Goto T, Christians E, Monk M
Molecular Embryology Unit, Institute of Child Health, London, England.
Mol Reprod Dev. 1998 Apr;49(4):356-67. doi: 10.1002/(SICI)1098-2795(199804)49:4<356::AID-MRD2>3.0.CO;2-M.
Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females, a process involving a regulatory gene, Xist (X-inactive specific transcript). Xist maps to the X-inactivation centre and is expressed from the inactive X chromosome in female somatic cells and at the time of X inactivation during spermatogenesis in the male. In female preimplantation embryos, Xist demonstrates imprinting in that the paternal allele inherited from the sperm is preferentially expressed. This preferential paternal Xist expression is correlated with paternal X inactivation in the extraembryonic lineages at the blastocyst stage. We have analysed a 233-bp Xist promoter fragment (nt -220 to +13) for its ability to direct appropriate expression and its regulation by DNA methylation. This minimal promoter sequence directs expression of the luciferase reporter gene following injection of the construct into one-cell embryos. In vitro methylation of the construct before injection represses transcription. In six different transgenic lines, expression of the Xist promoter-luciferase transgene occurs only in the testis of the males (as for the endogenous Xist gene). The testis-specific expression is correlated with hypomethylation of the transgene, although to different extents in different lines. Following paternal transmission, expression of the Xist promoter-luciferase construct in preimplantation embryos is correlated with degree of hypomethylation in the testis and the degree of hypomethylation of the transgene in embryos at the morula stage. It is concluded that the patterns of methylation of the transgene in sperm (and in microinjected transgenes) can regulate the activity of the Xist promoter in the preimplantation embryo and thus support the hypothesis that gametic methylation patterns govern imprinted expression of the endogenous Xist gene in development.
哺乳动物中X连锁基因的剂量补偿是通过使雌性两条X染色体中的一条失活来实现的，这一过程涉及一个调控基因Xist（X染色体失活特异性转录物）。Xist定位于X染色体失活中心，在雌性体细胞的失活X染色体上以及雄性精子发生过程中X染色体失活时表达。在雌性植入前胚胎中，Xist表现出印记现象，即从精子继承的父本等位基因优先表达。这种父本Xist的优先表达与囊胚期胚外谱系中的父本X染色体失活相关。我们分析了一个233bp的Xist启动子片段（核苷酸-220至+13）指导适当表达的能力及其受DNA甲基化调控的情况。将该构建体注射到单细胞胚胎后，这个最小启动子序列可指导荧光素酶报告基因的表达。注射前对构建体进行体外甲基化会抑制转录。在六个不同的转基因品系中，Xist启动子-荧光素酶转基因仅在雄性的睾丸中表达（如同内源性Xist基因）。睾丸特异性表达与转基因的低甲基化相关，尽管在不同品系中程度不同。父本传递后，植入前胚胎中Xist启动子-荧光素酶构建体的表达与睾丸中的低甲基化程度以及桑椹胚期胚胎中转基因的低甲基化程度相关。得出的结论是，精子中转基因的甲基化模式（以及显微注射的转基因）可调节植入前胚胎中Xist启动子的活性，从而支持配子甲基化模式在发育过程中控制内源性Xist基因印记表达的假说。
