Trans-acting factors involved in heterochromatinization of the inactive X chromosome

哺乳动物X染色体失活是一种染色体范围内的现象，导致雌性体细胞中两条X染色体中的一条的转录沉默。失活的X染色体具有惊人的特性，包括异步DNA复制、通过X染色体长度定位的XIST RNA、核周围性染色质体的形成以及组蛋白H4的低乙酰化。然而，这些特性之间的关系及其相对重要性仍然未知。在目前的研究中，我们试图分离参与失活的人类X染色体异染色化的反式作用因子。人类单染色体杂交，CF150具有一个或多个无活性的人类X染色体（Xi）作为其唯一的人类元素。在CF150细胞中，引入的Xi表现异常；(1) Xi在细胞核外周没有形成性染色质体，(2)Xi和其他小鼠染色体一样被中度乙酰化，(3)Xi没有被XIST RNA包被。从CF150转移到HeLa细胞中的相同的Xi表现为典型的失活X，这表明这些特性的充分表现需要某种在CF150中耗尽的人类特定因子。通过微细胞融合，进行了基因互补试验，以确定哪条人类染色体携带相关基因。当从CF150中引入Xi到小鼠胚胎癌（EC）细胞系时，失活发生逆转，但XIST持续表达。尽管其活性染色质状态，重新激活的人类X染色体被XIST RNA包被。这些单染色体EC杂交体的细胞分化导致XIST RNA从人类X染色体分离。将同一条重新激活的X染色体从单染色体EC杂交种转移到HeLa细胞中，重新激活的X染色体被XIST RNA包被。总之，这些数据表明，在小鼠细胞中存在两种不同的因素（分别为未分化细胞和体细胞类型）来定位Xist RNA。少

Mammalian X chromosome inactivation is a chromosome-wide phenomenon that leads to transcriptional silencing of one of the two X chromosomes in female somatic cells. The inactivated X chromosome shares striking properties, including asynchronous DNA replication, XIST RNA localization through the length of the X chromosome, sex chromatin body formation at nuclear periphery, and hypoacetylation of histone H4. Relationships among these properties and their relative importance, however, remain unknown. In the present study, we have tried to isolate trans-acting factors involved in heterochromatinization of the inactive human X chromosome.A human monochromosome hybrid, CF150 has one or more inactive human X chromosomes (Xi) as its only human element. In CF150 cells, the introduced Xi behaved abnormally ; (1) Xi did not form sex chromatin body at nucleus peripherally, (2) Xi was moderately acetylated as other mouse chromosomes, and (3) Xi was not coated with XIST RNA.The same Xi transferred f … More rom CF150 into HeLa cells behaved normally as the typical inactive X, suggesting full manifestation of these traits requires a certain human specific factor (s) that is depleted in CF150. A genetic complementation assay has been performed to determine which human chromosome bears the related genes, by microcell fusion.When Xi was introduced from CF150 into mouse embryonal carcinoma (EC) cell lines, the reversal of inactivation occurred, but XIST expressed persistently. Despite of its active chromatin state, the reactivated human X chromosome was coated with XIST RNA.Cell differentiation of these monochromosome EC hybrids leaded the dissociation of XIST RNA from the human X chromosome. When the same reactivated X chromosome was transferred from monochromosome EC hybrids into HeLa cells, the reactivated X chromosome was coated with XIST RNA.Together, these data suggest that two different factors (an undifferentiated and a somatic cell types, respectively) for Xist RNA localization exist in mouse cells. Less