Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus.
Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus.
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DOI：
10.1093/nar/gki729
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发表时间：
2005
影响因子：
14.9
通讯作者：
Dante R
中科院分区：
文献类型：
--
作者：
Auriol E;Billard LM;Magdinier F;Dante R
甲基 CpG 结合域 (MBD) 蛋白是解释导致基因沉默的 DNA 甲基化信号的关键分子。我们研究了它们在 CpG 岛的组成型甲基化区域的结合特异性，该岛含有乳腺癌易感基因 1、BRCA1 和近 BRCA1 2 (NBR2) 基因的双向启动子。在 HeLa 细胞中，定量染色质免疫沉淀分析表明 MBD2 与甲基化区域相关，而在该位点未检测到 MeCP2 和 MBD1。由表达小干扰 RNA (siRNA) 的转基因介导的 MBD2 耗竭 (∼90%) 不会诱导 MeCP2 或 MBD1 在甲基化区域结合。此外，BRCA1-NBR2 CpG 岛缺乏 MBD2 与 NBR2 转录物水平升高以及诱导的 DNA 低甲基化反应显着减少相关。在 MBD2 敲除细胞中，Mbd2 cDNA 的瞬时表达（难以抵抗 siRNA 介导的衰减）将 NBR2 mRNA 水平下调至未修饰的 HeLa 细胞中观察到的水平。 MBD2 水平的变化并不影响 BRCA1 的表达，尽管它受到 DNA 低甲基化的刺激。总的来说，我们的数据表明 MBD2 具有特定的靶点，并且它在这些靶点上的存在对于基因抑制是必不可少的。
The methyl-CpG binding domain (MBD) proteins are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitutively methylated region of a CpG island containing the bidirectional promoter of the Breast cancer predisposition gene 1, BRCA1, and the Near BRCA1 2 (NBR2) gene. In HeLa cells, quantitative chromatin immunoprecipitation assays indicated that MBD2 is associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. MBD2 depletion (∼90%), mediated by a transgene expressing a small interfering RNA (siRNA), did not induce MeCP2 or MBD1 binding at the methylated area. Furthermore, the lack of MBD2 at the BRCA1-NBR2 CpG island is associated with an elevated level of NBR2 transcripts and with a significant reduction of induced-DNA-hypomethylation response. In MBD2 knockdown cells, transient expression of a Mbd2 cDNA, refractory to siRNA-mediated decay, shifted down the NBR2 mRNA level to that observed in unmodified HeLa cells. Variations in MBD2 levels did not affect BRCA1 expression despite its stimulation by DNA hypomethylation. Collectively, our data indicate that MBD2 has specific targets and its presence at these targets is indispensable for gene repression.
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