Epigenetics refers to the study of heritable changes in genome function that occur without a change in the primary DNA sequence of an organism. One such change is DNA methylation, most frequently characterized by an enzymatic modification at the fifth position of cytosine (5-mC), present abundantly in the context of CpG dinucleotides. The formation and maintenance of 5-mC is catalyzed by cellular DNA (cytosine-5) methyltransferases (DNMTs).
The biology of 5-mC and its role in epigenetic inheritance and gene expression are well known. In addition to 5-mC, mammalian DNA also contains a variety of other modified nucleobases at low levels, which arise by DNA damage, normal metabolism, and other environmental factors. These modifications are generally removed during the onset of a new cell cycle.
Recent observations involving mouse Purkinje and granule neuron cells identified an additional modified cytosine, 5-hydroxymethylcytosine (5-hmC). This modified base was also found in the genome of undifferentiated embryonic stem (ES) cells. A family of genes encoding Tet (ten eleven translocation) proteins (Tet1, Tet2, and Tet3) catalyze the oxidation reaction of 5-mC to 5-hmC.
In addition to this mechanism, the transfer of formaldehyde to cytosine by DNMTs may be another cause of 5-hmC formation. The biological role of 5-hmC is the subject of much current speculation. A popular hypothesis is that 5-hmC may be an intermediate between 5-mC and cytosine in the mammalian genome. For example, conversion of 5-mC to 5-hmC and subsequent deamination of 5-hmC to 5-hydroxymethyluracil (5-hmU) would generate a mismatch between two opposite bases (5-hmU:G), resulting in activation of mismatch repair pathways.
In another study, a reversible enzymatic reaction catalyzed by DNA (cytosine-5) methyltransferases led to the release of formaldehyde from 5-hmC, producing unmodified cytosine. Therefore, through this mechanism, 5-hmC may participate directly in DNA demethylation.
Furthermore, disruption of the Tet1 and Tet2 genetic loci has been reported to associate with hematologic malignancies. A fusion of human Tet1 with the histone methyltransferase MLL has been identified in several cases of acute myeloid leukemia (AML) associated with the t(10;11)(q22;q23) translocation.
Homozygous null mutations and chromosomal deletions of the Tet2 locus have been discovered in myeloproliferative disorders, suggesting that Tet2 may function as a tumor-suppressor protein.
The cytosine nucleobase can reside as either unmethylated or methylated, including hydroxymethylated (5-mC and 5-hmC) forms. Methylated cytosine is generally associated with gene repression.
Several biomarkers rely on methylation status as a disease prognostic and/or diagnostic marker (e.g., methylation of Sept9 as a biomarker for colon cancer). However, neither gene expression nor biomarker studies have taken the presence of 5-hmC into account as, currently, there are no routinely used technologies that can distinguish 5-mC from 5-hmC.
The gold-standard approach to detect methylated cytosine is bisulphite conversion followed by DNA sequencing. Although this chemical method can reliably identify cytosine versus 5-methylcytosine, it does not distinguish between 5-mC and 5-hmC.