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UBC Theses and Dissertations

Transcriptional regulation of the XIST locus Chapman, Andrew Glen

Abstract

X-chromosome inactivation is a mechanism that has evolved in mammalian females allowing dosage compensation of X-linked genes. A region of the X chromosome called the Xinactivation centre (XIC) is required for X inactivation to occur. Within this region is a long noncoding RNA, XIST/Xist, which is upregulated on the future inactive X and initiates silencing. A major questions in the field of X inactivation is how XIST/Xist is regulated, becoming expressed on the inactive X and silenced on the active X. Much of what we currently know about XIST/Xist regulation comes from studies using mice, however, differences in conservation of the XIC and expression patterns of the major mouse Xist regulator, Tsix, indicate that humans and mice may regulate XIST/Xist differently. The objective of this thesis was to identify regulatory elements that are important for regulation of XIST in humans. Since regulatory elements controlling XIST are believed to reside within the XIC, we searched the XIC and identified two inactive X specific regulatory elements within the 5’ end of XIST using DNase I hypersensitivity mapping. We found one of the hypersensitive sites to be acting as an alternative P2 promoter for XIST which contains an upstream antisense promoter, P2as. The second hypersensitive site was associated with alternative splicing and inclusion of two novel exons for XIST. Interestingly, both P2 and the novel alternative splicing result in transcripts that lack functional domains of XIST. An additional candidate regulator is the region 3’ of XIST due to the importance of Tsix in mice. We found that transcription 3’ of XIST in somatic cells is low level sense transcription so we believe this to be leaky XIST rather than TSIX. In human embryonic stem cells we found an antisense transcript that extends the full length of XIST providing the first evidence for mouse-like TSIX in humans but very low-levels of this transcript argue against regulatory ability. Taken together, our results highlight the differences between mouse and human X inactivation and indicate that XIST transcription is more complex than previously thought, generating XIST molecules that lack functional domains.

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