BRCA1 Methylation Figure 2. Methylation of BRCA1 in ex vivo breast tumor samples. (a) Solid breast tumor tissue was ground with a mortar and pestle in the presence of liquid nitrogen to create a powdered tissue. RIPA buffer was added to the powdered tissue, the sample vortexed for 60 seconds, and placed on ice for 45 minutes. Samples were homogenized with a syringe and needle, followed by centrifugation at 14,000 g for 10 minutes. Two milligram of whole cell protein extracts from four different breast tumor samples (BT1-4) 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 an anti-K methyl, anti-R methyl and anti-BRCA1 antibodies. Results are representative of two independent experiments. Characteristics of tumor tissues are as follows: BT1: infiltrating ductal carcinoma, age 55, African American, ER negative, PR negative, HER2 negative, p53 negative. BT2: infiltrating lobular carcinoma, age 83, Caucasian, ER positive, PR negative, HER2 negative, p53 negative. BT3: variant papillary serous type of ductal carcinoma, age 54, African American, ER negative, PR negative, HER2 negative, p53 negative. BT4: infiltrating ductal carcinoma, age 68, Caucasian, ER negative, PR negative, HER2 negative, p53 positive. (b) Matched breast tumor tissue was processed as described. Four milligram of whole cell protein extract 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 an anti-R methyl and anti-BRCA1 antibodies. Input represents 1/5 of immunoprecipitated material. doi:10.1371/journal.pone.0011379.g002 minimal methylation region. Of interest, MeMo-predicted methyl- ation site and flanking residues of human BRCA1 600IHNS- KAPKKNRLRRKSSTRH619 are highly conserved in all sequenc- es analyzed, even in rodent BRCA1 sequences which presented approximately 55 percent identity to human BRCA1 (Figure 3c). To confirm an interaction between PRMT1 and BRCA1, all the GST-BRCA1 constructs utilized for the methyltransferase assay were used for a pull-down assay with total cell lysates from HeLa cells. HeLa cells were chosen as they display high levels of PRMT1 expression in contrast to other breast cancer cell lines (data not shown). An anti-PRMT1 western blot of the GST pull- down revealed that the only detectable interaction between PRMT1 and BRCA1 was occurring at the previously identified region of 504–802 (Figure 3d). To further corroborate this interaction, whole cell extracts were prepared and immunopre- cipitated with BRCA1 or IgG and western blotted with anti- PRMT1. BRCA1-PRMT1 interaction was observed specifically with the BRCA1 immunoprecipitation and not with the IgG (Figure 3e). Collectively, these results indicate that PRMT1 methylates BRCA1 in vitro, that the region of 504–802 is methylated, and that physical interaction of PRMT1-BRCA1 is only detectable at the 504–802 region of BRCA1. Methylation of BRCA1 alters promoter binding in vivo The internal region of BRCA1 (aa 452–1079) contains two DNA binding domains, DB1 at aa 498–663 and DB2 at aa 936–1057 [39]. BRCA1 binds to branched DNA structures [40] as well as the TTC(G/T)GTTG consensus sequence [41]. Therefore, it can influence DNA damage repair pathways such as homologous recombination as well as being a transcriptional cofactor. We have previously shown that BRCA1 can bind to eight novel promoters containing the TTC(G/T)GTTG consensus site [42] and wondered whether methylation of BRCA1 influences the binding of BRCA1 to these promoters. To this end, MDA-MB-231 cells were treated with the methyltransferase inhibitor adenosine dialdehyde (AdOx) [43]. AdOx treatment resulted in decreased BRCA1 arginine methylation (Figure 4a), further verifying that BRCA1 is methylated and providing a system to test the influence of methylation on the function of BRCA1. ChIP assays from untreated and AdOx treated cells were performed using antibodies against histone H3-pS10 (positive control), IgG (negative control) and BRCA1. Interestingly, BRCA1 binding was increased at the AP endonuclease (APEX), Ras homolog gene family member G (ARHG) and the growth arrest and DNA damage inducible family member G (GADD45G) when BRCA1 is hypo- methylated (Figure 4b). Conversely, estrogen receptor beta (ESR2), G- PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11379