Summary DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

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During 3T3-L1 preadipocyte differentiation induction, the insulin-stimulated insulin-like growth factor-1 (IGF-1) receptor signal is responsible for the induction of adipocyte differentiation. Treatment with inhibitors of phosphatidylinositol 3-kinase, LY294002 or wortmannin, leads to the complete blockade of adipocyte differentiation in 3T3-L1 preadipocytes. Of the three factors (1-methyl-3-isobutylxanthine, dexamethasone, and insulin) inducing 3T3-L1 preadipocyte differentiation, only insulin was able to activate the phosphatidylinositol 3-kinase-protein kinase B/Akt signal cascade. In 3T3-L1 preadipocytes, protein kinase B/Akt 1 RNA interference not only suppressed the expression of protein kinase B/Akt 1 but also blocked hormone-induced adipocyte differentiation. In these protein kinase B/Akt 1 RNA interference cells, the signal molecules upstream of protein kinase B/Akt 1, such as IGF-1 receptor and insulin receptor substrate-1, were normally activated by insulin stimulation, whereas insulin-stimulated phosphorylation of forkhead transcription factor (FKHR), which is a downstream molecule of PKB/Akt 1, was blocked. Thus, protein kinase B/Akt 1 is an important signal mediator in IGF-1 receptor signal cascade for inducing adipocyte differentiation.Most of our current understandings on the cellular mechanisms of adipocyte differentiation regulation have come from the studies on established in vitro preadipocyte cell lines (1-3). These in vitro preadipocyte cell lines, e.g. 3T3-L1, 3T3-F442A, and TA1, can be induced to differentiate into adipocytes by insulin or in combination with other hormones. Subsequent studies on 3T3-L1 adipocyte differentiation induction indicate that the authentic hormone to induce the adipocyte differentiation is insulin-like growth factor-1 (IGF-1), 1 and insulin acts through the IGF-1 receptor on the cell membrane to induce the differentiation (4). In post-confluent 3T3-L1 preadipocytes, IGF-1 receptor signal initiates the adipocyte differentiation process. Immediately after the hormonal stimulation, postconfluent G 0 3T3-L1 preadipocytes reenter the cell cycle and start the adipocyte differentiation program. It is clear that the IGF-1 receptor signal plays an irreplaceable role in inducing the adipocyte differentiation process in 3T3-L1 cells.The signaling pathways or networks involved in mediating IGF-1 receptor signal for adipocyte differentiation are not fully understood. Two kinase systems, MAP kinases and phosphatidylinositol 3-kinase-protein kinase B/Akt, in 3T3-L1 cells can be activated by IGF-1 receptor signaling. The function of the MAP kinase system in IGF-1 receptor signal-induced 3T3-L1 cell adipogenesis is not clear. It is further complicated by the different effects exhibited by p38 MAP kinase and ERK1/2 on 3T3-L1 cell adipogenesis (5-12). More molecular studies are required in order to understand the relationship between MAP kinase system and 3T3-L1 cell adipogenesis.PI 3-kinase-PKB/Akt as an important intracellular signal cascade has been involved in the regulation o...