Methylation of the Tumor Suppressor Protein, BRCA1, Influences Its Transcriptional Cofactor Function Irene Guendel1,4, Lawrence Carpio1,4, Caitlin Pedati1, Arnold Schwartz2, Christine Teal3, Fatah Kashanchi1,4, Kylene Kehn-Hall4* 1 Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University Medical Center, Washington, D. C., United States of America, 2 Department of Pathology, The George Washington University Medical Center, Washington, D. C., United States of America, 3 Breast Care Center, The George Washington University Medical Center, Washington, D. C., United States of America, 4 Department of Molecular and Microbiology, National Center for Biodefense & Infectious Diseases, George Mason University, Manassas, Virginia, United States of America Abstract Background: Approximately half of hereditary breast cancers have mutations in either BRCA1 or BRCA2. BRCA1 is a multifaceted tumor suppressor protein that has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. This multifunctional nature of BRCA1 is achieved by exerting its many effects through modulation of transcription. Many cellular events are dictated by covalent modification of proteins, an important mechanism in regulating protein and genome function; of which protein methylation is an important posttranslational modification with activating or repressive effects. Methods/Principal Findings: Here we demonstrate for the first time that BRCA1 is methylated both in breast cancer cell lines and breast cancer tumor samples at arginine and lysine residues through immunoprecipitation and western blot analysis. Arginine methylation by PRMT1 was observed in vitro and the region of BRCA1 504–802 shown to be highly methylated. PRMT1 was detected in complex with BRCA1 504–802 through in vitro binding assays and co-immunoprecipitated with BRCA1. Inhibition of methylation resulted in decreased BRCA1 methylation and alteration of BRCA1 binding to promoters in vivo as shown through chromatin immunoprecipitation assays. 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. Conclusions/Significance: These results suggest that methylation may influence either the ability of BRCA1 to bind to specific promoters or protein-protein interactions which alters the recruitment of BRCA1 to these promoters. Thus, given the importance of BRCA1 to genomic stability, methylation of BRCA1 may ultimately affect the tumor suppressor ability of BRCA1. Citation: Guendel I, Carpio L, Pedati C, Schwartz A, Teal C, et al. (2010) Methylation of the Tumor Suppressor Protein, BRCA1, Influences Its Transcriptional Cofactor Function. PLoS ONE 5(6): e11379. doi:10.1371/journal.pone.0011379 Editor: Joseph Alan Bauer, Bauer Research Foundation, United States of America Received December 17, 2009; Accepted June 8, 2010; Published June 29, 2010 Copyright: 2010 Guendel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an American Cancer Society Seed Grant from The George Washington University’s Cancer Institute to KKH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kkehnhal@gmu.edu Introduction BRCA1 is a tumor suppressor protein that has implications in processes such as cell cycle, transcription, DNA damage response, and chromatin remodeling. One way to explain the multifaceted nature of BRCA1 is that it exerts its many effects through modulating transcription of various factors. BRCA1 was first implicated in transcription when the C-terminus (amino acid, aa, 1560–1863) fused to Gal4 was able to activate transcription [1], with aa 1760–1863 being the minimal transactivation domain (TAD). Within this TAD are two BRCA1 C-terminus (BRCT) motifs that are found in a large family of proteins important for DNA damage response, such as DNA ligase IV, p53BP1, and base excision response scaffold protein XRCC1 [2]. Since that time, numerous other findings have served to strengthen the connection between transcription and BRCA1. For instance, BRCA1 is part of the RNA polymerase II holoenzyme complex [3,4,5]. BRCA1 also interacts with multiple cofactors and transcription factors, including CBP/p300, Sp1, STAT1, Estrogen Receptor and BRG1 [6,7]. Among the genes found to be transactivated by BRCA1 are MDM2, BAX, p21/WAF1, p27/KIP1, and GADD45a with p21/ WAF1 and GADD45a transactivation being independent of p53 [8,9,10,11,12,13]. Proteins involved in transcription are often regulated through posttranslational modifications (PTMs), such as acetylation and methylation. Acetylation is broadly linked with activation, whereas methylation can have either activating or repressing effects depending on the amino acid that is modified and the nature of the modification (mono-, di-, or tri-methylation). One well known examples of this is the transcription factor p53, where K372 monomethylation and K370 dimethylation result in stabilization and activation of p53 versus K370 and K382 monomethylation, PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11379