POLYMERASE CHAIN REACTION-AIDED GENOMIC SEQUENCING OF AN X-CHROMOSOME-LINKED CPG ISLAND - METHYLATION PATTERNS SUGGEST CLONAL INHERITANCE, CPG SITE AUTONOMY, AND AN EXPLANATION OF ACTIVITY STATE STABILITY
POLYMERASE CHAIN REACTION-AIDED GENOMIC SEQUENCING OF AN X-CHROMOSOME-LINKED CPG ISLAND - METHYLATION PATTERNS SUGGEST CLONAL INHERITANCE, CPG SITE AUTONOMY, AND AN EXPLANATION OF ACTIVITY STATE STABILITY
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DOI：
10.1073/pnas.87.21.8252
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发表时间：
1990-11-01
影响因子：
11.1
通讯作者：
RIGGS, AD
中科院分区：
文献类型：
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作者：
PFEIFER, GP;STEIGERWALD, SD;RIGGS, AD
编码人X染色体连接磷酸甘油酸激酶1（PGK 1）的基因的5“区域是一个含有启动子的CpG岛，已知在仓鼠-人细胞系X8- 6 T2中无活性人X染色体上450个碱基对区域的121个CpG二核苷酸中的119个被甲基化。在这里，我们报告使用聚合酶链反应辅助基因组测序，以确定该区域的完整甲基化模式的克隆来自X8- 6 T2细胞后，与甲基化抑制剂5“-氮杂胞苷处理。我们发现（i）显示人磷酸甘油酸激酶完全表达的克隆在该区域中完全未甲基化;（ii）不表达人磷酸甘油酸激酶的克隆在约90 °处保持甲基化。50%的CpG位点，其中散布的甲基化（M）和未甲基化（U）位点的模式对于每个克隆不同;（iii）单个，定义为M-U-M或U-M-U，是常见的;和（iv）一些CpG位点是部分甲基化的。根据多个自主CpG位点的模型解释数据，并对两个关键参数进行估计，即维持效率（Em approxeq.每代每个位点99.9%）和从头甲基化效率（艾德. apprxeq. 5%）。这些参数值和几个独立的位点必须是非甲基化的转录的假设可以解释稳定的维持X染色体失活。我们还考虑了活性区域是如何保持甲基化的，并建议转录通过降低Em来抑制甲基化，从而使甲基化不能维持。因此，相对于动态甲基化系统独立的多个CpG位点可以稳定甲基化和转录的两种替代状态。
The 5'' region of the gene encoding human X chromosome-linked phosphoglycerate kinase 1 (PGK1) is a promoter-containing CpG island known to be methylated at 119 of 121 CpG dinucleotides in a 450-base-pair region on the inactive human X chromosome in the hamster-human cell line X8-6T2. Here we report the use of polymerase chain reaction-aided genomic sequencing to determine the complete methylation pattern of this region in clones derived from X8-6T2 cells after treatment with the methylation inhibitor 5''-azacytidine. We find (i) a clone showing full expression of human phosphoglycerate kinase is fully unmethylated in this region; (ii) clones not expressing human phosphoglycerate kinase remain methylated at .apprxeq. 50% of CpG sites, with a pattern of interspersed methylated (M) and unmethylated (U) sites different for each clone; (iii) singles, defined as M-U-M or U-M-U, are common; and (iv) a few CpG sites are partially methylated. The data are interpreted according to a model of multiple, autonomous CpG sites, and estimates are made for two key parameters, maintenance efficiency (Em .apprxeq. 99.9% per site per generation) and de novo methylation efficiency (Ed .apprxeq. 5%). These parameter values and the hypothesis that several independent sites must be unmethylated for transcription can explain the stable maintenance of X chromosome inactivation. We also consider how the active region is kept free of methylation and suggest that transcription inhibits methylation by decreasing Em so that methylation cannot be maintained. Thus, multiple CpG sites, independent with respect to a dynamic methylation system, can stabilize two alternative states of methylation and transcription.