4 research outputs found
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Investigating DNA G-quadruplex structure function through genome and epigenome engineering
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.Herchel Smith Fun
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G-quadruplex DNA structure is a positive regulator of MYC transcription
DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequenceHerchel Smit
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Transcription-coupled repair of DNA–protein cross-links depends on CSA and CSB
Funder: Cancer Research UK (CRUK); doi: https://doi.org/10.13039/501100000289; Grant(s): CRUK Discovery Award DRCPGM\100005, CRUK RadNet grant C17918/A28870, CRUK Discovery Award DRCPGM\100005, Discovery Award DRCPGM\100005, A:29580, C9681/A29214, C9545/A19836Funder: China Scholarship Council (CSC); doi: https://doi.org/10.13039/501100004543Covalent DNA–protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA–protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS
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Transcription-coupled repair of DNA-protein cross-links depends on CSA and CSB.
Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS