METHYLATION-RELATED CHROMATIN STRUCTURE IS ASSOCIATED WITH EXCLUSION OF TRANSCRIPTION FACTORS FROM AND SUPPRESSED EXPRESSION OF THE O-6-METHYLGUANINE DNA METHYLTRANSFERASE GENE IN HUMAN GLIOMA CELL-LINES
METHYLATION-RELATED CHROMATIN STRUCTURE IS ASSOCIATED WITH EXCLUSION OF TRANSCRIPTION FACTORS FROM AND SUPPRESSED EXPRESSION OF THE O-6-METHYLGUANINE DNA METHYLTRANSFERASE GENE IN HUMAN GLIOMA CELL-LINES
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DOI：
10.1128/mcb.14.10.6515
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发表时间：
1994-10-01
影响因子：
5.3
通讯作者：
PIEPER, RO
中科院分区：
文献类型：
--
作者：
COSTELLO, JF;FUTSCHER, BW;PIEPER, RO
由于MGMT是神经胶质瘤细胞对化疗药物1，3-双（2-氯乙基）-1-亚硝基脲反应的主要决定因素，因此，对确定O-6-甲基鸟嘌呤DNA甲基转移酶（MGMT）基因表达的因子有相当大的兴趣。最近，我们已经表明，MGMT的表达是在一个直接的，分级的方式与甲基化的MGMT基因的身体和在一个反向的，分级的方式与启动子甲基化在人类胶质瘤细胞系。为了确定启动子甲基化是否是MGMT表达的重要组成部分，本研究探讨了具有不同MGMT表达水平的胶质瘤细胞系的MGMT启动子中甲基化、染色质结构和体内转录因子占用率之间的复杂相互作用。我们的研究结果表明，在表达MGMT的胶质瘤细胞系中的基础启动子是100%未甲基化的，在所有测试的位点处都非常容易被限制性酶接近，这表明该区域可能是无核小体的。然而，在具有最小MGMT表达的神经胶质瘤细胞中，75%未甲基化的基础启动子更难接近，并且在非表达细胞中，50%未甲基化的基础启动子完全不能被限制性酶接近。尽管存在的相关转录因子在所有的细胞系检查，在体内足迹显示在6个Sp1结合位点和一个新的结合位点的MGMT表达细胞系的DNA-蛋白质相互作用，但在nonexpressors没有这样的相互作用。我们的结论是，在以前的体外研究结果相反，Sp1是MGMT转录的重要组成部分。这些相关性也强烈表明，甲基化和染色质结构，通过确定是否Sp1和其他转录因子可以访问MGMT启动子，设置MGMT基因的转录状态。
There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at ail sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined, in vivo footprinting showed DNA-protein interactions at six Sp1 binding sites and one novel binding site in MGMT-expressing cell lines but no such interactions in nonexpressors. We conclude that in contrast to findings of previous in vitro studies, Sp1 is an important component of MGMT transcription. These correlations also strongly suggest that methylation and chromatin structure, by determining whether Sp1 and other transcription factors can access the MGMT promoter, set the transcriptional state of the MGMT gene.