BRCA1 Methylation Figure 5. Decreased levels of PRMT1 alters BRCA1 promoter binding in vivo. (a) HeLa cells were transfected with different concentrations of PRMT1 siRNA (10, 25, 50 nM) following manufacturer’s instructions. Results are representative of two independent experiments. (b) HeLa cells transfected with 50 nM Luc or PRMT1 siRNA were collected for ChIP analysis. Anti-BRCA1 (10 mg), anti-IgG (10 mg), and anti-histone H3- phosphorylated at S10 (H3-pS10, 5 mg) antibodies were used for ChIP analysis. PCR products were run on a 2% agarose gel and visualized with ethidium bromide staining. Results are representative of two independent experiments. doi:10.1371/journal.pone.0011379.g005 Methylation of BRCA1 alters protein-protein interactions BRCA1 is known to participate in many protein-protein interactions. A schematic diagram of the BRCA1 504–802 protein region is displayed in Figure 6a to indicate protein-protein interactions that have been reported within the region and thus interactions that may be affected by the methylation status of BRCA1. These include transcriptional coactivator BRG1 [44], DNA repair protein RAD50 [45], regulators of nuclear BRCA1 transport importin-a and BRAP2 [46,47], centrosome and microtubule component c-tubulin [48], and transcription factors Sp1 and STAT1 [49,50]. To investigate whether hyper- or hypo- methylation status of BRCA1 interfered with protein-protein interactions, AdOx treated MDA-MB-231 whole cell protein extracts were immunoprecipitated with anti-BRCA1 or anti-IgG antibodies. Immunoblotting of electrophoresed proteins revealed Sp1 preferentially bound to hypo-methylated BRCA1 (Figure 6b, compare lanes 1 and 2). To determine if AdOx has an effect in the abundance of Sp1 (as observed in Figure 6b, lanes 5 and 6), MDA- MB-231 cells were subjected to AdOx treatment and immuno- blotted with anti-Sp1 (Figure 6c). Densitometry from three independent experiments indicates that Sp1 total levels tend to decrease in AdOx treated cells, highlighting the relevance of observed increase in BRCA1-Sp1 interaction in Figure 6b lanes 1 and 2. In contrast, we observed STAT1 preferentially associated with hyper-methylated BRCA1 (data not shown). These results suggest that methylation of BRCA1 affects protein-protein interactions. Discussion The molecular function of BRCA1 has been subject of focused studies since it was cloned in 1994 [51]. Extensive studies have characterized BRCA1 as a multifaceted tumor suppressor protein due to its role in cell cycle progression, DNA repair and DNA damage response processes, transcriptional pathway regulation and apoptosis [7]. BRCA1 is regulated through phosphorylation PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11379