Mechanisms and dynamics of targeted TET-mediated DNA demethylation

Hoehn, Sven 2025. Mechanisms and dynamics of targeted TET-mediated DNA demethylation. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

DNA methylation is one of the most extensively studied epigenetic modifications governing gene expression and cellular identity. Despite considerable progress in understanding how methylation patterns are established and maintained, the detailed dynamics and mechanisms of active DNA demethylation have remained unclear. This thesis examines the mechanisms and temporal dynamics of targeted TET-mediated DNA demethylation by adapting a CRISPR/dCas9-based platform for epigenetic editing. I constructed a modular system using the piggyBac transposon for stable genomic integration of doxycycline-inducible dCas9-TET3 fusions. This approach allowed targeted recruitment of different TET3 domains with varying degrees of catalytic processivity, enabling a detailed study of active demethylation dynamics with locus-specific and temporal resolution. Three model genes, ASCL1, EpCAM, and LIN28A, were selected based on their contrasting DNA methylation patterns and regulatory complexity. Analysis of targeted TET-mediated demethylation revealed context-dependent patterns of methylation loss. Demethylation consistently initiated at sgRNA-binding sites before spreading outward over time, yet the extent and stability of these changes varied between genes. This variation highlights that pre-existing epigenetic states influence the effectiveness of targeted TET interventions in altering methylation patterns. While some promoters underwent extensive demethylation, coupled with transcriptional activation, others exhibited minimal changes. My studies, employing different catalytic TET3 mutants, revealed that efficient demethylation requires oxidation progression beyond 5-hydroxymethylcytosine, with 5-formylcytosine functioning as a crucial oxidative intermediate for methylation loss and transcriptional activation. Use of the VPR transcriptional activator demonstrated that transcriptional activity can induce localised demethylation independently of TET recruitment, but is insufficient to drive spreading demethylation. My findings demonstrate that targeted TET-mediated demethylation operates through context-dependent mechanisms influenced by the existing epigenetic landscape. Although demethylation can induce transcription, crosstalk with additional regulatory ii mechanisms likely influences transcriptional outcomes. This work advances our understanding of the underlying mechanism and dynamics of DNA demethylation, its spreading, and its influence on gene regulation.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	29 May 2026
Last Modified:	01 Jun 2026 10:48
URI:	https://orca.cardiff.ac.uk/id/eprint/187276

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