Investigating DNA G-quadruplex structure function through genome and epigenome engineering

Abstract

The importance of DNA secondary structures, such as four-stranded DNA G-quadruplexes (G4s), in genome function remains a largely unanswered question. G4s are implicated in transcriptional regulation; however, earlier studies are mostly correlative and do not directly address the role of an individual G4 within its endogenous cellular context. Using CRISPR genome and epigenome engineering, I performed a series of genetic and epigenetic perturbations in human cells to specifically modulate G4 formation within the upstream regulatory region of the MYC oncogene. In combination with chromatin profiling, whole-genome and locus-specific sequencing, and biophysical assays, I investigated how G4 folding in cells regulates the local chromatin environment and gene expression. I found that the MYC G4 structure positively regulates MYC transcription. In particular, the MYC G4 regulates promoter choice by promoting transcription from the P1 promoter. I also found that G4s shape the local chromatin architecture to coordinate multiple molecular processes and regulate transcription. First, G4s act as anchors for the binding of transcription factors, including SP1 and CNBP. Second, G4s recruit histone modifiers which in turn dictate the local histone methylome. Third, G4s organise nucleosome positioning to influence chromatin accessibility and recruitment of RNA polymerase II. Cells edited to lack the MYC G4 lead to loss of MYC transcription from the P1 promoter and the deposition of a de novo nucleosome that interferes with normal RNA polymerase recruitment. Confirming the importance of structure rather than primary sequence, I demonstrate that replacing the endogenous MYC G4 with a different G4 sequence restores G4 folding and MYC transcription. Furthermore, through epigenome engineering, I show how cytosine methylation can modulate G4 formation in cells. My findings suggest a mechanism whereby G4s are central features that coordinate regulatory protein recruitment and establishment of epigenetic and nucleosome landscapes to modulate gene expression. Overall, the evidence presented in this thesis provides direct and robust support of the importance of DNA secondary structure rather than primary sequence in genome regulation.