Active and passive mechanisms of 5mC and 5hmC removal in human T lymphocytes

In mammals, the 5'-methylcytosine (5mC) modification in the genomic DNA contributes to the dynamic control of gene expression. 5mC erasure is required for the activation of developmental programs and occurs either by passive dilution through DNA replication, or by enzymatic oxidation of the methyl mark to 5-hydroxymethylcytosine (5hmC), followed by passive dilution or further oxidation and active removal. For most biological systems, the relative contribution of active and passive processes of 5mC and 5hmC erasure to the regulation of cell physiology remains poorly defined. Using primary human T lymphocytes as a model of activation-driven cell proliferation and differentiation, we found that DNA demethylation-dependent processes were central in the efficient acquisition of cytokine gene expression by T lymphocytes. As for the underlying mechanism, T cells appeared to rely primarily on the conversion of 5mC into 5hmC, followed by passive, replication-dependent dilution of the 5hmC mark, although active 5hmC removal also occurred during the early stages of activation of naïve, but not memory T lymphocytes. Our data are consistent with a model in which 5hmC is required in quiescent naive T lymphocytes for the establishment and maintenance of regulatory regions poised for rapid response to physiological stimuli. However, cell cycle progression is the primary mechanism to relieve epigenetic constraints in memory T cells. Overall design: Primary naïve and memory T lymphocytes were obtained from two independent donors and were either left resting or were stimulated for 3 days with anti-CD3 and anti-CD28 antibodies.