Investigating the function of DNA G-quadruplex structures in human enhancers
DNA molecules have the capacity to assemble and fold into topologically distinct molecular structures. DNA G-quadruplexes (G4s) are four-stranded DNA three-dimensional (3D) structures, that form in guanine-rich regions of the genome. G4s are enriched at sites of important regulatory potential in diverse genomes, including promoters and enhancers. Enhancers interact with target promoters through 3D folding of interphase chromosomes to regulate gene transcription. However, the functional relevance of G4 structures at non- coding enhancer elements remains poorly understood. In this thesis, I explore the folding of G4 structure in 3D chromatin interactions, and dissect the function of a G4 in the control of enhancer-promoter communication for accurate gene regulation at the human β-globin locus. First, a new methodology, viewpoint Hi-C on accessible regulatory DNA (ViCAR), was developed to map 3D chromatin interactions which contain a folded G4 structure for the first time. This allowed for the identification of G4s which may have important function in the process of enhancer-driven gene regulation. Subsequently, I employed this methodology to uncover G4-containing 3D enhancer-promoter interactions at the human β-globin locus. I identified G4 folding in the major enhancer element of this locus, and investigated its function through genetic perturbation of the G4 motif by genome editing. In this system, enhancer G4 perturbation disrupted 3D enhancer-promoter interactions and target gene expression. Lastly, I investigate the potential mechanisms of G4 function at enhancers. Through proteomic, biophysical and genomic profiling I uncover an interaction between G4 structure, RNA polymerase II and one of its associated helicases DDX5 that is required for G4 function in enhancer activity. Overall, this thesis advances our understanding of G4s as functional elements of enhancers and provides mechanistic insight into how enhancers control the critical process of gene expression.
Gene regulation, DNA G-quadruplex, Genome biology, Enhancer biology, Beta-globin gene regulation, 3D genomics
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