Open Collections

Ling, Yeou Mei

University of British Columbia

Methyl-CpG-binding protein 2 (MECP2) plays a key role in brain homeostasis. MECP2 function is sensitive to changes in its concentration, as both overexpression and reduction of MECP2 result in neurodevelopmental disorders in humans and mice. MEPC2 was originally discovered based on its ability to bind methylated-CpG DNA to repress gene expression. Later research suggested that MECP2 may also interact with unmethylated DNA. While there have been in vitro studies supporting this idea, there were few in vivo studies. In this thesis, we demonstrate that expression of the human MECP2 in an organism lacking DNA methylation, the budding yeast Saccharomyces cerevisiae, disrupts normal yeast growth, by inhibiting passage through S phase. Further investigation revealed that MECP2-expressing yeast had defects in their cell cycle, which may be the cause of the growth defect. We found that the phenotype of yeast expressing MECP2 with disease-associated mutations reflects disease severity in humans. Using single-cell techniques such as flow cytometry and fluorescence microscopy, we confirmed the expression of MECP2 in yeast and its nuclear localization. Disease-associated mutations that reversed the growth defect either impair nuclear localization or, as previously reported, disrupt MECP2 binding to DNA. In addition, we found differences between the two MECP2 isoforms in terms of protein stability, and impact on the cell cycle and viability. This suggests that the isoforms have different functions and potentially, differing interacting partners within cells. Collectively, our findings confirmed the feasibility of using yeast as a model organism to study MECP2 function in vivo. Moreover, using this system, we are able to rapidly screen for phenotypes of MECP2-disease-associated mutations that reflect disease severity in humans.

Text

2023-04-20

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/84367

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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