Bruniquel and Schwartz122 found that a region in a promoter of the IL-2 gene demethylates following activation in the absence of DNA replication and results in a profound increase in the production of IL-2. These two papers provide the initial evidence for an active, environmentally driven alteration in DNA methylation in postmitotic cells. Szyf and colleagues101,123 Inhibitors,research,lifescience,medical first proposed that DNA methylation is enzymatically reversible and that DNA methylation is dynamic in fully differentiated cells. This idea remains controversial. Active demethylation was nevertheless find more clearly demonstrated early in embryogenesis and the parental

genome undergoes replication independent, active demethylation hours after fertilization, well before the initiation of replication. Demethylation at

very early stages in development has Inhibitors,research,lifescience,medical been relatively accepted, but the possibility of postnatal demethylation, especially in fully differentiated somatic cells, has been hotly disputed. However, active replication demethylation was demonstrated in Epstein-Barr virus (EB V)-infected B cells and in HEK293 cells. The HEK293 studies suggest Inhibitors,research,lifescience,medical that active replication-independent demethylation takes place in differentiated somatic cells and that it is dependent on alterations in chromatin structure. Earlier studies from Szyf’s122 laboratory extracted active DNA demethylase activity from a human lung cancer cell line and identified a protein with demethylase activity, which was cloned concurrently Inhibitors,research,lifescience,medical by Bird’s group and named MBD2.123 Interestingly, the protein, MBD2, was found by Bird’s group and others to also associate with a chromatin remodeling complex containing HDAC, which is involved in silencing Inhibitors,research,lifescience,medical of gene expression through the recruitment of a repressor complex. The assignment of a demethylase function to a protein that was independently discovered as a recruiter of repressor complexes triggered the expected controversy in the field and reports that MBD2 failed to produce demethylase activity. However, the observation that MBD2/demethylase Carnitine dehydrogenase expression

produces the demethylation of some, but not all, promoters in a dose- and time-dependent manner has been confirmed.108,124 Clearly, the contextual factors that determine MBD2 demethylase activity remain to be fully explained. Interestingly, MBD2 increased gene expression in those instances where promoter demethylation occurred, suggesting that not all promoters respond in the same orderly manner. Indeed, the same is true for DNA methylation, which impedes the DNA binding of most, but not all transcription factors; SP1 binds to methylated DNA. Antisense knock down of MBD2 resulted in inhibition of active demethylation induced by valproate and caused hypermethylation and silencing of the prometastatic gene uPA in metastatic breast cancer cells.