The role of TET2 protein and oxidised forms of 5-methylcytosine in brain tumours

Eleftheriou, Maria (2020) The role of TET2 protein and oxidised forms of 5-methylcytosine in brain tumours. PhD thesis, University of Nottingham.

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Abstract

DNA methylation (5-methylcytosine, 5mC) is the major epigenetic modification involved in transcriptional regulation. Ten-eleven translocation (TET) proteins can enzymatically oxidise 5mC producing 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). According to several reports, 5hmC levels are reduced in human tumours; however, the distribution and the exact biological role of TET-dependent oxidation to 5fC and 5caC in cancers is poorly studied.

Here, using a range of techniques including, immunocytochemistry, 2 dimensional ultra-performance liquid chromatography coupled with tandem mass-spectrometry (2D-UPLC–MS/MS) and CRISPR/Cas9 gene editing we studied the presence and the distribution of 5caC/5hmC in brain tumours cell lines. Our ultimate goal was to examine the role of TET2 in the oxidation of 5mC to 5hmC/5caC in glioblastoma multiforme (GBM) pathogenesis and the role of 5caC potential readers in the interpretation of active DNA demethylation in medulloblastoma (MB) (paediatric brain tumour) cell lines.

We found that, while GBM cell lines exhibit low levels of 5hmC, they are, rather unexpectedly, characterised by detectable 5caC levels. Remarkably, 5caC content in GBM corresponds to elevated levels of TET2 transcript. We next used CRISPR/Cas9 to knockout (KO) TET2 in the LN18 GBM cell line. 5hmC levels were significantly reduced in TET2 KO cell line. Moreover, we showed significantly reduced tumorigenic ability of TET2 KO cells in parallel with the reduction of the transcript levels of key glial cancer stem cell markers. We also showed that SMARCC2 and RCOR2 transcription factors with unique roles in neurogenesis, are highly expressed in paediatric brain tumours, while their transient knockdown resulted at impaired 5caC levels in MB cell lines; indicating their role as 5caC readers. Our compiled data show the unique epigenetic signatures of adult and paediatric brain tumours, the role of TET2 in active DNA demethylation and its potential contribution to tumorigenesis.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Ruzov, Alexey
Coyle, Beth
Keywords: Brain tumours; DNA methylation; 5caC/5hmC; TET2; Pathogenesis; Tumorigenesis
Subjects: W Medicine and related subjects (NLM Classification) > WL Nervous system
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 63635
Depositing User: Eleftheriou, Maria
Date Deposited: 07 Jan 2021 14:04
Last Modified: 07 Jan 2021 14:15
URI: http://eprints.nottingham.ac.uk/id/eprint/63635

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