Involvement of DOT1L in the Remodeling of Heterochromatin Configuration During Early Preimplantation Development in Mice Running title: DOT1L IN PREIMPLANTATION EMBRYOS

The global chromatin configuration is dramatically remodeled during fertilization and early preimplantation development. Although the chromocenters, which are pericentromeric heterochromatin clusters, are observed in the nuclei of oocytes, they disappear after fertilization and then reappear at the 4-cell stage. To elucidate the mechanism of this reorganization of heterochromatin, we investigated the expression and nuclear localization of DOT1L, which is involved in the regulation of heterochromatin structure through histone H3 lysine 79 (H3K79) methyltransferase activity, during preimplantation development. The Dot1L mRNA level was low at the 2-cell stage. In the analysis by the immunocytochemistry, DOT1L protein was not observed in the nuclei at this stage. Microinjection of Flag-tagged Dot1L cRNA revealed that the DOT1L protein was localized in the nucleus of the embryos at the 1-cell and 4-cell stages but not at the 2-cell stage. However, C-terminus-truncated DOT1L was localized in the nucleus of 2-cell stage embryos. Expression of the truncated DOT1L caused hyper-methylation on H3K79 and the formation of chromocenter-like structures at the 2-cell stage. Intriguingly, the expression of catalytically inactive truncated DOT1L also caused the formation of chromocenter-like structures without an increase in H3K79 methylation. Most embryos expressing th