title: Tissue-Specific Differences in DNA Modifications (5-Hydroxymethylcytosine, 5-Formylcytosine, 5-Carboxylcytosine and 5-Hydroxymethyluracil) and Their Interrelationships.
abstract: BACKGROUND
  Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions.
  
  
  RESULTS
  Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine, as well as 5-hydroxymethyl-2'-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2'-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2'-deoxycytidine and its product 5-formyl-2'-deoxycytidine, as well as between 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively).
  
  
  CONCLUSIONS
  Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2'-deoxycytidine and/or 5-carboxyl-2'-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2'-deoxycytidine which defines the active demethylation process.
authors.affiliation: Department of Clinical Biochemistry, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gackowski
authors.affiliation: Department of Clinical Biochemistry, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Zarakowska
authors.affiliation: Department of Clinical Biochemistry, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Starczak
authors.affiliation: Department of Clinical Biochemistry, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland.
authors.forename: Martyna
authors.initials: M
authors.lastname: Modrzejewska
authors.affiliation: Department of Clinical Biochemistry, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland.
authors.forename: Ryszard
authors.initials: R
authors.lastname: Olinski
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism; D044127:Epigenesis, Genetic; D015870:Gene Expression; D007668:Kidney / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008168:Lung / Q000737:chemistry / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism; D009928:Organ Specificity; D010432:Pentoxyl / Q000031:analogs & derivatives* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013552:Swine; D053719:Tandem Mass Spectrometry; D013950:Thymus Gland / Q000737:chemistry / Q000378:metabolism
chemical_list: C560974:5-carboxylcytosine; C560973:5-formylcytosine; C011865:5-hydroxymethylcytosine; C008055:5-hydroxymethyluracil; D044503:5-Methylcytosine; D010432:Pentoxyl; D003596:Cytosine; D004247:DNA; C000601821:TET1 protein, rat; D049308:Dioxygenases