Open Collections

Navarro Cobos, Maria Jose

University of British Columbia

X-chromosome inactivation (XCI) is an essential process in eutherian mammals to compensate dosage between females and males. XCI is triggered by XIST, a long non-coding RNA that acts as a scaffold for the recruitment of the needed heterochromatic changes to keep one X chromosome inactivated. XIST has a modular structure, composed of domains containing repetitive sequences. Repeat A is highly conserved and important for silencing, repeat F overlaps regulatory elements, and repeat E is essential for XIST localization. In mouse, a 600 bp region rich in CCC motifs and composed of repeat B and part of repeat C, recruits the protein HNRNPK, which then recruits PRC1. To get a small fully functional XIST transgene, different inducible transgenes containing the A, F and E human repeats as backbone plus other human and/or mouse Polycomb interaction domain (PID) region were tested for induced silencing and recruitment of heterochromatic marks using pyrosequencing and RNA FISH combined with immunofluorescence. The AFE transgene could not induce silencing or recruit heterochromatic changes, however, when the PID mouse region was added (miniXIST transgene), the silencing and heterochromatic marks were partially rescued. This led to the hypothesis that CCC motifs were important for recruitment of PRC1, PRC2, and SETD8, and new transgenes were designed with the aim of reaching full XIST functionality levels. A transgene including the Bh and B human repeats twice was as functional as miniXIST, suggesting that this region in humans is equivalent to the mouse PID region. The BhB-PID transgene was not better than the PID-PID transgene, which contains two PID mouse regions and the highest number of CCC motifs, suggesting that the number of CCC motifs was the most important element in recruiting heterochromatic marks and silencing. The PID-PID transgene was not statistically different from full XIST and is only 5.8 kb long, therefore it could be used for chromosome therapy. Overall, this work allowed the discovery of a shorter functional XIST and contributed to the understanding of the molecular mechanisms in the XCI process.

Text

2024-10-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/89576

Medical Genetics

University of British Columbia

UBCV

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