Four stranded G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures that are primarily found in nucleosome-depleted promoters and mark sites of elevated transcription within the human genome. Although numerous studies have indicated an association between G4 formation and active transcription, the causal nature of this relationship remains unknown. The work in this thesis first addresses whether promoter G4s form as a result of transcription or whether G4 formation enhances transcription. In addition, this thesis queries the relationship between the folding of G4s and chromatin accessibility at promoters.

The dependence of promoter G4 formation on transcription was tested following Inhibition of transcription elongation and initiation by 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside and triptolide respectively. Using G4 ChIP-seq, it was found that promoter G4 formation was not diminished upon transcriptional inhibition, refuting the dogma that folding of promoter G4s depends on transcriptional activity. Since endogenous G4s form primarily within sites of accessible chromatin, the impact of rapid chromatin compaction on promoter G4 folding was then assessed. Chromatin compaction was found to cause a depletion of some promoter G4s (~20%), which accompanied a reduction in RNA polymerase II (RNA Pol II) loading. This indicates that chromatin accessibility, as opposed to transcriptional activity, is a determinant of G4 formation. To assess whether G4s function to promote transcription, the small molecule ligand pyPDS was used to stabilise G4s in compacted chromatin. This was found to diminish the loss of G4s and mitigated loss of RNA Pol II at promoters upon chromatin compaction, indicating a role of G4s in promoting polymerase occupancy.

Overall, these results show that G4 folding within promoters is fostered by the establishment of accessible chromatin and functions to enhance transcription by promoting Pol II loading. This builds on earlier work showing that presence of promoter G4s accompanies increased gene expression and provides evidence that the G4 structure itself may be sufficient to drive transcription by recruiting key components of the transcriptional machinery.