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Identification and characterization of CIS-acting regulatory elements for human x-inactive specific transcript

University of British Columbia

Dosage compensation in female mammals is achieved by XIST/Xist RNA mediated transcriptional silencing of one of the two X chromosomes. Several developmental specific cis-acting regulatory elements for mouse XIST have been demonstrated. One of these regulatory elements is the mouse Tsix locus, which is transcribed antisense to Xist and represses Xist on one of the X chromosomes at the onset of X inactivation. A transcript antisense to human XIST has been shown; however, its functional significance has been repeatedly challenged. My thesis aims to uncover cis-acting regulatory elements for human XIST and determine whether the elements are comparable to those found in mice. Currently, multi-copy integrations of a transgene containing human XIST into male mouse embryonic stem (ES) cells or into male human fibrosarcoma cells are the model systems of choice for studying the initiation of human X inactivation because the transcription antisense to human XIST can be detected by RT-PCR from the transgenes. Using DNase I hypersensitivity (HS) mapping, I found one HS site on the human transgene in mouse ES cells located approximately 11 kb downstream of XIST3’ end. While this HS site does not correspond to the transcription starts of TSIX previously described, it encompasses a small cluster of CTCF binding sites based on in silico search. Besides the downstream HS site, I discovered two HS sites in differentiated cell lines. One of the HS sites is immediately upstream of the XlST transcription start site. The other HS site (HS 101), located approximately 65 kb upstream of human XIST transcription start, resides within a region that shares above 70% sequence identity with cow and dog but not mouse. I analyzed these upstream HS sites and found that HS 101 exhibits bi-directional promoter and enhancer activity. My thesis revealed three previously unidentified HS sites flanking the human XIST locus; the presence of only one ES cell specific HS site downstream of XIST3’ end is in sharp contrast to the seven sites reported in mouse. The results suggest that mouse Xist and human XIST are regulated differently. To account for the differences in regulatory elements, I propose an alternative model for human XIST regulation.

Thesis/Dissertation

application/pdf

2009-11-27

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/15880

Medical Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/