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DNA G-quadruplex structures in human cancer cells 10.17863/CAM.90676 Hui, Wai In 0000000180410218 Balasubramanian, Shankar DNA structures alternative to the double helix have emerged as key features of the genome for the understanding of genetics and diseases. In particular, G-quadruplexes (G4s), four-stranded structures formed in guanine-rich sequences of cellular chromatin, are implicated in transcription, replication and genome stability. G4s may also present new opportunities for targeting in anti-cancer therapeutic interventions with small molecules. In this thesis, I expand the G4-profiling toolkit to better understand the biological roles of G4s, and potentially offer practical insights for G4-targeting drug development. First, quantitative G4-chromatin immunoprecipitation with sequencing (qG4-ChIP-seq), a method for mapping and comparing G4 landscapes between samples, was used to study G4s in cell lines and patient-derived tumour xenografts from different breast cancer subtypes. Differentially enriched G4s in each cancer model were associated with copy number aberrations and single-nucleotide variants, as well as common breast cancer driver regions, suggesting a link between cancer genome instability and G4 structure formation. Subsequently, to increase the versatility of G4 profiling, I developed G4-Cleavage Under Targets and Tagmentation (G4-CUT&Tag), a more efficient method to profile G4s with higher signal-to-noise ratio and 100-fold lower cellular input than G4-ChIP-seq. Further pushing the detection limit, I optimised G4-CUT&Tag for the first mapping of G4s at single-cell resolution. I demonstrated that individual cell identity can be discerned within a mixed cellular population based solely on single-cell G4 profiles. This result demonstrates that G4 signatures in individual cells relate to the fundamental identity of a cell. Next, I developed single-nuclei G4&RNA-seq, a multiomic method to simultaneously profile G4s and poly(A)-tailed RNA within the same single nucleus. Preliminary data provides proof-of-principle to directly associate G4 formation at individual loci with their transcriptional output within individual cells. Using this approach, I then showed its potential applications in discerning G4 landscapes in different cellular states with reference to cell cycle transcriptomic data within a mixed cell population. My work now enables future genomic investigations on cell-to-cell variation of a DNA secondary structure relative to other chromatin features that were previously not possible. Overall, this thesis demonstrates advancements in G4-profiling methodologies and enables a high resolution and multi-dimensional exploration of the incidence of G4s and their functions. University of Cambridge 2022-07-01 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/343265 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/65f7ba5e-530f-4900-84a9-9cbebe3808de/download 0ab68efe32bb4295cf9594b1647ed38d https://www.rioxx.net/licenses/all-rights-reserved/ cancer G-quadruplex single-cell sequencing transcription