Investigating the effects of expressing MECP2 in Saccharomyces cerevisiae
department or school: Medicine, Faculty of
department or school: Biochemistry and Molecular Biology, Department of
data provider: DSpace
degree campus: UBCV
creator: Ling, Yeou Mei
issued: 2023-04-20T17:51:19Z
issued: 2023
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: 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.
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/84367
note: INVESTIGATING THE EFFECTS OF EXPRESSING MECP2 IN SACCHAROMYCES CEREVISIAE by Yeou Mei Ling B.Sc., The University of British Columbia, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2023 © Yeou Mei Ling, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: INVESTIGATING THE EFFECTS OF EXPRESSING MECP2 IN SACCHAROMYCES CEREVISIAE submitted by Yeou Mei Ling in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology Examining Committee: Dr. LeAnn Howe, Professor, Biochemistry and Molecular Biology, UBC Supervisor Dr. Annie Vogel Ciernia, Assistant Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr. Ivan Sadowski, Professor, Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr. Louis Lefebvre, Associate Professor, Medical Genetics, UBC Additional Examiner iii Abstract 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 defec…
has type: Thesis/Dissertation
date issued: 2023-05
is shown at: 10.14288/1.0431321
language: eng
degree discipline: Biochemistry and Molecular Biology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
contributor: Howe, LeAnn
type: Text
identifier URI: http://hdl.handle.net/2429/84367