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.