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Studies of XIST and macroH2A1.2 independent of gene silencing : subtitle a unique hybrid cell model system Kwok, Edmund

Abstract

X chromosome inactivation results in the transcriptional silencing of one of the two X chromosomes present in mammalian female cells. XIST expression from the inactive X (Xi), and subsequent coating of the Xi by the XIST RNA, marks the initial major observable characteristic of X-inactivation. The Xi then begins to exhibit features of heterochromatin such as gene silencing, late replication, histone modification, hypermethylation, and the recruitment of core histone variants. However, the exact mechanistic events between XIST expression and the final heterochromatic state of the Xi remain unclear. To better understand the relationship between XIST, heterochromatin, and silenced gene expression, the first goal of this study was to develop and characterize a model system suitable for examination of those features of heterochromatin which can be induced by XIST independent of gene silencing. Previous work using rodent/human hybrids (containing active human and mouse X chromosomes) that had been demethylated to reactivate XIST/Xist expression showed that while human XIST RNA fails to localize properly to the human Xi, the mouse Xist localizes normally. In both cases no silencing of X-linked genes was observed. To verify this hybrid cell system as a model for studying the effects of XIST/Xist independent of inactivation, I examined the DNA methylation, H3 acetylation, and gene expression statuses of multiple human and mouse X-linked genes in these hybrids. The results confirm both i) the reactivation of XIST/Xist due to demethylation, and ii) the lack of gene silencing despite the presence of XIST/Xist.

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