High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors.
High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors.
复制标题
活性和非活性 X 染色体上人次黄嘌呤磷酸核糖基转移酶基因 5 区域的高分辨率甲基化分析：与转录因子结合位点的相关性。
DOI：
10.1128/mcb.14.2.1419-1430.1994
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发表时间：
1994
影响因子：
5.3
通讯作者：
Yang,TP
中科院分区：
文献类型：
--
作者：
Hornstra,IK;Yang,TP
组成型表达的X连锁基因的富含GC启动子内的DNA甲基化与雌性哺乳动物中失活X染色体上的转录沉默相关。对于大多数X连锁基因，X染色体失活导致转录活性和非活性等位基因占据每个雌性细胞核。为了研究维持这种独特的差异基因表达系统的机制，我们分析了活性和非活性X染色体上人类次黄嘌呤磷酸核糖基转移酶（HPRT）基因5′ CpG岛上单个胞嘧啶残基的甲基化。使用胞嘧啶特异性Maxam和吉尔伯特DNA测序反应结合连接介导的PCR，对纯化的DNA进行基因组测序，对142个CpG二核苷酸进行甲基化分析。这些研究表明，活性和5-氮杂胞苷再激活等位基因的5′ CpG岛基本上是未甲基化的，而非活性等位基因是高甲基化的。无活性等位基因在几乎所有CpG二核苷酸处完全甲基化，除了在含有四个相邻GC盒的68-bp区域中，其中大多数CpG二核苷酸是未甲基化或部分甲基化的。奇怪的是，这些GC盒只在活性X染色体上显示体内足迹，而不在非活性X染色体上。失活的HPRT基因的甲基化模式与报道的失活的X-连锁人磷酸甘油酸激酶基因的甲基化模式显著不同，后者在5′ CpG岛的所有CpG位点均表现出甲基化。这些结果表明，甲基化CpG二核苷酸的位置，甲基化CpG的密度，甲基化区域的长度，和/或与甲基化DNA相关的染色质结构可能在抑制非活性X染色体上的管家启动子的活性中起作用。失活的人类HPRT基因上的DNA甲基化模式也可以提供对女性胚胎发生早期基因失活过程的深入了解。
DNA methylation within GC-rich promoters of constitutively expressed X-linked genes is correlated with transcriptional silencing on the inactive X chromosome in female mammals. For most X-linked genes, X chromosome inactivation results in transcriptionally active and inactive alleles occupying each female nucleus. To examine mechanisms responsible for maintaining this unique system of differential gene expression, we have analyzed the methylation of individual cytosine residues in the 5′ CpG island of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Methylation analysis of 142 CpG dinucleotides by genomic sequencing was carried out on purified DNA using the cytosine-specific Maxam and Gilbert DNA sequencing reaction in conjunction with ligation-mediated PCR. These studies demonstrate the 5′ CpG islands of active and 5-azacytidine-reactivated alleles are essentially unmethylated while the inactive allele is hypermethylated. The inactive allele is completely methylated at nearly all CpG dinucleotides except in a 68-bp region containing four adjacent GC boxes where most CpG dinucleotides are either unmethylated or partially methylated. Curiously, these GC boxes exhibit in vivo footprints only on the active X chromosome, not on the inactive X. The methylation pattern of the inactive HPRT gene is strikingly different from that reported for the inactive X-linked human phosphoglycerate kinase gene which exhibits methylation at all CpG sites in the 5′ CpG island. These results suggest that the position of methylated CpG dinucleotides, the density of methylated CpGs, the length of methylated regions, and/or chromatin structure associated with methylated DNA may have a role in repressing the activity of housekeeping promoters on the inactive X chromosome. The pattern of DNA methylation on the inactive human HPRT gene may also provide insight into the process of inactivating the gene early in female embryogenesis.