Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications
Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications
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DOI：
10.1186/1471-2407-10-297
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发表时间：
2010-06-17
期刊：
影响因子：
3.8
通讯作者：
Schwarzenbach, Heidi
中科院分区：
文献类型：
--
作者：
Mueller, Imke;Wischnewski, Frank;Schwarzenbach, Heidi
背景：本研究的目的是分析甲基 CpG 结合蛋白 (MBD) 和组蛋白修饰在不同细胞环境（例如前列腺癌细胞 DU145 和 LNCaP 以及乳腺癌细胞 MCF-7）中对 CD44、细胞周期蛋白 D2、GLIPR1 和 PTEN 的调节的参与。由于肿瘤中存在整体染色质变化，并且肿瘤相关基因区域受到表观遗传修饰的影响，这些可能构成恶性转化发病机制的重要调控机制。方法：通过定量RT-PCR测定DU145、LNCaP和MCF-7细胞中CD44、Cyclin D2、GLIPR1和PTEN的mRNA表达水平。 状态以及用去甲基化剂5-氮杂-2'脱氧胞苷和/或组蛋白脱乙酰酶抑制剂曲古抑菌素A处理后。此外，对基因组DNA进行亚硫酸氢盐转化并测序。使用 MBD1、MBD2 和 MeCP2 抗体以及 17 种不同的组蛋白抗体对刺激和未刺激的细胞进行染色质免疫沉淀。结果：不同启动子的比较表明，MeCP2 和 MBD2a 在所有细胞系中均特异性抑制启动子 Cyclin D2，而在 MCF-7 细胞中，MeCP2 特异性抑制所有甲基化的细胞 发起人。染色质免疫沉淀显示所有甲基化启动子与至少一种 MBD 相关。用去甲基化剂 5-aza-2'-脱氧胞苷 (5-aza-CdR) 处理细胞导致 MBD 从启动子上解离。只有 MBD1v1 独立地结合并抑制所有启动子的甲基化。由 17 种不同抗体免疫沉淀的 DNA 实时扩增显示，组蛋白 H3（H3K4me1、H3K4me2 和 H3K4me3）的甲基化赖氨酸在特定启动子处优先富集。值得注意的是，沉默启动子与未修饰的组蛋白相关，这些组蛋白在 5-aza-CdR 处理后被乙酰化。结论：这项研究是第一个揭示不同肿瘤细胞中 CD44、Cyclin D2、GLIPR1 和 PTEN 启动子的组蛋白密码和 MBD 谱以及甲基化抑制剂 5aza-CdR 刺激后相关变化的研究之一。
Background : The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs) and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation.Methods : In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'deoxycytidine and/ or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies.Results : Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3) at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR.Conclusions : This study is one of the first to reveal the histone code and MBD profile at the promoters of CD44, Cyclin D2, GLIPR1 and PTEN in different tumour cells and associated changes after stimulation with methylation inhibitor 5aza-CdR.