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Title: Biophysical and functional studies of potential G-quadruplex motifs from the proximal GATA4 gene promoter
Author: Klejevskaja, Beata
ISNI:       0000 0004 6348 0240
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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GATA4 is well known as a cardiac transcription factor and recently has also been implicated in several cancers. Therefore, a fundamental understanding of its gene regulation is currently of high interest. It has been suggested that guanine-rich motifs found in gene promoters could potentially adopt a G-quadruplex DNA structure and play a role in regulating gene expression. Two conserved-potential G-quadruplex motifs were identified in the human and mouse proximal GATA4 promoter region and it was hypothesised that they could be involved in regulation of GATA4 expression. The biophysical characterisation of the identified G-quadruplex motifs showed that the motif-1 found 72bp downstream from the transcription start site (TSS) adopts a stable G-quadruplex structure, whereas the motif-2 located 10 bp downstream from the TSS folds predominantly into a hairpin structure. To evaluate the functional potential of G-quadruplex structure formation in the proximal GATA4 promoter, the GATA4 promoter was cloned onto a luciferase expression vector and a series of G-quadruplex mutants was generated. Mutations of motif-1 or motif-2 -quadruplex had no significant effect on GATA4 promoter activity. Despite this, GATA4 G-quadruplex structure-stabilising ligands, such as pyridostatin and TMPyP4, were found to down-regulate GATA4 promoter activity, likely due to off-target effects. To more closely resemble the quadruplex environment in gene promoters, G-quadruplex formation within DNA mini-circles was investigated by single-molecule FRET. DNA mini-circles were found to be a promising novel tool for biophysical characterisation of G-quadruplexes from gene promoters, as well as for studies of G-quadruplex interaction with ligands. This work presents evidence that not all G-quadruplex motifs found in gene promoters are biologically relevant. It was also found that commonly used G-quadruplex ligands can affect promoter activity mediated via alternative mechanisms to G-quadruplex. Finally, the feasibility of studying G-quadruplex structures embedded within mini-circle DNA was demonstrated.
Supervisor: Vilar, Ramon ; Ying, Liming ; Schneider, Michael D. Sponsor: Engineering and Physical Sciences Research Council ; Institute of Chemical Biology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral