TY - JOUR
T1 - A cross-omics approach to investigate temporal gene expression regulation by 5-hydroxymethylcytosine via TBH-derived oxidative stress showed involvement of different regulatory kinases
AU - Briede, Jacob J.
AU - Deferme, Lize
AU - Wolters, Jarno E. J.
AU - Claessen, Sandra M. H.
AU - van den Beucken, Twan
AU - Wagner, Richard J.
AU - van Breda, Simone G.
AU - Kleinjans, Jos C. S.
PY - 2018/4/1
Y1 - 2018/4/1
N2 - Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.
AB - Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.
KW - tert-Butyl hydroperoxide
KW - 5-Hydroxymethylcytosine
KW - 5-Methylcytosine
KW - Differentially expressed genes
KW - Serine-threonine kinases
KW - HepG2 cells
KW - EMBRYONIC STEM-CELLS
KW - HEPATOCELLULAR-CARCINOMA
KW - DNA-DAMAGE
KW - LIVER CARCINOGENESIS
KW - DETOXIFYING ENZYMES
KW - TET PROTEINS
KW - 5-METHYLCYTOSINE
KW - METHYLATION
KW - SURVIVAL
KW - CANCER
U2 - 10.1016/j.tiv.2018.02.006
DO - 10.1016/j.tiv.2018.02.006
M3 - Article
C2 - 29432895
SN - 0887-2333
VL - 48
SP - 318
EP - 328
JO - Toxicology in Vitro
JF - Toxicology in Vitro
ER -