Mechanisms for XIST RNA cis-localisation

X inactivation is the process of silencing one of the two X chromosomes in mammalian female cells in order to equalise the dosage of X-linked genes with males. The process is initiated by the long noncoding RNA XIST, which is transcribed from the future inactive X and localises to it in cis. How XIST RNA is able to localise to the X chromosome is not well defined. The aim of the current study was to deduce mechanisms of XIST RNA localisation. This was addressed in various ways, including 1) testing the ability of an XIST transgene integrated into a variety of autosomes to localise to those autosomes, as opposed to the X chromosome; 2) assessing the ability of XIST transgenes with different regions deleted to localise, in order to identify sequences required for localisation; and 3) knocking down various proteins implicated in X inactivation in order to assess any effect on the ability of XIST to localise. We find that the XIST transgene is able to localise to a wide variety of different autosomes and furthermore, is able to direct the enrichment of the histone variant macroH2A on an autosome and the deposition of a repressive histone modification, H3K27me3, onto an autosome. We also find that a region of XIST encompassing repeats B and C, and sequences downstream of exon 1 are involved in localising XIST RNA, and that they do so in a redundant fashion. Lastly, we show that the knockdown of five proteins - YY1, hnRNP-U, SPOP, CUL3 and ASH2L - prevent the formation of an intact XIST focus. The results presented here add to the limited knowledge of how XIST RNA is able to localise, an essential step in the process of X chromosome inactivation. Medicine, Faculty ofMedical Genetics, Department ofGraduat