PROJECT SUMMARY Project title: Dnmt3b activities in mouse development DNA methylation is an epigenetic modification involved in the transcriptional regulation of genes involved in development and differentiation, and its deregulation contributes to human pathogenesis. It is catalyzed by the family of DNA methyltransferases including catalytically active Dnmt1, Dnmt3a, and Dnmt3b. DNA methylation plays a major role in preimplantation development in mice. To establish a new epigenome, the mouse zygotic genome undergoes epigenetic reprogramming, including global DNA demethylation at the 8-cell stage. Upon implantation, a wave of de novo methylation in epiblast cells mediated by de novo enzymes Dnmt3a and Dnmt3b results in new methylation patterns maintained by Dnmt1 that form a basis for tissue-specific expression and differentiation. Dnmt3b regulates developmental and imprinted genes, X chromosome inactivation, pericentromeric regions, gene bodies, and other genomic regions. Its importance in mouse development was demonstrated by the embryonic lethality of Dnmt3b-/- mice. We recently found that Dnmt3bCI/CI mice expressing catalytically inactive Dnmt3bCI protein survived both pre-and postnatal development. Molecular analysis suggested that the accessory function - the ability to recruit other Dnmts to proper genomic loci – of Dnmt3b rather than its catalytic activity, is important for methylation and survival. Here we hypothesize that Dnmt3b is a multifaceted protein whose various activities affect pre-and postnatal development and disease formation in mice. In Aim 1, we analyze global methylation and expression at different stages of development in mice lacking various Dnmt activities to determine the scope of Dnmt3b’s accessory function in Dnmt3a-mediated de novo methylation in vivo. In Aim 2, will test the ability of Dnmt3b to complex with other Dnmts and contribute to de novo methylation induced by other Dnmts in Dnmt1-/-;Dnmt3a-/-;Dnmt3b-/- mouse embryonic stem cells. In addition, we will test the importance of Dnmt3a and Dnmt3l for Dnmt3b’s accessory function and validate our data in a human cell line. In Aim 3, we will assess disease development in mice conceived through the use of in vitro fertilization techniques to analyze disease development, Dnmt levels, the rate of methylation and gene expression errors, as well as their persistence over time. Collectively, our studies will reveal the physiological relevance of Dnmt3b activities in mouse development, and uncover basic mechanisms utilizing Dnmt3b functions and their involvement in IVF. Our results could result in a changed focus of preventive care for individuals conceived by Assisted Reproductive Technologies.