Functional Analysis of TET-Family 5-Methylcytosine Dioxygenases in Non-CpG DNA Demethylation

Abstract

One of the most widely studied epigenetic marks in DNA is 5-methylcytosine (5mC). Research has indicated that in the mammalian genome around 5-6% of all cytosines are methylated (5mC), with a strong preference for the CpG dinucleotide context. In addition, several recent studies have uncovered extensive C5 cytosine methylation (5mC) at non-CpG (5mCpH, where H = A/C/T) sites. 5mC plays an important function in tissue-specific gene expression, human development, X-chromosome inactivation, genomic imprinting, and as a biomarker of ageing. The dynamic regulation of 5mC epigenetics is also essential for various stages of pluripotency, differentiation, and development. In humans, demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation of dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair (TDG/BER).

To study the functional analysis of TET-family 5-methylcytosine dioxygenases in non-CpG DNA demethylation, efficient purification of enzymatically active untagged human TET2 and His-Tagged mouse TET1 using cation exchange chromatography and affinity chromatography was established. In addition, using the positive and negative mode of the mass spectrometer we have developed an improved and highly sensitive positive/negative ion-switching-based liquid chromatography-tandem mass spectrometry (LC–MS/MS) method that can separate and quantify modified cytosine bases produced by TET-family 5-methylcytosine dioxygenases. The substrate specificity of the TET enzymes has been explored in the CpG context, but little is known about the enzyme responsible for the active demethylation of 5mCpH sites. Initial studies have reported that the human TET2 can rapidly oxidize 5mCpG sites, but oxidation of both the 5mCpA and 5mCpC sites was negligible. Using the established high-sensitive method, we demonstrated that the human TET2 (including mouse TET1) can oxidize 5mCpH sites in double-stranded DNA in-vitro.

In addition, we have demonstrated the substrate specificity of the human TET2/mouse TET1 dioxygenase and the role of DNA flanking sequences in the demethylation of methylated CpG and CpA sites in DNA. Our results showed that human TET2/mouse TET1 does have DNA sequence preference like other DNA modifying enzymes. Our findings also indicated that TET-mediated mCpA oxidation has a higher flanking sequence preference compared to mCpG oxidation. These results may help elucidate the novel role of TET2-mediated mCpG and mCpA demethylation in the genome. And finally, in the last set of experiments, the activity of ten different TET2 clinical mutations was studied. Over the last decade, hundreds of frame-shift, nonsense, and missense mutations in the TET2 gene have been identified in patients. TET2 is one of the most frequently mutated genes in myelodysplastic syndromes (MDS), MDS-myeloproliferative neoplasms (MDS-MPNs) and acute myeloid leukemia derived from MDS and MDS-MPN (sAML). However, our understanding of biochemical abnormalities associated with TET2 mutations is very limited because all the study has been focused on the CpG context. In this project, the activity of ten different TET2 clinical mutations was studied in both the CpG and CpA contexts for a comparative analysis to better understand the biochemical abnormalities associated with TET2 mutation.