The methylation of CpG dinucleotides represents an epigenetic mark that is crucial for regulating the normal progression of numerous biological processes including development and cell differentiation. During the last decade, it became increasingly clear that methylation patterns are not static but may adapt to various cellular requirements. Regarding normal somatic cells, the dynamic of DNA methylation including its extent throughout the genome as well as its implication in cellular differentiation is largely unknown. In the context of the present thesis, it was demonstrated that several cell type or cell lineage specific genes harboured a specific methylation profile. Interestingly, those differences in DNA methylation were mostly confined to regions upstream or downstream of the core promoter and preferentially affected CpG poor DNA regions. The gene regulatory relevance of DNA sequences affected by dynamical alterations in the methylation pattern, may be studied by means of transient transfection assays. For this purpose, a novel CpG free luciferase reporter vector was designed that provides a simple and robust tool for analysing effects of DNA methylation within CpG poor as well as CpG rich DNA stretches on gene expression.
As particularly the regulated and active removal of methyl CpG marks still remains controversial, the major aim of the present work was the characterization of this epigenetic phenomenon in a natural setting of post mitotic cells: the proliferation independent differentiation of human peripheral blood monocytes into dendritic cells or macrophages, respectively. Using a global, comparative CpG methylation profiling approach that was directed to detect differentially methylated regions in CpG rich as well as CpG poor DNA stretches, 45 examples for active demethylation were identified. The validation by a bisulfite conversion based technique and the characterization of a selected subset revealed that DNA demethylation was not restricted to promoter regions and that the time course varied for individual CpGs. Irrespective of their location, the removal of methylated cytosines strictly coincided with the appearance of activating histone marks indicating the presence of cis acting elements. Since demethylation events were highly reproducible between monocyte derived dendritic cells from distinct donors, the present data suggest that active demethylation is a precisely targeted process. The comparison of the global methylation data with the genome wide mRNA expression profiles demonstrated that active DNA demethylation is not always directly followed by transcriptional activation. Probably, gene activation is a multilevel process that is dependent on various genetic and epigenetic factors. Thereby, CpG demethylation seems to be a necessary prerequisite for priming the chromatin structure for transcription factor binding
