Epigenetic inheritance is defined as cellular information, other than the DNA sequence itself, which is heritable during cell division. Epigenetic aberrations can lead to diseases such as childhood cancer and developmental defects and because heritable nature of epigenetic information, the epigenetic diseases display a pattern of inheritance similar to "traditional" genetic diseases. However, little is known about the molecular mechanisms that control this epigenetic information and therefore identification of factors that would allow prediction, or early diagnosis of pathology is not possible. A particularly intriguing type of DNA epigenetic regulation is imprinting. Imprinting, also called parent-of-origin-effect, is a mechanism by which certain genes are epigenetically marked in a parent-specific manner. The most prominent epigenetic mark on imprinted loci is DNA methylation and this imprinted methylation affects gene expression, resulting in either gene silencing or activation. As a result, imprinted genes are expressed mono-allelic and in a parental-origin dependent fashion. Misregulation of imprinted gene expression can lead to a variety of diseases including Beckwith-Wiedemann syndrome, Angelmann syndrome and Prader-Willi syndrome. In addition, recent data demonstrate that aberrant imprinting can be a causative event in tumorigenesis. Imprinted gene regulation is tightly associated with the germline, since it is during germ cell development that imprints are established. Several key enzymes have been identified that can mediate DNA methylation at imprinted sites. These are members of the DNA methyl-transferase family (Dnmt) and mutations in some of these genes lead to aberrant DNA methylation and loss of imprinting. Much less is known about the mechanisms by which DNA can loose its methylation mark. To date, there have been no reports on genes that can actively remove DNA methylation, despite evidence that such activity exists and plays an important role during early development and in the regulation of imprinted gene expression. Using a novel method of in vitro germ cell differentiation, we have identified members of the Methyl-binding-domain gene family as candidate genes regulating DNA methylation and show that disruption of their expression results in changes in DNA methylation at imprinted loci. We propose to investigate the mechanism by which Mbd genes orchestrate DNA methylation and will analyze a possible mechanism by which these genes cooperate to cause epigenetic reprogramming of imprinted genes during germline differentiation. Deciphering the molecular components involved in the regulation of global DNA methylation and in particular methylation at imprinted loci is critical for developing urgently needed strategies to predict, diagnose and interfere with epigenetic disease.