Does G-quadruplex DNA have a functional role in regulation of the transcriptome?

Myers, Paul
Does G-quadruplex DNA have a functional role in regulation of the transcriptome? PhD thesis, University of Liverpool.

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All DNA transactions involving overtwisting or undertwisting of the double helix alter the topological state of DNA, yielding distortions known as DNA supercoiling or DNA torsional stress. Under conditions of negative supercoiling susceptible sequences within B-form DNA may be transformed to stable, non-B-DNA structures such as G-quadruplex. G-quadruplex DNA has the potential to create or destroy transcription factor binding sites or influence nucleosome positioning and so may contribute to gene regulation, making it an attractive therapeutic target. Although in vitro and in silico studies support the notion that G-quadruplex is regulatory, a compelling in vivo demonstration of G-quadruplex function is lacking. Using computational and experimental approaches previously developed to identify DNA segments that actually adopt non-B DNA conformations in vivo, I have identified sequences with a high probability of secondary structure formation. Identified sequences are located in the promoters of three genes central to cancer biology where much of the work in G-Quadruplex has been focused. I have expanded this focus into the field of neuroscience by identifying a further three sequences in the promoters of genes implicated in a range of neurological disorders. Preliminary biochemical analysis of these regions assessed methylation status and transcription factor binding profiles corresponding to a dynamic model of G-quadruplex formation at the well characterised CT element in the c-MYC promoter. In order to provide definitive structure function analysis of G-quadruplex I designed a gene editing strategy to reengineer the well characterized CT element of c-MYC. The initial strategy used AAV- targeting vectors to introduce strategic mutations that destabilize G-quadruplex DNA conformations in the CT element of c-MYC in the HCT-116 cell line via homologous recombination. In parallel to these structure-destabilizing mutations, neutral base changes were targeted to the 5´-UTRs of the targeted alleles, distinguishing them from their respective unmodified homologous partners. qPCR and ChIP analysis of the relative expression and factor binding profiles of targeted and wild-type alleles will enable a definitive structure function analysis of basal or induced expression; determining whether these secondary structures have a functional role in regulating gene expression and the factors involved. Low targeting efficiencies of the AAV gene editing strategy prompted me combine the recent technology of the CRISPR-Cas system with traditional AAV targeting to produce a highly efficient, high fidelity and streamlined approach to gene editing.

Item Type: Thesis (PhD)
Additional Information: Date: 2014-09-30 (completed)
Subjects: ?? Q1 ??
Depositing User: Symplectic Admin
Date Deposited: 19 Aug 2015 09:35
Last Modified: 17 Dec 2022 01:34
DOI: 10.17638/02007083