{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/343265"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/343265","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"DNA G-quadruplex structures in human cancer cells","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Hui, Wai In"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Balasubramanian, Shankar"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-07-01","date_published":"2022-07-01","updated_at":"2026-07-22T22:24:30Z","subjects":["cancer","G-quadruplex","single-cell sequencing","transcription"],"languages":["eng"],"rights":[],"rights_urls":["https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000180410218"],"render_values":[{"text":"0000-0001-8041-0218","href":"https://orcid.org/0000-0001-8041-0218","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.90676","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Balasubramanian, Shankar"]},{"key":"dc:creator","label":"Author","values":["Hui, Wai In"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000180410218"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-07-01"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/343265"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["cancer","G-quadruplex","single-cell sequencing","transcription"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.90676"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/65f7ba5e-530f-4900-84a9-9cbebe3808de/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["DNA structures alternative to the double helix have emerged as key features of the genome for the understanding of genetics and diseases. 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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. 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