The recognition, generation and detection of 5-hydroxymethylcytosine

Thesis

5-hydroxymethylcytosine (5hmC) is a DNA modification generated from the oxidation of 5-methylcytosine (5mC) by ten-eleven translocation (TET) enzymes. 5hmC is enriched at promoters, gene bodies and enhancers. The enrichment of 5hmC at the gene body is positively correlated with gene expression. Low levels of 5hmC are observed in various solid tumours and haematological malignancies. The distribution of 5hmC in the genome is strand asymmetric at CG sequences. There are high levels of adjacent and opposing hydroxymethylated and methylated CpG (H/M) sites in the genome. 5hmC in H/M sites account for around 60% of all 5hmC. In this thesis, proteins that specifically recognise H/M sites were identified using DNA pull down followed by LC-MS/MS. However, the binding specificity requires more biochemical validations. The enzymatic pathway that generates H/M site was studied and preliminarily results showed that TET2 have strand selective activity on double strand DNA, which might be a possible way to generate H/M sites. To better understand the distribution of 5hmC in the genome, a 5hmC selective oxidation reaction was applied to map 5hmC on DNA and RNA. The method can be further expended to 5mC mapping in RNA in combination with TET oxidation of 5mC to 5hmC.

Authors: Bi, Y

• DOI: 10.5287/ora-nzbqxakzr
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: DNA modifications; 5-methylcytosine; 5-hydroxymethylcytosine
• Subjects: Genomics; Epigenetics; Biochemistry