can act on. In NgTET, significant drop in catalytic efficiency is observed by altering
CpG site to CpA (~1.75-fold), CpT (~3.80-fold) and CpC (~5.80-fold) [112]. The
crystal structure of NgTET1 indicates H-bonding interactions between the guanosine
in the CpG pair and the NgTET protein which can account for the preference for
CpG [112]. Note that 5-C modifications of the CpG on the reverse strand do not
influence the catalytic activity.
The alkylation repair protein (AlkB) homologs, subfamily of 2OG oxygenases,
are closely related to the TET oxygenases and use Fe(II) catalysis with 2OG (3) and
O 2 to oxidise DNA or RNA. This subfamily contains ABHs and fat mass and
obesity-associated (FTO) proteins. In particular, ABH2 and ABH3, demethylases
involved in repair processes, are structurally similar to hTET2. The preferred substrates for ABHs or FTO are 1N
0 -methyl-adenine (1 mA), 3N
0 -methyl thymidine
(3mT), 3N
0 -methyl cytosine (3mC) and 6N
0 -methyl adenosine (6 mA), and their
oxidation results in unstable hemiaminal intermediate which decomposes to formaldehyde and demethylated base [114].
4.3 TETs in Development and in Disease
5hmC, 5fC and 5caC are found in many cells, but their concentrations vary
depending on the cell types. Interestingly, while the levels of 5mC remain relatively
even across different cell types at approximately ~3.5–4.5% of all cytosine in the
genomic DNA, this is not observed for 5hmC, 5fC or 5caC. The levels of 5hmC can
range from 0.7% in the central nervous system to 0.03% in the spleen [124]. High
levels of 5hmC are commonly found in the brain and neurons. Purkinje neurons, for
example, contain up to 40% of 5hmC abundance relative to the 5mC levels in the
cell [5].
While 5fC and 5caC are stable modifications and believed to be part of signalling
pathways, 5fC and 5caC are significantly less abundant than 5mC or 5hmC. Levels
of 5fC can range between 0.2 parts per million (ppm) in the lungs and 12 ppm in the
brain in mice. Interestingly, there is no direct correlation between 5mC/5hmC and
5fC in cells or among the age of the tissue, suggesting that these marks have
independent roles and are actively generated over time [125]. Postnatal mice have
undetectable levels of 5caC (<0.1 ppm), but in 12-week-old mice, some tissues,
such as the liver, can have elevated levels of 5caC (up to 2.0 ppm), while in others
such as the kidney or brain, 5caC levels remain below the detection limit [125].
Aside from their role as intermediates for demethylation, the function of 5hmC,
5fC and 5caC, is not fully understood. Recent studies suggest that each mark has
multiple implications. 5hmC formation is detected in active genes and enriched in
the promotor regions [126]. While 5fC has been proposed to influence the helical
structure of DNA [127], this may be context dependent [128]. Raiber et al. demonstrated that additional anchoring of the nucleosome to the DNA can occur in vitro
and in vivo, as a result of histone lysine reacting with the 5fC to form an imine
derivative [129]. This would provide enhanced nucleosome organisation within the
Chemical Compounds Targeting DNA Methylation and Hydroxymethylation
271
CpG site to CpA (~1.75-fold), CpT (~3.80-fold) and CpC (~5.80-fold) [112]. The
crystal structure of NgTET1 indicates H-bonding interactions between the guanosine
in the CpG pair and the NgTET protein which can account for the preference for
CpG [112]. Note that 5-C modifications of the CpG on the reverse strand do not
influence the catalytic activity.
The alkylation repair protein (AlkB) homologs, subfamily of 2OG oxygenases,
are closely related to the TET oxygenases and use Fe(II) catalysis with 2OG (3) and
O 2 to oxidise DNA or RNA. This subfamily contains ABHs and fat mass and
obesity-associated (FTO) proteins. In particular, ABH2 and ABH3, demethylases
involved in repair processes, are structurally similar to hTET2. The preferred substrates for ABHs or FTO are 1N
0 -methyl-adenine (1 mA), 3N
0 -methyl thymidine
(3mT), 3N
0 -methyl cytosine (3mC) and 6N
0 -methyl adenosine (6 mA), and their
oxidation results in unstable hemiaminal intermediate which decomposes to formaldehyde and demethylated base [114].
4.3 TETs in Development and in Disease
5hmC, 5fC and 5caC are found in many cells, but their concentrations vary
depending on the cell types. Interestingly, while the levels of 5mC remain relatively
even across different cell types at approximately ~3.5–4.5% of all cytosine in the
genomic DNA, this is not observed for 5hmC, 5fC or 5caC. The levels of 5hmC can
range from 0.7% in the central nervous system to 0.03% in the spleen [124]. High
levels of 5hmC are commonly found in the brain and neurons. Purkinje neurons, for
example, contain up to 40% of 5hmC abundance relative to the 5mC levels in the
cell [5].
While 5fC and 5caC are stable modifications and believed to be part of signalling
pathways, 5fC and 5caC are significantly less abundant than 5mC or 5hmC. Levels
of 5fC can range between 0.2 parts per million (ppm) in the lungs and 12 ppm in the
brain in mice. Interestingly, there is no direct correlation between 5mC/5hmC and
5fC in cells or among the age of the tissue, suggesting that these marks have
independent roles and are actively generated over time [125]. Postnatal mice have
undetectable levels of 5caC (<0.1 ppm), but in 12-week-old mice, some tissues,
such as the liver, can have elevated levels of 5caC (up to 2.0 ppm), while in others
such as the kidney or brain, 5caC levels remain below the detection limit [125].
Aside from their role as intermediates for demethylation, the function of 5hmC,
5fC and 5caC, is not fully understood. Recent studies suggest that each mark has
multiple implications. 5hmC formation is detected in active genes and enriched in
the promotor regions [126]. While 5fC has been proposed to influence the helical
structure of DNA [127], this may be context dependent [128]. Raiber et al. demonstrated that additional anchoring of the nucleosome to the DNA can occur in vitro
and in vivo, as a result of histone lysine reacting with the 5fC to form an imine
derivative [129]. This would provide enhanced nucleosome organisation within the
Chemical Compounds Targeting DNA Methylation and Hydroxymethylation
271