Chemically induced DNA hemimethylation and active demethylation

Abstract

The inheritance of epigenetic signals involves DNA methylation. DNA methylation patterns are inherited through cell generations by the action of DNA methyltransferases (DNMTs). During DNA replication DNMTs recognise hemimethylated DNA and, by the addition of methyl groups to the newly synthesised DNA, maintain methylation. However, the explanation of the cellular methylation machinery is far from completion and the current model of DNA methylation is controversial. Answering the question of how a cell maintains its individual methylation patterns is one of the central questions of epigenetics. The aim of this thesis was to attain deeper knowledge of the dynamics of DNA methylation by direct comparison of complementary DNA strands. I expanded the hairpin bisulfite sequencing method by several technical improvements to compare methylation symmetry of complementary strands of individual DNA molecules in synchronised, dividing Jurkat cells. The method was validated by numerous control and repeat experiments, and provided extensive methylation maps of several Jurkat gene promoters. Mammalian active DNA demethylation is a potentially important, but largely unexplored field. Genome-wide active DNA demethylation in cell lines has only been described in zygotes and embryonic stem cells, and a small number of studies have observed gene-specific demethylation which occurred as a response to physiological signals in somatic cells. I documented and visually mapped patterns of genome-wide active demethylation occurring in dividing Jurkat cell lines triggered by an oxidative treatment. Based on a mechanism proposed more than twenty years ago, most experiments employ Decitabine as a specific inhibitor of DNMT1. I observed actively demethylated strands in the heavily methylated Jurkat gene promoters PCDHGA12 and RASSF1 as early as 2 hours after Decitabine treatment; such demethylated strands were never observed in untreated cells. The observations of this study indicated that DNMT1 inhibition was not the only mechanism by which Decitabine affected DNA methylation and that research employment of Decitabine as a specific DNMT1 inhibitor should be critically reappraised. TET family proteins, which catalyse the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), have been associated with mammalian active demethylation. The involvement of TET enzymes was investigated by assessing promoter hydroxymethylation status during Decitabine induced active DNA demethylation using the exploratory method of hairpin oxidative-bisulfite sequencing. The results of this experiment indirectly demonstrated the involvement of TET proteins in active DNA demethylation. A surprising finding was that the oxidative compound glycine chloramine caused patterns of active demethylation similar to those observed after Decitabine treatment. The experiments documented active demethylation occurring in response to an environmental, oxidative trigger, which phenomenon has not been described before. The findings of this study point to an alternative model of DNA methylation dynamics.