BRCA1 Methylation Figure 6. BRCA1 methylation status alters protein-protein interactions at the 504-802 region. (a) Schematic of BRCA1 504-802 primary sequence depicting important protein-protein interactions and domains that could be affected by the methylation of this region. (b) 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 or anti-IgG antibodies, separated on a 4-20% gel by SDS-PAGE, and western blotted using antibodies against Sp1 and BRCA1 proteins. Input represents 1/10 of immunoprecipitated material. Results are representative of two independent experiments. (c) MDA-MB-231 cells were treated with AdOx (30 mM) and whole cell extract separated on a 4-20% gel by SDS-PAGE, and probed with anti-Sp1 antibody. Densitometry was averaged from three independent immunoblots. doi:10.1371/journal.pone.0011379.g006 by the DNA damage response kinases, hCds1/Chk2, ATM, and ATR, following DNA damage produced by ionizing radiation, UV, or DNA damaging inducing chemicals such as mitomycin C [52,53,54,55,56]. Our in vitro and in vivo analyses indicate another avenue for BRCA1 regulation through arginine methylation, and PRMT1 as a cellular arginine methyltransferase candidate for this methylation. Interestingly, methylation of BRCA1 by PRMT1 indicates a regulatory mechanism for BRCA1 binding to particular promoters as well as protein-protein interactions. We have demonstrated that BRCA1 is methylated both in breast cancer cell lines and breast cancer tumor samples. Both arginine and lysine methylation was detected. Interestingly, lysine methylation was only detected in MDA-MB-231 cells but not MCF-7, while arginine methylation was detected in both. Both cell lines were obtained from pleural effusions, but differ in their characteristics. In vitro, MDA-MB-231 cells display a highly invasive phenotype in contrast to MCF-7 cells, while they both have the ability to form in vivo tumors in mice [57,58]. In correlation with breast cancer, MDA-MB-231 cells are triple negative and posses a mutant p53. MCF-7 cells are double positive, negative for HER2 and posses wild-type p53 [58,59,60]. It is tempting to speculate that methylation patterns for both lysine and arginine may be linked to phenotypical characterization of breast cancer types. However, a much larger sample size is needed to draw a clear conclusion in this regard. Arginine methylation by PRMT1 was observed in vitro and the region of BRCA1 504–802 was highly methylated. One well known PRMT consensus methylation sequence is the arginine and glycine-rich (GAR) motif (i.e. repeating RGG sequences), which is recognized by PRMT1, 3, 5, 6, and 8 [61]. However, more recently a focused peptide library screen was used to identify additional sequences methylated by PRMT1 [62]. The authors demonstrated that additional sequences such as ‘‘RLG’’, ‘‘RYG’’, ‘‘RFG’’, ‘‘RTG’’, and ‘‘RKG’’ were substrates for PRMT1. In addition, other PRMTs, such as PRMT4 have no known consensus site, which hinders the identity of arginine methylated proteins. The predicted methylation site at residue 610 harbors a ‘‘RXR’’ sequence, where X is occupied by a leucine, making it the most likely candidate for methylation within that area. Upon methylation inhibition, in vivo BRCA1 binding to the APEX, ARHG and GADD45G promoters was increased. BRCA1 binding to the ESR2, SREB and FGF9 gene promoters was hindered. In addition, BRCA1 binding to RYBP, SST and pS2 gene promoters was unaffected. These results suggest that methylation may influence either the ability of BRCA1 to bind to specific promoter or protein-protein interactions which alters the recruitment of BRCA1 to these promoters. As can be observed in Figure 4a, AdOx treatment abolished detectable levels of PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11379