Laboratoire de Biologie Moléculaire et Cellulaire de Cancer, Hôpital Kirchberg, Luxembourg.
Biochem Pharmacol. 2011 Jun 1;81(11):1329-42. doi: 10.1016/j.bcp.2011.03.014. Epub 2011 Mar 29.
Glutathione-S-transferase P1 (GSTP1) gene is commonly silenced by CpG island promoter hypermethylation in prostate, breast, and liver cancers. However, mechanisms leading to GSTP1 repression by promoter hypermethylation in leukemia and its relationship with pathological alterations of the chromatin structure remain poorly understood. A panel of leukemia cell lines was analyzed for their GSTP1 expression, revealing cell lines with high, moderate or no detectable GSTP1 expression. Bisulfite sequencing, methylation-specific PCR and combined bisulfite restriction analysis revealed that GSTP1 promoter was completely methylated in transcriptionally inactive RAJI and MEG-01 cell lines. In contrast, cell lines expressing GSTP1 exhibited an unmethylated and transcriptionally active promoter. Furthermore, histone marks and effector proteins associated with transcriptional activity were detected by chromatin immunoprecipitation in the GSTP1 expressing hypomethylated K-562 cell line. However, repressive chromatin marks and the recruitment of silencing protein complexes were found in the non-expressing hypermethylated RAJI and MEG-01 cell lines. Finally, we provide evidence that treatment of RAJI and MEG-01 cells with the DNA demethylating agent, 5-aza-2'-deoxycytidine, resulted in GSTP1 promoter demethylation, drastic changes of histone modifications and promoter associated proteins and GSTP1 gene activation. In contrast, treatments with HDAC inhibitors failed to demethylate and reactivate the GSTP1 gene. Our study extends the knowledge on leukemia-specific epigenetic alterations of GSTP1 gene. Furthermore, we are showing the correlation of DNA methylation and histone modifications with the positive/negative GSTP1 transcriptional expression state. Finally, these data support the concept of the dominance of DNA methylation over HDAC inhibitor-sensitive histone deacetylation in gene silencing.
谷胱甘肽 S-转移酶 P1（GSTP1）基因通常在前列腺癌、乳腺癌和肝癌中因 CpG 岛启动子超甲基化而沉默。然而，导致白血病中启动子超甲基化导致 GSTP1 抑制的机制及其与染色质结构的病理改变的关系仍知之甚少。分析了一组白血病细胞系，以检测其 GSTP1 表达，揭示了 GSTP1 表达高、中或低的细胞系。亚硫酸氢盐测序、甲基特异性 PCR 和联合亚硫酸氢盐限制分析显示，转录不活跃的 RAJI 和 MEG-01 细胞系中 GSTP1 启动子完全甲基化。相比之下，表达 GSTP1 的细胞系表现出未甲基化和转录活跃的启动子。此外，通过 GSTP1 表达的低甲基化 K-562 细胞系中的染色质免疫沉淀检测到与转录活性相关的组蛋白标记和效应蛋白。然而，在非表达的高甲基化 RAJI 和 MEG-01 细胞系中发现了抑制性染色质标记和沉默蛋白复合物的募集。最后，我们提供的证据表明，用 DNA 去甲基化剂 5-氮杂-2'-脱氧胞苷处理 RAJI 和 MEG-01 细胞导致 GSTP1 启动子去甲基化、组蛋白修饰和启动子相关蛋白的剧烈变化，并激活 GSTP1 基因。相比之下，用 HDAC 抑制剂处理未能使 GSTP1 基因去甲基化和重新激活。我们的研究扩展了关于白血病中 GSTP1 基因特异性表观遗传改变的知识。此外，我们正在显示 DNA 甲基化与组蛋白修饰与 GSTP1 转录表达阳性/阴性状态的相关性。最后，这些数据支持 DNA 甲基化在基因沉默中优于 HDAC 抑制剂敏感的组蛋白去乙酰化的概念。
