Evidence that silencing of the HPRT promoter by DNA methylation is mediated by critical CpG sites

被引:41
作者
Chen, C
Yang, MCK
Yang, TP
机构
[1] Univ Florida, Coll Med, Dept Biochem & Mol Biol, Gainesville, FL 32610 USA
[2] Univ Florida, Coll Med, Ctr Mammalian Genet, Gainesville, FL 32610 USA
[3] Univ Florida, Coll Med, Div Pediat Genet, Gainesville, FL 32610 USA
[4] Univ Florida, Coll Med, Dept Stat, Gainesville, FL 32610 USA
关键词
D O I
10.1074/jbc.M007096200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The strong correlation between promoter hypermethylation and gene silencing suggests that promoter methylation represses transcription. To identify methylation sites that may be critical for maintaining repression of the human HPRT gene, we treated human/hamster hybrid cells containing an inactive human X chromosome with the DNA demethylating agent 5-azadeoxycytidine (5aCdr), and we then examined the high resolution methylation pattern of the HPRT promoter in single cell-derived lines. Reactivation of HPRT correlated with complete promoter demethylation, In contrast, the 61 BaCdr-treated clones that failed to reactivate HPRT exhibited sporadic promoter demethylation. However, three specific CpG sites remained methylated in all unreactivated clones, suggesting these sites may be critical for maintaining transcriptional silencing of the HPRT gene. Re-treatment of partially demethylated land unreactivated) clones with a second round of 5aCdr did not increase the frequency of HPRT reactivation. This is consistent with mechanisms of methylation-mediated repression requiring methylation at specific critical sites and argues against models invoking overall levels or a threshold of promoter methylation. Treatment of cells with the histone deacetylase inhibitor, trichostatin A, failed to reactivate HPRT on the inactive X chromosome, even when the promoter was partially demethylated by 5aCdr treatment, suggesting that transcriptional repression by DNA methylation is unlikely to depend upon a trichostatin A-sensitive histone deacetylase.
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页码:320 / 328
页数:9
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