In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes.
In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes.
复制标题
活性和非活性 X 染色体上小鼠次黄嘌呤磷酸核糖转移酶基因 5 区域的体内足迹和高分辨率甲基化分析。
DOI：
10.1128/mcb.16.11.6190
复制
发表时间：
1996
影响因子：
5.3
通讯作者：
Yang,TP
中科院分区：
文献类型：
--
作者：
Litt,MD;Hornstra,IK;Yang,TP
为了探讨X染色体失活对次黄嘌呤磷酸核糖转移酶（HPRT）基因调控的潜在机制，我们对小鼠HPRT基因5′端的活性和失活等位基因进行了体内足迹法和高分辨率DNA甲基化分析，并与人类HPRT基因的结果进行了比较。我们在活跃的小鼠HPRT等位基因上发现了多个足迹位点，而在不活跃的等位基因上没有足迹。小鼠和人HPRT基因的足迹模式的比较表明，这些物种之间的调节蛋白的体内结合通常是保守的，但不相同。在小鼠和人类基因的足迹区域的详细的核苷酸序列比较揭示了一种新的9-bp序列与转录因子结合附近的两个基因的转录位点，这表明一个新的保守的启动子元件的识别。连接介导的PCR基因组测序表明，所有的CpG二核苷酸检查的活性等位基因是未甲基化的，而大多数的非活性等位基因上的CpG是甲基化的，并散布着一些低甲基化的网站。失活小鼠等位基因上的这种甲基化模式与失活人类基因的不寻常甲基化模式明显不同，后者在GC盒处表现出强烈的低甲基化。这些研究与X连锁基因的其他基因组测序研究相结合，证明（i）活性等位基因基本上是未甲基化的，（ii）非活性等位基因是高甲基化的，（iii）高甲基化的非活性等位基因的高分辨率甲基化模式不是严格保守的。无活性等位基因上甲基化位点的模式与活性等位基因上转录因子结合位点的模式之间没有明显的相关性。这些结果进行了讨论的关系，X染色体失活的转录调控的潜在机制。
To investigate potential mechanisms regulating the hypoxanthine phosphoribosyltransferase (HPRT) gene by X-chromosome inactivation, we performed in vivo footprinting and high-resolution DNA methylation analysis on the 5′ region of the active and inactive mouse HPRT alleles and compared these results with those from the human HPRT gene. We found multiple footprinted sites on the active mouse HPRT allele and no footprints on the inactive allele. Comparison of the footprint patterns of the mouse and human HPRT genes demonstrated that the in vivo binding of regulatory proteins between these species is generally conserved but not identical. Detailed nucleotide sequence comparison of footprinted regions in the mouse and human genes revealed a novel 9-bp sequence associated with transcription factor binding near the transcription sites of both genes, suggesting the identification of a new conserved initiator element. Ligation-mediated PCR genomic sequencing showed that all CpG dinucleotides examined on the active allele are unmethylated, while the majority of CpGs on the inactive allele are methylated and interspersed with a few hypomethylated sites. This pattern of methylation on the inactive mouse allele is notably different from the unusual methylation pattern of the inactive human gene, which exhibited strong hypomethylation specifically at GC boxes. These studies, in conjunction with other genomic sequencing studies of X-linked genes, demonstrate that (i) the active alleles are essentially unmethylated, (ii) the inactive alleles are hypermethylated, and (iii) the high-resolution methylation patterns of the hypermethylated inactive alleles are not strictly conserved. There is no obvious correlation between the pattern of methylated sites on the inactive alleles and the pattern of binding sites for transcription factors on the active alleles. These results are discussed in relationship to potential mechanisms of transcriptional regulation by X-chromosome inactivation.