adding 20 ml of 5M NaCl and incubating elutes at 65uC overnight. The next day, proteinase K (100 mg/ml final concentration) was added and samples incubated at 55uC for 1 hour. Samples were extracted with phenol:chloroform twice and ethanol precipitated overnight. Pellets were then washed with 70% ethanol, dried, resuspended in 50 ml of TE and assayed by PCR. Thirty-five cycles of PCR were performed in 50 ml with 10 ml of immuno- precipitated material, 0.1 mM of primers, 0.2 mM dNTPs, and 1 unit of Taq DNA polymerase. Finally, PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. References 1. Monteiro AN, August A, Hanafusa H (1996) Evidence for a transcriptional activation function of BRCA1 C-terminal region. Proc Natl Acad Sci U S A 93: 13595–13599. 2. Glover JN, Williams RS, Lee MS (2004) Interactions between BRCT repeats and phosphoproteins: tangled up in two. Trends Biochem Sci 29: 579–585. 3. Scully R, Anderson SF, Chao DM, Wei W, Ye L, et al. (1997) BRCA1 is a component of the RNA polymerase II holoenzyme. Proc Natl Acad Sci U S A 94: 5605–5610. 4. Krum SA, Miranda GA, Lin C, Lane TF (2003) BRCA1 associates with processive RNA polymerase II. J Biol Chem 278: 52012–52020. 5. Anderson SF, Schlegel BP, Nakajima T, Wolpin ES, Parvin JD (1998) BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A. Nat Genet 19: 254–256. 6. Pao GM, Janknecht R, Ruffner H, Hunter T, Verma IM (2000) CBP/p300 interact with and function as transcriptional coactivators of BRCA1. Proc Natl Acad Sci U S A 97: 1020–1025. 7. Rosen EM, Fan S, Pestell RG, Goldberg ID (2003) BRCA1 gene in breast cancer. J Cell Physiol 196: 19–41. 8. Ouchi T, Monteiro AN, August A, Aaronson SA, Hanafusa H (1998) BRCA1 regulates p53-dependent gene expression. Proc Natl Acad Sci U S A 95: 2302–2306. 9. Somasundaram K, Zhang H, Zeng YX, Houvras Y, Peng Y, et al. (1997) Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiP1. Nature 389: 187–190. 10. Jin S, Zhao H, Fan F, Blanck P, Fan W, et al. (2000) BRCA1 activation of the GADD45 promoter. Oncogene 19: 4050–4057. 11. Harkin DP, Bean JM, Miklos D, Song YH, Truong VB, et al. (1999) Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expres- sion of BRCA1. Cell 97: 575–586. 12. Williamson EA, Dadmanesh F, Koeffler HP (2002) BRCA1 transactivates the cyclin-dependent kinase inhibitor p27(Kip1). Oncogene 21: 3199–3206. 13. Zhang H, Somasundaram K, Peng Y, Tian H, Bi D, et al. (1998) BRCA1 physically associates with p53 and stimulates its transcriptional activity. Oncogene 16: 1713–1721. 14. Sims RJ, 3rd, Reinberg D (2008) Is there a code embedded in proteins that is based on post-translational modifications? Nat Rev Mol Cell Biol 9: 815–820. 15. Herrmann F, Pably P, Eckerich C, Bedford MT, Fackelmayer FO (2009) Human protein arginine methyltransferases in vivo–distinct properties of eight canonical members of the PRMT family. J Cell Sci 122: 667–677. 16. McBride AE, Silver PA (2001) State of the arg: protein methylation at arginine comes of age. Cell 106: 5–8. 17. Pahlich S, Zakaryan RP, Gehring H (2006) Protein arginine methylation: Cellular functions and methods of analysis. Biochim Biophys Acta 1764: 1890–1903. 18. Tang J, Frankel A, Cook RJ, Kim S, Paik WK, et al. (2000) PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells. J Biol Chem 275: 7723–7730. 19. Gary JD, Lin WJ, Yang MC, Herschman HR, Clarke S (1996) The predominant protein-arginine methyltransferase from Saccharomyces cerevisiae. J Biol Chem 271: 12585–12594. 20. Pahlich S, Bschir K, Chiavi C, Belyanskaya L, Gehring H (2005) Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide. Proteins 61: 164–175. 21. Boisvert FM, Rhie A, Richard S, Doherty AJ (2005) The GAR motif of 53BP1 is arginine methylated by PRMT1 and is necessary for 53BP1 DNA binding activity. Cell Cycle 4: 1834–1841. 22. Boisvert FM, Dery U, Masson JY, Richard S (2005) Arginine methylation of MRE11 by PRMT1 is required for DNA damage checkpoint control. Genes Dev 19: 671–676. 23. Le Romancer M, Treilleux I, Leconte N, Robin-Lespinasse Y, Sentis S, et al. (2008) Regulation of estrogen rapid signaling through arginine methylation by PRMT1. Mol Cell 31: 212–221. 