which results in transcriptional repression [14]. To date, the known PTMs of BRCA1 include phosphorylation and ubiquiti- nylation [7]. The phospho-BRCA1 predominates in S phase and subsequently becomes dephosphorylated after M phase [7]. This cell cycle dependent phosphorylation occurs in the absence of DNA damage and is accomplished by multiple protein kinases including cyclin A/cdk2, cyclin E/cdk2, cyclin D/cdk4, and aurora-A [7]. In addition, BRCA1 is phosphorylated following DNA damage by ATR and ATM kinases [7]. Although epigenetic changes to the BRCA1 gene have been extensively researched at the DNA-level, proteome research has focused on phosphorylation of BRCA1 by DNA damage protein kinases, while little information is known about the effects of other PTM events. Protein methylation can occur on both lysine and arginine residues. Arginine methylation is carried out by a family of protein arginine methyltransferases (PRMT), which contains eleven family members to date [15]. These enzymes utilize S-adenosyl methionine (AdoMet) as a methyl donor [16], and can be further subdivided into type I and type II enzymes. Type I enzymes form monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) and type II enzymes catalyze the formation of MMA and symmetric dimethylarginine (sDMA) [17]. PRMT1, PRMT2, PRMT3, PRMT4/CARM1, PRMT6, and PRMT8 are type I enzymes; while PRMT5, PRMT7, and PRMT9 are type II enzymes. PRMT1 is the most predominant methyltransferase in mammalian cells [18] and is responsible for the majority of arginine methylation, thus the vast majority of research has been focused on this enzyme [19]. A number of PRMT1 substrates have been identified including 53BP1, histone H4, MRE11, nucleolin, RNA helicase A, SAM68, and ERa [20,21,22,23]. Arginine methylation can regulate multiple cellular processes including transcription [24,25,26,27,28,29], protein-protein interactions [30], nuclear trafficking [31,32], transcriptional elongation, DNA damage response [22], and cell cycle checkpoints [22]. Lysine methylation occurs mainly through the SET domain family of proteins. These enzymes were originally termed histone methyl- transferases due to their ability to methylation various histone protein residues [33], but in light of the identification of many non-histone protein substrates they are now referred to as protein lysine methyltransferases (PKMTs). The exception to this is the DOT1 family of lysine methyltransferases. Lysine residues can be mono-, di-, or tri-methylated [34,35]. Here we have identified for the first time the arginine and lysine methylation of BRCA1 in tissue culture cell lines as well as breast tumor tissue samples. We show that BRCA1 is methylated by PRMT1 in vitro within the 504–802 region. PRMT1 was detected in complex with BRCA1 504–802 through in vitro binding assays and co-immunoprecipitated with BRCA1. Inhibition of methyla- tion resulted in decreased BRCA1 methylation and alteration of BRCA1 binding to promoters in vivo. Knockdown of PRMT1 also resulted in increased BRCA1 binding to particular promoters in vivo. Finally, following methylation inhibition, Sp1 was found to preferentially associate with hypo-methylated BRCA1 and STAT1 was found to preferentially associate with hyper-methylated BRCA1. These findings indicate that BRCA1 is posttranslationally modified through methylation by PRMT1 and that this methyl- ation influences its ability to bind to different promoters in vivo. Results BRCA1 is methylated in breast cancer cell lines Methylation of proteins can have either activating or repressive effects depending on the amino acid that is modified (arginine-R or lysine-K) and whether it is mono-, di-, or tri-methylated [36]. For BRCA1 Methylation example, K4 methylation on histone H3 is an indicator of actively transcribed genes [36]. In contrast, methylation of histone H3K9, H3K27, and H4K20 is involved in heterochromatin formation and gene silencing [36]. To date there are no reports of BRCA1 protein methylation. Based on the role of BRCA1 in transcription and the influence of methylation on various transcription factors, we speculated that BRCA1 may be methylated. In silico analysis revealed a total of seven R and ten K residues in BRCA1 that could potentially be methylated (Figure 1a). Interestingly, two of these residues, R1076 and R1751 have known BRCA1 mutations, R1076T, R1751Q and R1751P, according to the Breast Cancer Information Core (http://research.nhgri.nih.gov/bic/). To deter- mine if methylated BRCA1 could be detected in breast cancer cell lines, two cell lines, MCF-7 and MDA-MB-231, were analyzed. BRCA1 was immunoprecipitated and western blot analysis performed with anti-K methyl, anti-R methyl and anti-BRCA1 antibodies. Results in Figure 1b indicated that BRCA1 is methylated on both K and R residues in MDA-MB-231 cells, but only R methylation could be detected in MCF-7 cells. These cell lines have very distinct characteristics, with MDA-MB-231 being triple negative and MCF-7 being ER and PR positive. In addition, MDA-MB-231 is a highly metastatic cell line, whereas MCF-7 is not. To demonstrate that the methyl band observed is specific to BRCA1, BRCA1 was immunoprecipitated from MDA-MB-231 and UWB1.289 BRCA1 negative cells (Figure 1c). No methyl band was detected in UWB1.289 cells, indicating that the methylated band is specific to BRCA1. To further characterize BRCA1 methylation status, MDA-MB-231 cells were synchronized by nocodazole treatment and cell populations collected at various stages of the cell cycle. Results in Figure 1d indicate that R methylation can be detected throughout the cell cycle with no drastic changes, suggesting that this PTM may not be cell cycle regulated. Synchronization was verified by western blotting for cyclin B, cyclin D1, cyclin E (Figure 1e), with cdk4 and actin serving as controls. Collectively, these results indicate that BRCA1 is methylated in breast cancer cell lines. This is the first time to our knowledge that BRCA1 has been shown to be methylated. BRCA1 is methylated in ex vivo patient samples To determine if BRCA1 is also methylated in ex vivo patient samples, four different breast tumor tissue samples (BT1-4) were tested. Interestingly, 3 of the 4 samples were triple negative breast cancers (BT1, 3, and 4), which are extremely difficult to treat. BT3 was ER positive, PR negative and does not have HER-2 overexpression. We immunoprecipitated BRCA1 from BT1-4 and western blotted with anti-K methyl, anti-R methyl and anti- BRCA1 antibodies (Figure 2a). Our results indicate that BRCA1 is methylated at both K and R residues (lanes 1–4) in all four breast tumor patient samples, while no methylation was observed with the negative control IgG IP (lane 5). To investigate methylation status in normal tissue, a matched tumor sample was equally processed and immunoblotted with anti-R methyl antibody. BRCA1 appears to be expressed and methylated in higher amounts in the one normal tissue examined (Figure 2b, compare lanes 2 and 3). Of interest, BRCA1 levels were not detected when western blotting the non-immunoprecipiated material, indicating that these tissues samples express very low levels of BRCA1. Tumor samples 11559-1 is an ER positive, PR negative, non- overexpressed HER-2 invasive ductal carcinoma, similar to BT2 in panel 2a. Larger sample sizes are necessary to determine if BRCA1 methylation occurs more frequently in particular types of breast cancer or in normal breast tissue. These preliminary results demonstrate that both K- and R-methylation are detected in normal and breast tumor tissue samples. PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11379