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Functionality of domains of XIST and their role in establishing chromosome inactivation Dixon-McDougall, Thomas

Abstract

X-chromosome inactivation establishes dosage compensation between the sexes of eutherian mammals through the long non-coding RNA gene, XIST. During development one of the female X chromosomes up-regulates XIST, which coats that chromosome and causes the large-scale silencing of genes. The X chromosome being inactivated by XIST is repositioned within the nucleus, condensed and enriched with heterochromatin associated factors. Understanding the mechanisms of how XIST functions has provided insights into how non-coding RNAs regulate cellular biology, the process of X-chromosome inactivation and novel tools for regulating the epigenome. The function of XIST was investigated using an inducible XIST cDNA construct integrated into the autosome of a male fibrosarcoma cell line, HT1080. The chromatin domain surrounding the XIST loci had a significant effect on its activity, and of all the autosomal loci the 8p integration site functioned most effectively and was thus used for further studies. A series of isogenic inducible partial XIST constructs were created by modifying the Full length XIST construct in 8p to study the importance of individual regions of XIST. The functions dependent on each region of XIST were identified and the relationships between these identified processes were then examined through the use of chemical inhibitors. XIST silencing of genes was demonstrated to depend upon two distinct regions at the extreme ends of the transcript, but the internal sequences spanning these regions were dispensable. Silencing occurred without obvious dependence on chromatin modifications such as those established by the two polycomb group complexes, that in turn relied on ¬distinct regions of XIST suggesting entirely independent mechanisms. Both polycomb complexes were crucial, along with additional elements, for the recruitment of additional heterochromatin factors. This study in human differentiated cells yielded important insights beyond those seen in mouse differentiating cells. The results of this thesis revealed the regions of XIST that were both crucial and dispensable for its activity, and offer novel insights into the mechanisms that lead to chromosome inactivation.

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