BRCA1 Methylation Figure 1. BRCA1 is methylated at both arginine and lysine residues in breast cancer cell lines. (a) Predicted BRCA1 methylation sites generated by Memo: Methylation Modification Prediction Server (http://www.bioinfo.tsinghua.edu.cn/,tigerchen/memo.html) R = Arginine and K = Lysine. (b) Two milligram of whole cell protein extracts from MCF-7 and MDA-MB-231 cells were immunoprecipitated with either BRCA1(C-20) or rabbit normal IgG antibody, separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against K-methyl, R-methyl and BRCA1(C- 20). Input represents 1/10 of immunoprecipitated material. Results are representative of three independent experiments. (c) One milligram of whole cell protein extracts from MDA-MB-231 and BRCA1-null UWB1.289 cells were immunoprecipitated with either BRCA1(C-20) or rabbit normal IgG antibody, separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against R-methyl and BRCA1(C-20) in order to validate observed methylated bands. Densitometry of R-methylation was normalized to amount of detected immunoprecpipitated BRCA1, background subtracted based on IgG counts. (d) One milligram of whole cell protein extracts from synchronized MDA-MB-231 cells were immunoprecipitated with either BRCA1(C-20) or rabbit normal IgG antibody, separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against R- methyl and BRCA1(C-20). Densitometry of R-methylation was normalized to amount of detected immunoprecpipitated BRCA1, background subtracted based on IgG counts. Input represents 1/10 of immunoprecipitated material. (e) Whole cell protein extracts from synchronized MDA-MB- 231 cells were separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against Cyclin B(H-433), Cyclin D1(M-20), Cyclin E(C-19), cdk4(H-303) and actin(C-11). doi:10.1371/journal.pone.0011379.g001 PRMT1 methylates and associates with BRCA1 Results above indicate that BRCA1 protein is methylated in both cancer cell lines and patient samples. We were interested in the site of BRCA1 arginine methylation and for that purpose, GST- BRCA1 constructs spanning BRCA1 protein were used for in vitro methyltransferase assays. Due to the lack of a previously identified enzyme, GST-PRMT1 was chosen for this analysis because PRMT1 is responsible for approximately 85% of all arginine methylation [19]. GST-BRCA1 constructs spanning the entire protein (Figure 3a) were incubated in a reaction mixture containing GST-PRMT1 enzyme, buffer, and S-Adenosyl-L [methyl-3H] methionine as a source of radio-labeled methyl groups. Core histones were used as a positive control, as PRMT1 has been shown to methylate histone H4 [17,37] and GST was used as negative control. BRCA1 fragment 504–802 was consistently the most highly methylated in repeated experiments, while no methylation was detected for GST-BRCA1 1501–1861(Figure 3a). Lower levels of methylation were observed with BRCA1 1–500, 452–1079, and 1021–1552. As BRCA1 504–802 overlaps with GST-BRCA1 697– 1276, which exhibited no methylation, amino acids 504–696 are the minimal area necessary for the observed methylation. According to the predicted methylation sites in Figure 3a, the only arginine predicted to be methylated within the 504–802 region is R610 (highlighted in red in Figure 3b). R610 is not the typical GAR motif present in many PRMT1 substrates, but is an RXR sequence, which is methylated in Poly(A)-binding protein II (PABPII) by PRMT1 [38]. Therefore, it is possible that other arginine residues within BRCA1 504–802 are methylated (highlighted in blue in Figure 3b). As BRCA1 is a poorly conserved protein, we were interested to determine if the predicted methylation site was a conserved residue. Full length BRCA1 sequences from human, four non-human primates and two rodents were aligned using Clustal W in order to analyze conservation of the predicted site within our identified PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11379