BRCA1 Methylation Figure 3. PRMT1 methylates and associates with BRCA1. (a) GST-BRCA1 constructs (0.5 mg) and core histones (1 mg) were incubated with purified recombinant PRMT1 enzyme (0.2 mg) in the presence of 0.55 mCi SAdenosyl- L-[methyl-3H] methionine. GST was used as negative control and all GST-BRCA1 construct methylation levels normalized to background GST methylation levels. An average result of two independent experiments run in triplicates is shown. (b) The amino acid sequence of BRCA1 504–802 with arginine (R) residues within the 540–696 minimal region highlighted in blue and bolded, and the predicted methylated arginine residue highlighted in red, underlined and bolded. (c) Multiple sequence alignment generated by Clustal W of four non-human primates and two rodent BRCA1 full length sequences. Score represents pairwise alignment against full length human BRCA1. Amino acid residues are color-coded according to physical properties: basic (magenta), small hydrophobic (red) and hydroxyl + amine + basic (green). Consensus symbols for degree of conservation observed is represented by ‘‘*’’ (residues in column are identical in all seven sequences), ‘‘:’’ (conserved substitutions observed), and ‘‘.’’ (semi-conserved substitutions observed). (d) Two milligram of HeLa whole cell protein extract was incubated with 0.5 mg GST-BRCA1 constructs, beads were washed twice with TNE150 + 0.1% NP-40 and once with TNE50 + 0.1% NP-40, separated on a 4–20% gel by SDS-PAGE, and probed with an antibody against PRMT1. Input represents 1/10 of immunoprecipitated material. (e) Two milligram of HeLa whole cell protein extract were immunoprecipitated with anti-BRCA1 and anti-IgG antibodies, beads were washed twice with TNE150 + 0.1% NP-40 and once with TNE50 + 0.1% NP-40, separated on a 4–20% by SDS-PAGE, and probed with an anti-PRMT1 antibody. Input represents 1/10 of immunoprecipitated material. Results are representative of two independent experiments. doi:10.1371/journal.pone.0011379.g003 protein coupled receptor 85 (SREB2) and the mesothelial fibroblast growth factor 9 (FGF9) exhibited a marked decrease of BRCA1 binding upon protein methylation inhibition. Lastly, the RING1 and YY1 binding protein (RYBP) promoter, somatostatin (SST) and the estrogen regulated gene, pS2 displayed no change in promoter binding between BRCA1 methylation states (Figure 4b). These results indicate that methylation of BRCA1 could play an important role in BRCA1 DNA binding at specific promoters in vivo. Decreased levels of PRMT1 alters BRCA1 promoter binding in vivo To further characterize the specific impact of BRCA1 methylation by PRMT1, PRMT1 cellular levels were decreased by means of RNAi. A titration of PRMT1 siRNA indicated that at 50 nM, PRMT1 protein levels were decreased by 90% (Figure 5a). Therefore, 50 nM PRMT1 siRNA was utilized for ChIP assays to determine the influence of PRMT1 on BRCA1 DNA binding. Consistent with ChIP results from the AdOx treated cells, PRMT1 knockdown resulted in a dramatic increase of BRCA1 DNA binding to the APEX and GADD45G promoters (Figure 5b, lanes 7–8). A modest increase in binding was also observed at the ARHG gene. ORC4L was used as an internal control for no known interaction with BRCA1 [42]. No change in H3-pS10 was observed following PRMT1 knockdown. These results further suggest that PRMT1 methylates BRCA1 and that its capacity to methylate affects binding of BRCA1 to its responsive promoters. Figure 4. Methylation status of BRCA1 alters BRCA1-DNA interactions at specific promoters in vivo. (a) MDA-MB-231 cells were treated with AdOx (30 mM) in order to observe BRCA1 methylation inhibition upon treatment. Two milligram of MDA-MB-231 whole cell protein extract was immunoprecipitated with anti-BRCA1 and anti-IgG antibodies, separated on a 4–20% gel by SDS-PAGE, and probed with anti-K methyl antibody. Blot was stripped and reprobed with anti-BRCA1 antibody. Input represents 1/10 of immunoprecipitated material. Results are representative of two independent experiments. (b) MDA-MB-231 cells were treated with AdOx (30 mM) for 48 hours prior to being collected for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 mg), anti-IgG (10 mg), and anti-histone H3-phosphorylated at S10 (H3-pS10, 5 mg). PCR products were run on a 2% agarose gel and visualized with ethidium bromide staining. Results are representative of two independent experiments. doi:10.1371/journal.pone.0011379.g004 PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11379