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Title: Targeting G-quadruplex DNA in promoters of cardiac function-related genes
Author: Zhou, Wenhua
ISNI:       0000 0004 2724 3168
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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G-quadruplexes (G4) are four-stranded DNA secondary structures, which are involved in a diverse range of biological processes. Although the anti-cancer potential of G4s in promoters of oncogenes has been thoroughly investigated, the functions of promoter G4s in non-cancer-related genes are not well understood. The aims of this thesis are (i) to investigate the biological importance of promoter G4s in cardiac function-related genes, and (ii) to explore the feasibility and potential of multitargeting strategy at the single gene level. These aims have been achieved by revealing the prevalence of G4s in TRRs (transcription regulatory regions) of cardiac function-related genes via bioinformatics approaches, and then investigating potential biological significance of G4s in promoters of human MEF2D and TnIc genes via a combination of biophysical, molecular, and cell biology approaches. By using bioinformatics approaches, TRRs of cardiac function-related genes were found to be enriched by potential G4-forming sequences. According to these results, G4s from the promoters of human MEF2D and TnIc were chosen and subjected to biophysical characterisations in solution. By using EMSA, DMS footprinting, CD and smFRET spectroscopy, the formation, thermodynamic stability, and unfolding kinetics of MEF2D and TnIc G4s were investigated. Briefly, in 100 mM K+, the MEF2DG4 can adopt a hybrid of very stable parallel/anti-parallel G4(s), while the TnIc MNSG4 and -80G4 are able to adopt anti-parallel and parallel structures, respectively, with comparable stability as compared to other oncogene G4s. Subsequently, cooperative regulatory roles of TnIc G4s as enhancers once stabilised by a G4-binding ligand (di-copper complex) were observed in vitro mainly by dual luciferase reporter assays in HEK293 cells. Three possible G4-mediated mechanisms have been proposed. This work provides the first indication about the biological significance of promoter G4s in cardiac function-related genes, and the feasibility to target multiple G4s at the single gene level.
Supervisor: Ying, Liming ; Ferenczi, Mike Sponsor: National Heart and Lung Institute
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral