Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University Medical Center, Washington, DC, United States of America.
PLoS One. 2010 Jun 29;5(6):e11379. doi: 10.1371/journal.pone.0011379.
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.
大约一半的遗传性乳腺癌有 BRCA1 或 BRCA2 的突变。BRCA1 是一种多方面的肿瘤抑制蛋白，它在细胞周期、转录、DNA 损伤反应和染色质重塑等过程中都有影响。BRCA1 的这种多功能性质是通过调节转录来发挥其许多作用来实现的。许多细胞事件都是由蛋白质的共价修饰决定的，这是调节蛋白质和基因组功能的重要机制；其中蛋白质甲基化是一种重要的翻译后修饰，具有激活或抑制作用。
方法/主要发现：在这里，我们首次证明 BRCA1 在乳腺癌细胞系和乳腺癌肿瘤样本中通过免疫沉淀和 Western blot 分析在精氨酸和赖氨酸残基上被甲基化。体外观察到 PRMT1 介导的精氨酸甲基化，并且 BRCA1 的 504-802 区域被证明高度甲基化。通过体外结合实验检测到 BRCA1 504-802 与 PRMT1 复合物的形成，并与 BRCA1 共免疫沉淀。抑制甲基化导致 BRCA1 甲基化减少，并改变体内 BRCA1 与启动子的结合，如通过染色质免疫沉淀实验所示。PRMT1 的敲低也导致体内 BRCA1 与特定启动子的结合增加。最后，在抑制甲基化后，发现 Sp1 优先与低甲基化的 BRCA1 结合，而 STAT1 优先与高甲基化的 BRCA1 结合。
结论/意义：这些结果表明，甲基化可能影响 BRCA1 与特定启动子结合的能力，或改变 BRCA1 与这些启动子结合的蛋白质-蛋白质相互作用。因此，鉴于 BRCA1 对基因组稳定性的重要性，BRCA1 的甲基化可能最终影响 BRCA1 的肿瘤抑制能力。
