MARSUPIAL X INACTIVATION

X染色体失活是一种染色体范围的基因调控形式，其导致雌性哺乳动物中两条X染色体之一的沉默，从而恢复雄性和雌性之间相等剂量的基因表达。哺乳动物群体之间的失活过程存在显著差异，这可能反映了其进化。最显著的区别之一是，在后兽目哺乳动物（有袋动物）中，只有父方X染色体失活，而真兽目（胎盘）哺乳动物在大多数组织中表现出随机失活。在真兽目中，X失活起始于XIST位点，但在有袋类动物中尚未发现同源位点。X失活在有袋类动物中比在真兽目动物中更不稳定，并且是不完全的和组织特异性的。分子基础也可能不同。虽然组蛋白乙酰化的差异已被证明在有袋动物和真兽目动物的活跃和不活跃的X染色体之间，不活跃基因的启动子区域的DNA甲基化还没有，也许占在这个哺乳动物组中不太稳定的失活。有袋类和真兽目哺乳动物的X染色体失活过程之间的这些差异表明，有袋类可能保留了一种不那么复杂和更原始的X染色体失活形式。群体之间的差异提供了一个独特的机会，在染色体水平上解剖出基因沉默过程的层次。我们的目标是通过一系列的分子方法来研究这些差异。我们计划首先从有袋动物中分离出XIST基因座，这是基于我们使用富含有袋动物X染色体DNA的探针初步发现的女性特异性RNA信号。我们将比较真兽目和后兽目的XIST序列和甲基化状态，以检查有袋动物中可能的XIST印记。其次，我们将通过测量单个细胞中活性和非活性X染色体的表达来研究有袋动物基因与小鼠相比X失活的稳定性。第三，我们将把一个有袋动物基因插入小鼠X染色体，以确定该基因在体内是否对真兽目基因的表观遗传变化敏感或抵抗。

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.