been fully elucidated, and its relevance in eukaryotes remains to be established. In
order to better understand the biological functions of 4mC, it is necessary to map this
epigenetic mark along the genome.
Epigenetic deregulation is increasingly recognized as a critical event in the
development of cancer and other diseases [34]. Changes in DNA methylation
patterns can lead to silencing of tumor suppressor genes and activation of protooncogenes, which represents an early critical event in tumor development
[34]. Silencing of gene expression is correlated with hypermethylation of promoter
regions [3]. Aberrant cytosine methylation is implicated in many other human
diseases including asthma, autism, and amyotrophic lateral sclerosis [35–
37]. DNMT inhibitors such as 5-azacytidine, 5-aza-2
0 -deoxycytidine (decitabine),
and pyrimidin-2-one ribonucleoside (zebularine) are widely used as single therapeutic agents and in combination with classical chemotherapeutic agents in treatment of
various tumors [38–41].
On the other hand, the levels of 5hmC are substantially lowered in human
cancers, with a 50–90% reduction of 5hmC in breast cancer, liver cancer, lung
cancer, and several other tumor types [42]. In contrast, elevated levels of 5hmC
have been observed in patients with systemic lupus erythematosus [43], autism [36],
and bronchial asthma [44]. Recently, N6MedA concentrations in mouse brain have
been shown to be mediated by environmental stress [45], although the biological
significance of this finding remains to be established.
2 Methods for Detection and Quantification of Epigenetic
Modifications Within the Genome
2.1 Initial Detection of 5mC and Its Oxidized Forms via
Paper Chromatography and Thin-Layer
Chromatography
Paper chromatography and thin-layer chromatography experiments played a key role
in the initial discovery of epigenetically modified DNA bases [46]. In paper chromatography, a compound mixture is spotted on a strip of chromatography paper,
which is then hung in a development chamber, and dipped into a solution of volatile
alcohols in which to develop the chromatogram. Compounds that are more soluble in
the alcohol mobile phase move up the paper strip during development. Paper
chromatography was used for the initial detection of 5mC in eukaryotic DNA
[46]. Calf thymus DNA was hydrolyzed with acid, and the resulting free bases
were separated by paper chromatography using butanol. The resulting chromatographic spots were extracted with alcohol and identified by comparing their UV
spectra to those of standard nitrogenous bases. Paper chromatography has since been
replaced by newer techniques such as thin-layer chromatography (TLC).
492
C. L. Seiler et al.
order to better understand the biological functions of 4mC, it is necessary to map this
epigenetic mark along the genome.
Epigenetic deregulation is increasingly recognized as a critical event in the
development of cancer and other diseases [34]. Changes in DNA methylation
patterns can lead to silencing of tumor suppressor genes and activation of protooncogenes, which represents an early critical event in tumor development
[34]. Silencing of gene expression is correlated with hypermethylation of promoter
regions [3]. Aberrant cytosine methylation is implicated in many other human
diseases including asthma, autism, and amyotrophic lateral sclerosis [35–
37]. DNMT inhibitors such as 5-azacytidine, 5-aza-2
0 -deoxycytidine (decitabine),
and pyrimidin-2-one ribonucleoside (zebularine) are widely used as single therapeutic agents and in combination with classical chemotherapeutic agents in treatment of
various tumors [38–41].
On the other hand, the levels of 5hmC are substantially lowered in human
cancers, with a 50–90% reduction of 5hmC in breast cancer, liver cancer, lung
cancer, and several other tumor types [42]. In contrast, elevated levels of 5hmC
have been observed in patients with systemic lupus erythematosus [43], autism [36],
and bronchial asthma [44]. Recently, N6MedA concentrations in mouse brain have
been shown to be mediated by environmental stress [45], although the biological
significance of this finding remains to be established.
2 Methods for Detection and Quantification of Epigenetic
Modifications Within the Genome
2.1 Initial Detection of 5mC and Its Oxidized Forms via
Paper Chromatography and Thin-Layer
Chromatography
Paper chromatography and thin-layer chromatography experiments played a key role
in the initial discovery of epigenetically modified DNA bases [46]. In paper chromatography, a compound mixture is spotted on a strip of chromatography paper,
which is then hung in a development chamber, and dipped into a solution of volatile
alcohols in which to develop the chromatogram. Compounds that are more soluble in
the alcohol mobile phase move up the paper strip during development. Paper
chromatography was used for the initial detection of 5mC in eukaryotic DNA
[46]. Calf thymus DNA was hydrolyzed with acid, and the resulting free bases
were separated by paper chromatography using butanol. The resulting chromatographic spots were extracted with alcohol and identified by comparing their UV
spectra to those of standard nitrogenous bases. Paper chromatography has since been
replaced by newer techniques such as thin-layer chromatography (TLC).
492
C. L. Seiler et al.