MARSUPIAL X INACTIVATION

X染色体失活是一种染色体范围的基因调控形式，它导致雌性哺乳动物两条X染色体中的一条沉默，从而恢复雄性和雌性之间同等剂量的基因表达。哺乳动物群体之间在这种失活过程中存在显著差异，这可能反映了它的进化。最显著的区别之一是，在变态哺乳动物(有袋类)中，只有父亲的X染色体被灭活，而真兽类(胎盘)哺乳动物在大多数组织中表现出随机的失活。真兽类中X基因失活发生在XIST基因座，但在有袋类动物中尚未发现相应的基因座。有袋类动物的X失活不如真兽类动物稳定，而且是不完整的，具有组织特异性。分子基础也可能不同。尽管在有袋类和真兽类动物中，活性和非活性X染色体在组蛋白乙酰化方面存在差异，但非活性基因启动子区域的DNA甲基化没有差异，这可能是该哺乳动物群体中不稳定失活的原因。有袋类动物和真兽类动物的X失活过程之间的这些差异表明，有袋类动物可能保留了一种更简单、更原始的X失活形式。这两个群体之间的差异为在染色体水平上剖析基因沉默过程的各个层面提供了一个独特的机会。我们的目标是通过一系列分子方法来研究这些差异。我们计划首先从有袋类动物中分离出XIST基因座，这是基于我们使用富含有袋类X染色体DNA的探针初步发现了雌性特有的RNA信号。我们将比较真动物和变态动物的XIST序列和甲基化状态，以检查有袋动物中可能存在的XIST印记。其次，我们将通过测量单个细胞中活跃和非活跃的X染色体的表达来研究有袋类基因失活的稳定性。第三，我们将把有袋类基因插入到小鼠的X染色体中，以确定该基因在体内是否对表观遗传变化敏感或抵抗，表观遗传变化是真核生物基因的特征。

X chromosome inactivation is a form of chromosome-wide gene regulation which results in the silencing of one of the two X chromosomes in female mammals, thereby restoring equal dosage of gene expression between males and females. There are significant differences in this inactivation process between mammal groups, which probably reflects its evolution. One of the most striking difference is that in metatherian mammals (marsupials) only the paternal X chromosome is inactivated, whereas eutherian (placental) mammals show random inactivation in most tissues. X inactivation is initiated at the XIST locus in eutherians, but no homologous locus has yet been demonstrated in marsupials. X inactivation is less stable in marsupials than in eutherians, and is incomplete and tissue-specific. The molecular basis, too may be different. Although differences in histone acetylation have been demonstrated between active and inactive X chromosomes in marsupials as well as eutherians, DNA methylation of the promoter region of inactive genes have not, perhaps accounting for the less stable inactivation in this mammalian group. These differences between the X inactivation processes in marsupials and eutherian mammals suggest that marsupials may have retained a less complex and more primitive form of X inactivation. The differences between the groups offer a unique opportunity to dissect out the layers of the process of gene silencing at a chromosome level. Our goal is to investigate these differences by a series of molecular approaches. We plan first to isolate the XIST locus from marsupials, based on our preliminary finding of a female-specific RNA signal using a probe enriched in marsupial X chromosome DNA. We will compare eutherian and metatherian XIST sequence and methylation status to examine possible XIST imprinting in marsupials. Secondly, we will investigate the stability of X inactivation of marsupial genes in comparison to mouse by measuring expression from the active and inactive X chromosomes in individual cells. Thirdly, we will insert a marsupial gene into the mouse X chromosome to determine whether the gene is susceptible or resistant in vivo to epigenetic changes which characterize eutherian genes.