Lock L F, Melton D W, Caskey C T, Martin G R
Mol Cell Biol. 1986 Mar;6(3):914-24. doi: 10.1128/mcb.6.3.914-924.1986.
It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt gene.
有人提出，DNA甲基化参与了X染色体失活的机制，在雌性（XX）和雄性（XY）哺乳动物中，通过这个过程可实现X连锁基因产物水平的等同。在本研究中，对用甲基化敏感的限制性内切酶消化的雌性和雄性DNA进行Southern印迹分析，并与克隆的小鼠hprt基因的不同部分杂交，确定了小鼠hprt基因内雌性和雄性细胞中甲基化存在差异的位点。通过对由两种小鼠（小家鼠和卡罗来鼠）杂交产生的雌性胚胎建立的克隆细胞系的DNA进行类似分析，直接测定了这些位点在活性和失活X染色体上的甲基化程度。结果揭示了小鼠hprt基因中两个甲基化差异区域。一个区域在基因的第一个内含子中，包含四个位点，这些位点在活性X染色体上时完全未甲基化，而在失活X染色体上时则高度甲基化。在自发重新激活或经5-氮杂胞苷处理后重新激活的hprt基因中，这些相同的位点会大量去甲基化。第二个区域包括从外显子3延伸到外显子9的基因3'端20千碱基中的几个位点，呈现相反的模式；即它们在活性X染色体上时完全甲基化，而在失活X染色体上时完全未甲基化。这些位点中至少有一个在基因重新激活后不会甲基化。本研究的结果，连同其他人先前对人类hprt基因的研究结果，表明活性和失活X染色体上这些甲基化差异区域在哺乳动物物种之间是保守的。此外，这里描述的数据与这样一种观点一致，即至少基因5'区域的位点在X染色体失活现象和小鼠hprt基因表达调控中发挥作用。
