Mechanism Of Histone H3K27me3 Demethylase UTX In Zygotic Genome Activation

Following fertilization,the zygotic genome is activated through a process termed zygotic genome activation(ZGA),which enables zygotic gene products to replace the maternal products and initiates early embryonic development.During the ZGA period,the embryonic epigenome including H3K27me3 experiences extensive remodification.The H3K27me3 demethylase UTX(Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome)plays an important role in regulating post-implantation embryonic development,cell reprogramming and cell differentiation.However,it remains unclear whether UTX participates in preimplantation development,especially during the ZGA process.In the present study,we designed three parts of the experiment: Firstly,the pcs2-UTX overexpression vector was constructed by conventional molecular experimental techniques,and the effect of its substrate protein H3K27me3 was detected.At the same time,Utx transgenic mice(Utx+ mice)model were prepared and bred to F3 generation.Secondly,Utx+ mice embryos(in vivo model)and UtxmRNA injected embryos(in vitro model)were used to explore the effects of Utx gene on reproductive performance and embryonic development.Finally,the mechanism of Utx gene in zygotic genome activation was analyzed in detail by injection of UtxsiRNA and embryonic "rescue" experiment.The main results are as follows:(1)The coding sequences of Utx gene was cloned in vitro and was constructed the overexpression vector pcs2-UTX by genetic engineering.Then,the vector pcs2-UTX was transfected into mouse embryonic fibroblasts(MEF)and the expression of the substrate protein H3K27me3 was detected by IF and WB.The results showed that the expression of H3K27me3 was significantly reduced after transfection of the pcs2-UTX.In addition,Utx-mRNA was obtained by in vitro transcription from the pcs2-UTX and used for cytoplasmic injection of zygotes;IF results showed that the level of substrate protein H3K27me3 was also significantly reduced.Furthermore,Utx transgenic mice(Utx+ mice)were prepared by pronuclear injection,and the positive rate of F0 generation was 28.6%.After continuous breeding,it was propagated to the F3 generation.(2)To understand the effects of UTX on reproductive ability in Utx+ mice,we conducted a series of evaluation.It was found that the number of newborn pups and ovulations of Utx+ mice were significantly reduced.Furthermore,the size of their ovaries was significantly smaller than that of the controls,and the ovary/body weight ratio also being significantly decreased.The developmental rate of the Utx+ embryos was significantly reduced,especially at the 2-cell to 4-cell stage.The 12 ZGAassociated genes(MuERVL,Zscan4 d,Tcstv1,Tcstv3,Cdc2,eIF-1a,Rif1,Rpl23,U2afbp-rs,Ube2 a,Mt1a,Wee1)and 4 maternal effector genes(Lin28a,Stella,Gdf9,Brg1)were detected by qPCR.The results indicated that the expressions of Zscan4 d,Tcstv3 and Gdf9 were significantly up-regulated.We then simulated in vivo experiments(Utx+ mice)and investigated directly injected Utx-mRNA into the embryos that found that the expression of MuERVL,Zscan4 d,Tcstv1,Tcstv3 and Lin28 a was significantly up-regulated.Based on above results,UTX was mainly targeted to Zscan4 d gene.(3)To further explore the role of Utx gene in preimplantation embryonic development,we designed and constructed Utx-siRNA.The results of qPCR,IF and WB revealed that the UTX expression level decreased by 90% in the si-Utx-injected embryos compared to that in the si-control-injected embryos.Consistent with the Utx+ mice,the embryonic development of si-Utx-injected embryo was decreased.We examined the expression of 12 ZGA-associated genes and 4 maternal effector genes in embryos injected with Utx-siRNA,and the expression of Zscan4 d,Tcstv1 and Stella was significantly decreased.This validates the conclusion that UTX targets the Zscan4 d gene.(4)Combining the Utx+ mice,Utx-mRNA and Utx-siRNA results,we found that Zscan4 d is a downstream gene of UTX.Using the ChIP-qPCR assay,we demonstrated that UTX positively regulates the expression of Zscan4 d by binding to the promoter region of the Zscan4 d gene.To further examine the relationship between UTX and Zscan4 d,we constructed an in vitro transcription vector pcs2-ZSCAN4 D,and further designed the embryonic "rescue" experiment,which simultaneously injected UtxsiRNA and Zscan4d-mRNA into the zygotes.Through the "rescue" experiment,Zscan4 d can partially rescue the impaired embryonic development upon UTX depletion.In this study,the Utx+ mice,Utx-mRNA and Utx-siRNA assays were used to reveal the important role of histone H3K27me3 demethylase UTX in preimplantation embryonic development.We found that abnormal UTX expression leads to ZGA retardation;UTX can directly bind and activate the key gene Zscan4 d in ZGA events to facilitate preimplantation embryonic development.