The Role Of Dux Gene In The Zygotic Genome Activation Of Mouse Early Embryos

After fertilization in mammals,zygotic genome transcription remains silent,and the development of embryo is controlled by maternal genetic substance.With the gradual degradation of maternal substances,embryo unlock the transcription of zygotic genome to achieve ZGA(zygotic genome activation).As a key link of maternal to zygotic transition,ZGA is an important biological event after fertilization and is essential for the development of embryos.Further study of ZGA will help to address the problem of embryo development retardation in vitro culture and establish a foundation for optimization of assisted reproductive technology.To investigate the roles of Dux in early mouse embryonic development,especially in ZGA.We first monitored Dux and ZGA-related genes(e.g.Zscan4?MERVL and Eif1a)expression during preimplantation development.Although the results based on RT-qPCR and immunofluorescence showed that Dux gene could be detected the early embryonic development,the highest expression of Dux was 2-cell stage.Unlike RNA,DUX proteins could be detected at 2-cell and 4-cell stages;Interestingly,the expression pattern of Dux was similar to the ZGA related genes,such as Zscan4,MERVL,Gm6763.To further study the role of Dux,we designed and synthesized siRNA which specifically target to the transcript of Dux.The data indicated that the siRNA effiency was about 60%.Compared to the control group,the morula and blastocyst formation rates in siRNA injected group were significantly lower than those in the control group(31% vs.83%,P < 0.001;28% vs 76%,P < 0.001).In addition,2-cell block occurred in the embryos of the interfering group.To explain this phenomenon,we compared ZGA-related genes expression between control group and siRNA injected group.RT-qPCR result presented that the expression of ZGA-related genes is decreased in Dux knock-down embryo compared with those in control group.Based on the above results,we speculated that Dux regulate the expression of Zscan4 directly.To verify this hypothesis,we performed dual luciferase reporter assay.The results showed that DUX binds to LTR(156-163 bp)of Zscan4 upstream promoter and directly regulates the expression of Zscan4.Due to the vital role of Dux,we constructed Dux overexpression mice for studying the function of Dux in vivo,which provides relevant experimental materials for future research.