chromatin remodelling for activation and silencing. Furthermore, 5fC and 5caC can
affect the activity of proteins that associate with these marks. For example, with
RNA polymerase II, 5fC and 5caC result in a lower rate of incorporation of pairing G
[130]. Additionally, modifications at 5mC are distinctly recognised by an array of
reader proteins [131] providing additional level of epigenetic regulation mediated by
TET oxidation.
TET1 was first identified as a translocation partner of MLL gene in patients with
acute myeloid leukaemia (AML) [105]. Subsequently, it was demonstrated that MLL
fusion protein directly binds to TET1 promoter, causing upregulation of TET1 and
global increase in 5hmC levels in MLL-rearranged leukaemia [132]. TET1 plays a
critical role in the oncogenesis of MLL-rearranged leukaemia in vitro and in vivo
through co-activation of the Hoxa9/Meis1/Pbx3 signalling pathway [132]. In glioblastomas, TET1-mediated 5hmC production plays a critical role in tumorigenicity
[133]. These studies highlight the therapeutic potential for targeting TET1 in certain
cancers. In contrast, TETs have been shown to also have important tumour suppressor
roles in multiple cancers. Mutations in TET2 have been linked to haematopoietic
malignancies [134]. In myelodysplastic syndrome (MDS), TET2 is the most highly
mutated gene [135], and in AML patients, multiple TET2 mutations (including
frameshift, nonsense and missense within the protein creating mutated or truncated
proteins) are found with reduced or abolished catalytic activity [136]. Furthermore,
abnormalities have been observed in lymphoid malignancies, such as hTET2 B/TCell lymphoma [137]. Rare occurrences of mutations are found in hTET1/3 in chronic
lymphocytic leukaemia (CLL) [138]. TETs are also linked to various solid tumours
including gastric, breast, lung, liver and prostate cancer [139]. Downregulation of
TET gene expression has been observed in multiple solid tumours, with decrease in
5hmC levels and increasing rate of proliferation [140].
Taken together, the evidence suggests that TETs can have both oncogenic and
tumour suppressor roles, depending on the cellular context. Chemical probes for
TETs are thus needed to understand their biological functions in development and in
diseases.
5 TET Enzyme Assay and Inhibitor Development
The availability of robust and quantitative assays is prerequisite for the biochemical and
functional studies of enzymes. For the TET enzymes, there are now a wide range of
methodologies available to detect and quantitate oxidised 5mC levels, including global
genome-wide mapping at base resolution [141–143]. Analysis techniques of modified
nucleosides/oligonucleotides include (1) antibody-based detection of oxidised 5mC,
(2) analytical methods using thin-layer chromatography (TLC) [5, 110, 112, 144] or
liquid chromatography coupled with mass spectrometry (LC-MS, LC-MS/MS)
[107, 124, 145] and (3) chemical conversion or enzymatic labelling of modified
cytosines (including glucosylation of 5hmC) [146–153]. These methodologies have
enabled studies on tissue and genomic distribution and dynamics of oxidised 5mC in
272
R. Belle et al.
affect the activity of proteins that associate with these marks. For example, with
RNA polymerase II, 5fC and 5caC result in a lower rate of incorporation of pairing G
[130]. Additionally, modifications at 5mC are distinctly recognised by an array of
reader proteins [131] providing additional level of epigenetic regulation mediated by
TET oxidation.
TET1 was first identified as a translocation partner of MLL gene in patients with
acute myeloid leukaemia (AML) [105]. Subsequently, it was demonstrated that MLL
fusion protein directly binds to TET1 promoter, causing upregulation of TET1 and
global increase in 5hmC levels in MLL-rearranged leukaemia [132]. TET1 plays a
critical role in the oncogenesis of MLL-rearranged leukaemia in vitro and in vivo
through co-activation of the Hoxa9/Meis1/Pbx3 signalling pathway [132]. In glioblastomas, TET1-mediated 5hmC production plays a critical role in tumorigenicity
[133]. These studies highlight the therapeutic potential for targeting TET1 in certain
cancers. In contrast, TETs have been shown to also have important tumour suppressor
roles in multiple cancers. Mutations in TET2 have been linked to haematopoietic
malignancies [134]. In myelodysplastic syndrome (MDS), TET2 is the most highly
mutated gene [135], and in AML patients, multiple TET2 mutations (including
frameshift, nonsense and missense within the protein creating mutated or truncated
proteins) are found with reduced or abolished catalytic activity [136]. Furthermore,
abnormalities have been observed in lymphoid malignancies, such as hTET2 B/TCell lymphoma [137]. Rare occurrences of mutations are found in hTET1/3 in chronic
lymphocytic leukaemia (CLL) [138]. TETs are also linked to various solid tumours
including gastric, breast, lung, liver and prostate cancer [139]. Downregulation of
TET gene expression has been observed in multiple solid tumours, with decrease in
5hmC levels and increasing rate of proliferation [140].
Taken together, the evidence suggests that TETs can have both oncogenic and
tumour suppressor roles, depending on the cellular context. Chemical probes for
TETs are thus needed to understand their biological functions in development and in
diseases.
5 TET Enzyme Assay and Inhibitor Development
The availability of robust and quantitative assays is prerequisite for the biochemical and
functional studies of enzymes. For the TET enzymes, there are now a wide range of
methodologies available to detect and quantitate oxidised 5mC levels, including global
genome-wide mapping at base resolution [141–143]. Analysis techniques of modified
nucleosides/oligonucleotides include (1) antibody-based detection of oxidised 5mC,
(2) analytical methods using thin-layer chromatography (TLC) [5, 110, 112, 144] or
liquid chromatography coupled with mass spectrometry (LC-MS, LC-MS/MS)
[107, 124, 145] and (3) chemical conversion or enzymatic labelling of modified
cytosines (including glucosylation of 5hmC) [146–153]. These methodologies have
enabled studies on tissue and genomic distribution and dynamics of oxidised 5mC in
272
R. Belle et al.