24. Kwak YT, Guo J, Prajapati S, Park KJ, Surabhi RM, et al. (2003) Methylation of SPT5 regulates its interaction with RNA polymerase II and transcriptional elongation properties. Mol Cell 11: 1055–1066. BRCA1 Methylation Acknowledgments We would like to thank Dr. Wainberg for generously providing the GST- PRMT1 construct. Author Contributions Conceived and designed the experiments: IG KKH. Performed the experiments: IG LC CP KKH. Analyzed the data: IG FK KKH. Contributed reagents/materials/analysis tools: AS CT FK. Wrote the paper: IG KKH. 25. Rezai-Zadeh N, Zhang X, Namour F, Fejer G, Wen YD, et al. (2003) Targeted recruitment of a histone H4-specific methyltransferase by the transcription factor YY1. Genes Dev 17: 1019–1029. 26. Cuthbert GL, Daujat S, Snowden AW, Erdjument-Bromage H, Hagiwara T, et al. (2004) Histone deimination antagonizes arginine methylation. Cell 118: 545–553. 27. Chen D, Ma H, Hong H, Koh SS, Huang SM, et al. (1999) Regulation of transcription by a protein methyltransferase. Science 284: 2174–2177. 28. Wang Y, Wysocka J, Sayegh J, Lee YH, Perlin JR, et al. (2004) Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306: 279–283. 29. Wang H, Huang ZQ, Xia L, Feng Q, Erdjument-Bromage H, et al. (2001) Methylation of histone H4 at arginine 3 facilitating transcriptional activation by nuclear hormone receptor. Science 293: 853–857. 30. Bedford MT, Frankel A, Yaffe MB, Clarke S, Leder P, et al. (2000) Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains. J Biol Chem 275: 16030–16036. 31. Yu MC, Bachand F, McBride AE, Komili S, Casolari JM, et al. (2004) Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors. Genes Dev 18: 2024–2035. 32. Yun CY, Fu XD (2000) Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae. J Cell Biol 150: 707–718. 33. Dillon SC, Zhang X, Trievel RC, Cheng X (2005) The SET-domain protein superfamily: protein lysine methyltransferases. Genome Biol 6: 227. 34. Kakimoto Y (1971) Methylation of arginine and lysine residues of cerebral proteins. Biochim Biophys Acta 243: 31–37. 35. Kakimoto Y, Akazawa S (1970) Isolation and identification of N-G,N-G- and N- G,N’-G-dimethyl-arginine, N-epsilon-mono-, di-, and trimethyllysine, and glucosylgalactosyl- and galactosyl-delta-hydroxylysine from human urine. J Biol Chem 245: 5751–5758. 36. Bhaumik SR, Smith E, Shilatifard A (2007) Covalent modifications of histones during development and disease pathogenesis. Nat Struct Mol Biol 14: 1008–1016. 37. Paik WK, Kim S (1967) Enzymatic methylation of protein fractions from calf thymus nuclei. Biochem Biophys Res Commun 29: 14–20. 38. Smith JJ, Rucknagel KP, Schierhorn A, Tang J, Nemeth A, et al. (1999) Unusual sites of arginine methylation in Poly(A)-binding protein II and in vitro methylation by protein arginine methyltransferases PRMT1 and PRMT3. J Biol Chem 274: 13229–13234. 39. Mark WY, Liao JC, Lu Y, Ayed A, Laister R, et al. (2005) Characterization of segments from the central region of BRCA1: an intrinsically disordered scaffold for multiple protein-protein and protein-DNA interactions? J Mol Biol 345: 275–287. 40. Paull TT, Cortez D, Bowers B, Elledge SJ, Gellert M (2001) Direct DNA binding by Brca1. Proc Natl Acad Sci U S A 98: 6086–6091. 41. Cable PL, Wilson CA, Calzone FJ, Rauscher FJ, 3rd, Scully R, et al. (2003) Novel consensus DNA-binding sequence for BRCA1 protein complexes. Mol Carcinog 38: 85–96. 42. Kehn K, Berro R, Alhaj A, Bottazzi ME, Yeh WI, et al. (2007) Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells. Oncogene 26: 5060–5069. 43. Chen DH, Wu KT, Hung CJ, Hsieh M, Li C (2004) Effects of adenosine dialdehyde treatment on in vitro and in vivo stable protein methylation in HeLa cells. J Biochem 136: 371–376. 44. Bochar DA, Wang L, Beniya H, Kinev A, Xue Y, et al. (2000) BRCA1 is associated with a human SWI/SNF-related complex: linking chromatin remodeling to breast cancer. Cell 102: 257–265. 45. Zhong Q, Chen CF, Li S, Chen Y, Wang CC, et al. (1999) Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response. Science 285: 747–750. 46. Li S, Ku CY, Farmer AA, Cong YS, Chen CF, et al. (1998) Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs. J Biol Chem 273: 6183–6189. 47. Feng Z, Kachnic L, Zhang J, Powell SN, Xia F (2004) DNA damage induces p53-dependent BRCA1 nuclear export. J Biol Chem 279: 28574–28584. PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11379