arginine methylation in BRCA1. AdOx inhibits activity of all cellular methyltransferases, thus its effect regarding PRMT1 is non-specific. However, BRCA1-DNA interaction at the APEX, ARHG and GADD45G promoters upon siRNA-mediated knockdown of PRMT1 mimicked the results observed when cells were treated with the methyltransferase inhibitor. The difference between the levels of increased binding observed in AdOx-treated versus PRMT1 knockout samples may be due to compensatory mechanisms by other PRMTs or lysine methylation. Preliminary studies demonstrated that SETDB1, a PKMT member of the SUV39 family of SET-domain containing proteins, methylated BRCA1 in vitro (data not shown). Neither the site of methylation nor the influence of lysine methylation on BRCA1 is known, but will be the focus of future studies. Protein-protein interactions at the BRCA1 504–802 region involve several proteins that are important for transcription pathways and in particular, protein localization. These include BRAP2, importin-a, BRG1, Sp1, STAT1 and c-tubulin. BRCA1 localization also plays an important role in protein function, with both cytoplasmic and nuclear targets. Subsequently, BRCA1 has been found to posses two nuclear localizations signals (NLS) as well as one nuclear export sequence (NES) that guide the shuttling process of BRCA1 [63,64,65]. Albeit, mechanisms for this shuttling process are not clearly understood. Interestingly, both NLS are located in the vicinity of the identified BRCA1 region that is being methylated. Specifically, NSL1 is located at residues 501–507 and NSL2 at residues 606–615. Furthermore, phosphor- ylation of T508 at the Akt concensus phosphorylation motif immediately adjacent NSL1 resulted in cytoplasmic accumulation of BRCA1 [66], suggesting that methylation of this region may possess similar shuttling regulatory mechanisms. PRMT1 is also regulated through nucleo-cytoplasmic shuttling [67,68]. Impor- tantly, enzymatic activity is required for this shuttling process where a catalytically inactive mutant of PRMT1, rapidly accumulates in the nucleus. The nuclear export of PRMT1 is dependent on the release of the enzyme from its substrates following methylation [67]. These findings suggest a dynamic mechanism for the regulation of substrate methylation that is dependent on the methylation status of its substrates, in this case, BRCA1. Upon hypomethylation of BRCA1, increased binding to Sp1 protein was observed. The Sp1 transcription factor is a potent transactivator of the insulin-like growth factor-I receptor (IGF-I-R) gene. Initially, the functional interaction between BRCA1 and Sp1 was suggested to regulate the IGF-I-R, a receptor overexpressed in most breast cancers that serves as an antiapoptotic factor [69,70]. Later, the same group showed that BRCA1 itself does not exhibit any specific binding to the IGF-I-R promoter but instead, it prevented Sp1 binding the promoter by BRCA1-Sp1 interaction at the BRCA1 260–802 region [50]. Moreover, it has been found that BRCA1 gene expression is regulated by the IGF-I signaling pathway where IGF-I enhances BRCA1 promoter activity and that Sp1 is directly involved in BRCA1 gene transactivation [71]. Thus, it is possible that the status of BRCA1 methylation plays a role in the transcriptional regulation of the IGF-I-R gene by BRCA1 and Sp1. Because BRCA1 has an inhibitory control of the IGF-I-R promoter as well as repressing the Sp1-induced transactivation of the IGF-I-R gene, this suggests that methylated BRCA1 state (as observed in our cancer cell lines and breast tumor tissues) allows for decreased BRCA1-Sp1 binding which could be part of a regulatory mechanism of the IGF-I-R gene expression. The in vivo interaction of Sp1 with the IGF-I-R promoter in MDA- MB-231 cells treated or untreated with AdOx still needs to be characterized. We have however, observed decreased BRCA1 BRCA1 Methylation binding at APEX promoter, which is regulated by Sp1. APEX participates in base excision repair through the recognition and the initial step toward removing abasic sites [72]. The APEX promoter contains Sp1 binding sites both upstream and down- stream of the transcriptional start site [73,74,75]. Sp1 binding to the downstream site regulates the expression of APEX in a cell cycle dependent fashion [75]. Interestingly, our results indicate that hypomethylated BRCA1 binds to the APEX promoter and that this form of BRCA1 has increased binding to Sp1. Therefore it is possible that BRCA1 is acting as a transcriptional coactivator for Sp1 mediated APEX transcription. The precise role of BRCA1 in APEX transcription will be the focus of future studies. In our current study we have identified a novel posttranslational modification of BRCA1, namely methylation. We have only begun to elucidate the role of arginine methylation in terms of regulating BRCA1 protein-protein interactions and its transcriptional coactivator function. Future studies will be focused on the identification of the methylated residues, study of lysine methyl- ation, and determining if there is a correlation between BRCA1 methylation and tumor progression. As was the case for BRCA1 phosphorylation, this study is the beginning and multiple future research efforts are needed to uncover the intricate workings of BRCA1 methylation. Materials and Methods Ethics Statement The breast tumor samples were previously collected frozen additional extraneous tissue that resulted following testing of breast cancer samples for the presence of hormone receptors. Samples were collected between 1985 and 1995 for pathologic examination and diagnosis. Existing discarded tissue samples were stripped of all identifying information. Ethical approval for the study was given by the George Washington University IRB Committee based on Federal Regulation 45 CFR 46.101(b) (4). The IRB committee review indicated that informed consent was not required as this was additional tissue left over from routine pathological examination. In silico BRCA1 analysis Predicted methylation sites from the full length primary amino acid sequence of BRCA1 were generated by the freeware MeMo: Methylation Modification Prediction Server (http://www.bioinfo. tsinghua.edu.cn/,tigerchen/memo.html). Sequence analysis was performed using the Clustal W multiple sequence alignment program [76]. Pan troglodytes (NCBI AAR04849), Macaca mulatta (NCBI AAT44833), Pongo pygmaeus (NCBI AAT44834), Gorilla gorilla (NCBI AAT44835), Mus musculus (NCBI AAD00168) and Rattus norvegicus (NCBI AAC36493) sequences were compared to human full length BRCA1 (NCBI NP_009225). Cell culture and breast tumor tissue samples MDA-MD-231 and MCF-7 are epithelial breast carcinoma cells derived from pleural effusions [59]. HeLa is a cervical carcinoma cell line commercially available from the ATCC. All cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 1% L-glutamine, and 1% streptomycin/penicillin. All cells were incubated at 37uC and 5% CO2. Cells were cultured to confluency, washed and pelleted at 4uC for 15 minutes at 3,000 rpm. Pellets were lysed in a buffer containing Tris-HCl pH 7.5, 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM Na3VO4, 1 mM DTT and one tablet complete protease inhibitor cocktail per 50 ml. Lysis was performed under ice-cold PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11379