| In mammalian genomes,DNA methylation is an epigenetic mechanism that transfers methyl groups to the C5 site of cytosine to form 5-methylcytosine(5m C).DNA methylation regulates gene expression by recruiting inhibitory transcription factors or proteins that bind to DNA and are involved in gene expression inhibition.During embryonic development,active demethylation of sperm genome occurs mainly in the male prokaryote during the zygotic stage,while passive demethylation of maternal genome is adopted in a DNA replication-dependent manner.Ten-eleven translocation(TET)family(TET1/ 2/ 3),which continuously oxidized 5-methyl-cytosine(5m C)to 5-hydroxymethylcytosine(5hm C),5-formylcytosine(5f C)and 5-carboxycytosine(5ca C),It is then removed by thymine glycosylase(TDG)and base excision repair mechanism(BER)to produce unmethylated cytosine at the target site and achieve genomic demethylation.TET3 is mainly expressed in oocytes and early embryos,and its mediated DNA demethylation is critical for genome reprogramming and gene regulation during gametogenesis and zygotic activation.In this study,immunofluorescence staining(IF),immunocoprecipitation(Co-IP),Western Blot,Dot Blot,mouse oocyte and embryo culture were used to study the regulation of AKT’s protein function through phosphorylation of TET3.This study revealed the regulation of AKT activity on the methylation level of cell genome and the mechanism of its influence on oocyte maturation and early embryonic development.The results are as follows:1.Mouse GV-stage oocytes were treated with the AKT inhibitor MK2206(40μM),and the maturation rate was reduced by about 50%.In addition,most oocytes could not pass the GVBD stage,and a few oocytes developed to the M Ⅱ stage showed abnormal division and could not expel the first polar body normally.MK2206(20 μM)had a significant effect on the mouse early embryonic development.The embryos in the treatment group could not go beyond the 2-cell stage to the 4-cell stage.The morula stage was still in the 2-cell state after 86 h,but no embryo death was observed.The 5hm C immunofluorescence staining experiment showed that the level of 5hm C in the genome of PN4 stage fertilized eggs was significantly increased after MK treatment.The average fluorescence intensity of male and female prokaryotes in the control group and the experimental group was statistically analyzed by Imag J,and it was found that compared with the control group,the level of 5hm C in male prokaryotes after MK2206 treatment was increased by 204%.The level of5 hm C in female prokaryotes increased by 308%,which was more significant.The methylation level of 2-cell was almost unaffected by MK2206.TET3 is a 5m C demethylase mainly expressed in oocytes and early embryos.Immunostaining experiments showed that the subcellular localization of TET3 protein was changed and its distribution in the male and female prokaryotes increased after MK2206(20μM)treatment of fertilized eggs(PN4 stage).2.By 5hm C immunofluorescence staining,it was found that MK2206 could increase the 5hm C level of cell genome by inhibiting AKT activity.The subcellular localization of TET3 protein tends to be localized in the nucleus.Activation of AKT increased cytoplasmic localization of endogenous TET3.Protein stability experiments showed that inhibition of AKT activity can maintain the stability of TET3 protein,while activation of AKT activity can promote the degradation of TET3 protein,suggesting that AKT activity also plays a role in regulating the stability of TET3 protein.3.These results suggest that AKT phosphorylates TET3 and regulates its subcellular localization and stability.Western Blotting(WB)experiments showed that the total Thr phosphorylation level of TET3 protein was significantly decreased after AKT activity was inhibited,suggesting that TET3 might be phosphorylated by AKT.To further demonstrate this,direct physical interaction between TET3 and AKT was found by Co-IP assay,and AKT mainly binds to the DR And JBP domains of TET3 by constructing functional domain intercepts of TET3.Immunofluorescence assay(IF)showed that after MK2206 treatment,only the subcellular localization of TET3-JBP was changed,and the subcellular localization of other domains was not regulated by the activity of AKT.Therefore,it was speculated that the phosphorylation site of AKT might be located in the JBP domain.As predicted by GPS5.0,Thr1191 in the JBP domain was consistent with the AKT phosphorylation substrate characteristics,and the TET3-T1191 A mutant was localized in the nucleus,and the overall Thr phosphorylation level was significantly decreased,which was not regulated by AKT activity.The stability of the T1191 A mutant was significantly improved by protein stability test.The 5hm C immunostaining results showed that the level of 5hm C in the genome of cells transfected with TET3-T1191 A was increased.These results reveal the mechanism by which AKT regulates the function of TET3 protein by phosphorylating T1191.In conclusion,AKT regulates nuclear localization and stability by phosphorylating TET3 at Thr1191,thereby regulating demethylation of the mouse zygotic genome.We speculate that this regulatory mechanism is particularly important for inhibiting the entry of TET3 into the female prokaryote of mouse zygote and avoiding active demethylation of the maternal genome.However,the present results do not explain the enrichment of TET3 in male prokaryotes,and further studies are needed.In conclusion,our results suggest a new regulatory mechanism for the differences in demethylation patterns of male and female prokaryotic DNA during the zygotic phase. |
