 or 120 min. (D) Representative western analysis of global histone H3K4me1 and H3K9me2 modifications in LNCaP cells transiently transfected with 25 and 50 nM N/S or SET9 siRNAs. (E) An anti-AR antibody was used in ChIP analysis as in (B). Data represents three independent repeats ± standard error (A and E).

Mentions: Having established a co-activator role for SET9 in AR-mediated transcription, we next sought to identify if SET9 was recruited to androgen-regulated genes by ChIP analysis. LNCaP cells grown in steroid-depleted media were treated with 10 nM DHT over a time-course of 0–120 min prior to ChIP analysis using an anti-SET9 antibody or an anti-AR antibody control. As expected, AR recruitment to the proximal (ARE I) and distal enhancer (ARE III) regions of the PSA promoter (illustrated in Figure 6A) was androgen-dependent and showed a more robust recruitment to the ARE III region compared to ARE I as described previously (46) (Supplementary Figure S4). In contrast, we found SET9 associated with similar magnitude to both ARE I and III regions of the PSA promoter in the absence of androgen (Figure 6A). After 15 min of hormone treatment, SET9 levels at the PSA promoter rapidly decreased, remained low at 30 min and then increased gradually between 60 and 120 min of androgen stimulation to levels equivalent to the inactive promoter. This dynamic association–disassociation profile of SET9 is very similar to that seen for the binding of SET9 to the IL-8 promoter in response to TNF-α treatment (34), suggesting a common recruitment mechanism for the HMT. Importantly, SET9 was not present on the same promoter elements in the AR PC3 cell line indicating a dependency on the AR signalling cascade for promoter binding (Supplementary Figure S5).Figure 6.


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 recruited to the AR-regulated PSA promoter to methylate histone H3K4 and facilitate AR recruitment. (A) LNCaP cells grown in steroid-depleted media for 72-h were treated with 10 nM DHT over a time-course of 0–120 min and then subject to ChIP analysis using an anti-SET9 antibody, or non-specific isotype control, followed by quantitative PCR analysis using primers specific to the proximal (ARE I) and distal (ARE III) regions of the PSA promoter. (B) Representative SET9 knockdown and AR levels from the stable doxycycline-inducible SET9 knockdown LNCaP cell line (LNCaP-SET9K/D) compared to the equivalent non-silencing (N/S) cell line (LNCaP-N/SK/D). (C) ChIP analysis of histone H3K4 mono-methylation (H3K4me1) at ARE III in LNCaP-SET9K/D cells treated with and without doxycycline for 48 h and 10 nM DHT for 0 or 120 min. (D) Representative western analysis of global histone H3K4me1 and H3K9me2 modifications in LNCaP cells transiently transfected with 25 and 50 nM N/S or SET9 siRNAs. (E) An anti-AR antibody was used in ChIP analysis as in (B). Data represents three independent repeats ± standard error (A and E).
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Figure 6: SET9 is recruited to the AR-regulated PSA promoter to methylate histone H3K4 and facilitate AR recruitment. (A) LNCaP cells grown in steroid-depleted media for 72-h were treated with 10 nM DHT over a time-course of 0–120 min and then subject to ChIP analysis using an anti-SET9 antibody, or non-specific isotype control, followed by quantitative PCR analysis using primers specific to the proximal (ARE I) and distal (ARE III) regions of the PSA promoter. (B) Representative SET9 knockdown and AR levels from the stable doxycycline-inducible SET9 knockdown LNCaP cell line (LNCaP-SET9K/D) compared to the equivalent non-silencing (N/S) cell line (LNCaP-N/SK/D). (C) ChIP analysis of histone H3K4 mono-methylation (H3K4me1) at ARE III in LNCaP-SET9K/D cells treated with and without doxycycline for 48 h and 10 nM DHT for 0 or 120 min. (D) Representative western analysis of global histone H3K4me1 and H3K9me2 modifications in LNCaP cells transiently transfected with 25 and 50 nM N/S or SET9 siRNAs. (E) An anti-AR antibody was used in ChIP analysis as in (B). Data represents three independent repeats ± standard error (A and E).
Mentions: Having established a co-activator role for SET9 in AR-mediated transcription, we next sought to identify if SET9 was recruited to androgen-regulated genes by ChIP analysis. LNCaP cells grown in steroid-depleted media were treated with 10 nM DHT over a time-course of 0–120 min prior to ChIP analysis using an anti-SET9 antibody or an anti-AR antibody control. As expected, AR recruitment to the proximal (ARE I) and distal enhancer (ARE III) regions of the PSA promoter (illustrated in Figure 6A) was androgen-dependent and showed a more robust recruitment to the ARE III region compared to ARE I as described previously (46) (Supplementary Figure S4). In contrast, we found SET9 associated with similar magnitude to both ARE I and III regions of the PSA promoter in the absence of androgen (Figure 6A). After 15 min of hormone treatment, SET9 levels at the PSA promoter rapidly decreased, remained low at 30 min and then increased gradually between 60 and 120 min of androgen stimulation to levels equivalent to the inactive promoter. This dynamic association–disassociation profile of SET9 is very similar to that seen for the binding of SET9 to the IL-8 promoter in response to TNF-α treatment (34), suggesting a common recruitment mechanism for the HMT. Importantly, SET9 was not present on the same promoter elements in the AR PC3 cell line indicating a dependency on the AR signalling cascade for promoter binding (Supplementary Figure S5).Figure 6.

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