Limits...
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.

Show MeSH

Related in: MedlinePlus

The AR is methylated by SET9 in vitro and in vivo. (A) An in vitro 3H-SAM methylation assay containing purified SET9 and N- and C-terminal fragments of the AR was incubated for 30 min and samples subject to PAGE, dried and exposed to X-ray film. (B) LNCaP cells grown in steroid-depleted media were treated for 6 h with 10 nM DHT prior to IP using an anti-AR antibody and western analysis using an anti-pan-methyl-lysine antibody (α-pan-methyl-K). (C) LNCaP cells grown in serum-containing media were transiently transfected with either non-silencing (N/S) or SET9 siRNAs for 72 h prior to IP and western analysis as in (B). (D) HEK293T cells were transiently transfected with AR and either SET9 or SET9H297A for 48 h prior to IP and western analysis as in (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045589&req=5

Figure 2: The AR is methylated by SET9 in vitro and in vivo. (A) An in vitro 3H-SAM methylation assay containing purified SET9 and N- and C-terminal fragments of the AR was incubated for 30 min and samples subject to PAGE, dried and exposed to X-ray film. (B) LNCaP cells grown in steroid-depleted media were treated for 6 h with 10 nM DHT prior to IP using an anti-AR antibody and western analysis using an anti-pan-methyl-lysine antibody (α-pan-methyl-K). (C) LNCaP cells grown in serum-containing media were transiently transfected with either non-silencing (N/S) or SET9 siRNAs for 72 h prior to IP and western analysis as in (B). (D) HEK293T cells were transiently transfected with AR and either SET9 or SET9H297A for 48 h prior to IP and western analysis as in (B).

Mentions: The binding of SET9 to the AR-DBD/H/LBD fragment that contains the 630KLKK633 motif, a sequence that resembles the SET9-methylation sites of p53 [KSK(me)K] (31) and ER [RSK(me)K] (33), suggested a potential role for SET9 in directly methylating the AR. To test this, purified AR-TD or AR-DBD/H/LBD proteins were incorporated into an in vitro methylation assay containing SET9, 3H-labelled S-adenosyl methionine (3H-SAM) and 10 nM DHT. As shown in Figure 2A, the AR-DBD/H/LBD fragment was directly and specifically methylated in the presence of SET9 (Figure 2A, lane 4), but not the AR-TD that remained unmodified (Figure 2A, lane 2), indicating a positive correlation between SET9-AR interaction and receptor methylation. We also detected SET9 auto-methylation as described in ref. (41).Figure 2.


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)

The AR is methylated by SET9 in vitro and in vivo. (A) An in vitro 3H-SAM methylation assay containing purified SET9 and N- and C-terminal fragments of the AR was incubated for 30 min and samples subject to PAGE, dried and exposed to X-ray film. (B) LNCaP cells grown in steroid-depleted media were treated for 6 h with 10 nM DHT prior to IP using an anti-AR antibody and western analysis using an anti-pan-methyl-lysine antibody (α-pan-methyl-K). (C) LNCaP cells grown in serum-containing media were transiently transfected with either non-silencing (N/S) or SET9 siRNAs for 72 h prior to IP and western analysis as in (B). (D) HEK293T cells were transiently transfected with AR and either SET9 or SET9H297A for 48 h prior to IP and western analysis as in (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: The AR is methylated by SET9 in vitro and in vivo. (A) An in vitro 3H-SAM methylation assay containing purified SET9 and N- and C-terminal fragments of the AR was incubated for 30 min and samples subject to PAGE, dried and exposed to X-ray film. (B) LNCaP cells grown in steroid-depleted media were treated for 6 h with 10 nM DHT prior to IP using an anti-AR antibody and western analysis using an anti-pan-methyl-lysine antibody (α-pan-methyl-K). (C) LNCaP cells grown in serum-containing media were transiently transfected with either non-silencing (N/S) or SET9 siRNAs for 72 h prior to IP and western analysis as in (B). (D) HEK293T cells were transiently transfected with AR and either SET9 or SET9H297A for 48 h prior to IP and western analysis as in (B).
Mentions: The binding of SET9 to the AR-DBD/H/LBD fragment that contains the 630KLKK633 motif, a sequence that resembles the SET9-methylation sites of p53 [KSK(me)K] (31) and ER [RSK(me)K] (33), suggested a potential role for SET9 in directly methylating the AR. To test this, purified AR-TD or AR-DBD/H/LBD proteins were incorporated into an in vitro methylation assay containing SET9, 3H-labelled S-adenosyl methionine (3H-SAM) and 10 nM DHT. As shown in Figure 2A, the AR-DBD/H/LBD fragment was directly and specifically methylated in the presence of SET9 (Figure 2A, lane 4), but not the AR-TD that remained unmodified (Figure 2A, lane 2), indicating a positive correlation between SET9-AR interaction and receptor methylation. We also detected SET9 auto-methylation as described in ref. (41).Figure 2.

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