Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.

insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner.

Although apoptosis occurs during myogenesis, its mechanism of initiation remains unknown. In a culture model, we demonstrate activation of caspase-12, the initiator of the endoplasmic reticulum (ER) stress-specific caspase cascade, during apoptosis associated with myoblast differentiation. Induction of ER stress-responsive proteins (BiP and CHOP) was also observed in both apoptotic and differentiating cells. ATF6, but not other ER stress sensors, was specifically activated during apoptosis in myoblasts, suggesting that partial but selective activation of ER stress signaling was sufficient for induction of apoptosis. Activation of caspase-12 was also detected in developing muscle of mouse embryos and gradually disappeared later. CHOP was also transiently induced. These results suggest that specific ER stress signaling transmitted by ATF6 leads to naturally occurring apoptosis during muscle development.