甲基化 CpG 结合蛋白和组蛋白修饰对 CD44、Cyclin D2、GLIPR1 和 PTEN 的启动子和细胞特异性表观遗传调控。
Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by methyl-CpG binding proteins and histone modifications.
机构信息
Department of Tumour Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
出版信息
BMC Cancer. 2010 Jun 17;10:297. doi: 10.1186/1471-2407-10-297.
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 5-aza-CdR.
背景
本研究旨在分析甲基化-CpG 结合蛋白（MBDs）和组蛋白修饰在不同细胞环境中对 CD44、Cyclin D2、GLIPR1 和 PTEN 的调控作用，这些细胞环境包括前列腺癌细胞 DU145 和 LNCaP 以及乳腺癌细胞 MCF-7。由于已经表明肿瘤中会发生全基因组染色质改变，并且肿瘤相关基因区域会受到表观遗传修饰的影响，因此这些可能是恶性转化发病机制的重要调控机制。
方法
在 DU145、LNCaP 和 MCF-7 细胞中，通过定量 RT-PCR 在基础状态以及用去甲基化剂 5-氮杂-2′-脱氧胞苷和/或组蛋白去乙酰化酶抑制剂 Trichostatin A 处理后，测定 CD44、Cyclin D2、GLIPR1 和 PTEN 的 mRNA 表达水平。此外，还对基因组 DNA 进行了亚硫酸氢盐测序。使用针对 MBD1、MBD2 和 MeCP2 以及 17 种不同组蛋白抗体的刺激和未刺激细胞进行染色质免疫沉淀。
结果
对不同启动子的比较表明，MeCP2 和 MBD2a 在所有细胞系中特异性地抑制 Cyclin D2 的启动子，而在 MCF-7 细胞中，MeCP2 特异性地抑制所有甲基化的启动子。染色质免疫沉淀显示，所有与甲基化相关的启动子都至少与一个 MBD 相关。用去甲基化剂 5-氮杂-2′-脱氧胞苷（5-aza-CdR）处理细胞会导致 MBD 从启动子上解离。只有 MBD1v1 结合并独立于甲基化抑制所有启动子。用 17 种不同抗体免疫沉淀的 DNA 的实时扩增显示，在特定启动子上，组蛋白 H3 的甲基化赖氨酸（H3K4me1、H3K4me2 和 H3K4me3）优先富集。值得注意的是，沉默的启动子与未修饰的组蛋白相关，这些组蛋白在经过 5-aza-CdR 处理后乙酰化。
结论
本研究是首次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 图谱以及用甲基化抑制剂 5-aza-CdR 刺激后的相关变化。
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