| H3K27me3,as an atypical imprinting method independent of the canonical imprinting,also enables the female pronucleus after fertilization to inherit the silencing mode of genes in the oocyte,thereby regulating gene expression;at the same time,H3K27me3 also interacts with the X chromosome in early embryos.Inactivation is closely related to the activation of zygotic genes.PGC7 is an important maternal effect protein in the development of oocytes and early embryos,and plays an important role in imprinting gene protection,cell cycle regulation,and chromatin condensation.We found that PGC7 is an inherently disordered protein,which functions mainly by interacting with other proteins.In the cell,PGC7 can interact with AKT and EZH2,respectively,and promote AKT phosphorylation to inhibit EZH2,thereby regulating H3K27me3 Level.On the basis of these results,this study mainly explored the effect of PGC7 on the level of H3K27me3 mediated by EZH2 during the zygotic phase of mice,and revealed the reasons for the early embryonic development defects of PGC7-deficient mice.PGC7 knockout mice were prepared by CRISPR/Cas9 technology,and the distribution of EZH2 and H3K27me3 in pronucleus was studied in PGC7 knockout or AKT-inhibited fertilized eggs using immunofluorescence,western blotting,and real-time fluorescent quantitative PCR.And the results of the study on the expression changes of the corresponding genes are as follows:1.In this study,PGC7 gene knockout mice were prepared.The sequencing results showed that there are multiple mutation methods.The mutants with a deletion of 13bp were selected for reproduction,and the PCR method was used for detection and offspring screening.The results of HE stained tissue sections of various organs of PGC7 knockout mice and control mice showed that the organs and tissues of knockout mice were normal,but the spleen and liver were more deeply stained.Early embryo culture of PGC7 knockout mice showed that the fertilized eggs(48.33±2.68%)were stagnated in 2?4 cells,and the blastocyst rate was only(2.33±0.47%).Statistics of the body weight of knockout mice showed that PGC7 biallelic knockout(Pgc-/-)mice developed slowly,and their body weight was significantly lower than that of wild-type(Pgc+/+)mice of the same age(19.70±2.05%)(P<0.05).2.Western Blot detected the activity of AKT in MII oocytes and fertilized eggs.It was found that p AKT-S473 decreased in PGC7 monoallelic knockout(Pgc7+/-)mouse oocytes,while in Pgc7-/-the fertilized egg may be recovered through some compensation mechanism.It was detected by immunofluorescence technology that the distribution of EZH2 in the pronucleus of Pgc7+/-fertilized eggs increased,the level of H3K27me3 increased(P<0.05),and the results in PGC7-/-fertilized eggs were more significant.It proves that the nuclear localization of EZH2 and the level of H3K27me3 may be affected by AKT activity.3.In order to verify that the localization of EZH2 in fertilized eggs is regulated by AKT activity,AKT inhibitor MK2206 and activator SC79 were used to treat fertilized eggs.The immunofluorescence results showed that MK2206 inhibited AKT and caused the prokaryotic localization of EZH2 and PGC7 to increase,correspondingly the level of H3K27me3 also increased.SC79 activation of AKT led to a decrease in the prokaryotic localization of EZH2and PGC7,and the corresponding level of H3K27me3 also decreased(P<0.05).4.The mimic phosphorylation mutant EZH2-S21D at position 21 of EZH2 and the non-phosphorylated mutant EZH2-S21A were constructed and transfected into 3T3 cells.Immunofluorescence staining revealed that the fluorescence signal of EZH2-S21D was mainly in the cytoplasm,while EZH2-S21A was mainly in the nucleus.Western Blot results showed that the levels of H3K27me3 in cells transfected with EZH2 and EZH2-S21A vectors were not significantly different,while the levels of H3K27me3 in cells transfected with EZH2-S21D mutant vectors were significantly reduced,indicating that phosphorylation of serine 21 of EZH2 would reduce EZH2 activity,thereby reducing the level of H3K27me3.Treatment with MK2206 will increase the level of H3K27me3,indicating that the inhibition of AKT will increase the level of H3K27me3.5.Screen out genes that are regulated by H3K27me3 and are activated in the zygotic phase,and use real-time fluorescent quantitative PCR to detect:the deletion of PGC7 leads to a significant decrease in the expression of Gab1,Jade1,Bbx and Etv6 in the 2-cell phase;MK2206 treatment results in the expression of the above genes However,the results were opposite after SC79 treatment(P<0.05).In summary,this study used the CRISPR/Cas9 system to construct a PGC7 knockout mouse model,which confirmed that PGC7 knockout has an important effect on early embryonic development and postnatal development of mice.It also proves that PGC7 may regulate EZH2 through AKT.The distribution in the pronucleus affects the level of H3K27me3 in the zygotic genome. |
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