Study Of The Regulatory Mechanisms Of Zygotic Genome Activation By ERK1/2 And Oxygen Concentration Via Histone H3 Methylation

Early embryos cultured in vitro would effectively improve the breeding efficiency of fine breeding livestock,which not only solves problems of the shortcoming of embryos in livestock but is also widely used in gene editing breeding,an important way to improve the fertility of livestock.However,due to factors such as oxygen concentration,early embryos cultured in vitro often fail to develop into morula or blastocysts,which restricts the application of early embryos in animal husbandry production.Zygotic genome activation(ZGA)is the driving force for the early embryonic regulation of its own development,which is essential for the establishment of embryonic developmental potential and pluripotency.The entire ZGA process consists of several overlapping stages,including clearance of maternal mRNA,protein phosphorylation,activation of "sternness" transcription factors(TF),initiation of metabolic processes,and reprogramming of chromatin modifications.Previous studies have shown that the transcription capacity of the genome during ZGA is established through extensive changes in chromatin and DNA methylation.The chromatin remodeling in ZGA is considered necessary for totipotency.However,how the precise structure of zygotic chromatin is remodeled to maintain totipotency and subsequent pluripotency,and the factors influencing the process remain largely unknown.Therefore,we use mouse embryos as research materials to explore the key factors affecting chromatin modification reprogramming during ZGA.The main contents are as follows:1.Effect of ERK1/2 levels on zygotic genome H3K9me2 during minor ZGAThis study aims to determine the effect of high mitogen-activated protein kinases(MAPKs)levels in mouse zygotes on the modification of parental H3K9me2.First,we used the small molecule inhibitors U0126,GDC-0994,SB203580,and SP600125 to inhibit the activities of mitogen-activated protein kinase kinase 1/2(MEK1/2),extracellular-signal-regulated kinasel/2(ERK1/2),p38 and c-Jun N-terminal kinase(JNK)in early zygotes,respectively,and the corresponding inhibition systems were established.Secondly,immunofluorescence was performed to measure the asymmetry of parental H3K9me2 following the inhibition of MEK1/2,ERK1/2,p38,or JNK.Finally,the small molecule inhibitors of G9a and LSD1,BIX-01294 and OG-L002,were added to the inhibition system of zygotic ERK1/2,and the role of G9a or LSD1 played in the regulation of the parental H3K9me2 were detected by the immunofluorescence.Our data reveal that(1)The optimal inhibitory concentrations of MEK1/2,ERK1/2,and p38 were 40 μM U0126,100 μM GDC-0994,100 μM SB203580,respectively.The result showed that protein activity in MEK1/2,ERK1/2,p38,and JNK was effectively inhibited under the treatment conditions of 40 μM U0126,100 μM GDC-0994,100 μM SB203580,and 100 μM SP600125.(2)Compared with the controls,parental H3K9me2 deposition was increased both in the U0126-treated and the GDC-treated group;the paternal/maternal signal ratio of H3K9me2 was greatly increased in ERK1/2-inhibited zygotes(P<0.01),but decreased in MEK1/2-inhibited zygotes.(3)In early PN3 zygotes,the paternal and maternal H3K9me2 deposition were both increased significantly following GDC-0994 treatment,and the paternal H3K9me2 deposited gradually from the PN3 stage to the PN5 stage.The asymmetry of parental H3K9me2 disappeared in the cleavage stage(17 hpf-24 hpf).Moreover,the expression of the H19 in the blastocysts which zygotic ERK1/2 inhibited was significantly downregulated.,but the expression of the Igf2 was significantly upregulated(P<0.01).(4)The inhibition of the catalytic activity of G9a caused the disappearance of paternal H3K9me2(P<0.01)induced by ERK inhibition,but did not affect the deposition of maternal H3K9me2.The pronuclear localization of G9a significantly increased in ERK1/2-inhibited zygotes.(5)Inhibition of the catalytic activity of LSD 1 further increased the level of maternal H3K9me2 in ERK1/2-inhibited zygotes,while did not affect the deposition of paternal H3K9me2.Our results are consistent with the notion that the high level of ERK1/2 activity in zygotes impedes the paternal localization of G9a,which contributes to the formation of H3K9me2 asymmetry between parental pronuclei.2.Effects of ERK1/2 levels on zygotic genome 5mC and its oxidation during minor ZGAIn this study,zygotic 5mC and 5-hydroxymethylcytosine(5hmC)levels were first detected in the same ERK1/2 inhibition system.Secondly,the small molecule inhibitors of DNA methyltransferases(DNMTs)and Ten-eleven translocation dioxygenases(TET)(Decitabine and DMOG,respectively)were added to the zygotic ERK1/2 inhibition system,to explore the roles of DNMTs and Tet3 in this system.Finally,to investigate the interaction between Tet3 and G9a proteins in zygotes,the pronuclear localization of Tet3 and G9a proteins and the corresponding catalytic products(5hmC and H3K9me2)were detected in the ERK1/2 inhibition system added with BIX-01294 and DMOG.Data in this study reveal that(1)Compared with the control group,the accumulation of paternal 5mC and its oxidation 5-carboxylcytosine(5caC)both increased in the 50 μM GDC-0994 treatment group(P<0.05,P<0.01).Notably,the asymmetries of 5mC and 5caC between the paternal and maternal genomes were all decreased following 100 μM GDC-0994 treatment.(2)In the 50 μM GDC-0994 treatment group,both paternal 5mC and 5hmC increased significantly in the early PN3 stage,while the maternal 5mC and 5hmC increased significantly in the late PN3 stage(P<0.01).(3)Following the joint inhibition of ERK1/2 and DNMTs,the accumulation of paternal 5mC and 5hmC induced by ERK inhibition decreased significantly(P<0.01),and the accumulation of paternal 5hmC induced by ERK inhibition also decreased significantly following the combined inhibition of ERK1/2 and Tet3(P<0.01).(4)Compared with the ERK1/2inhibited zygotes,the accumulation of 5mC and the decrease of 5hmC in paternal genomes following the combined inhibition of ERK1/2 and G9a,and the paternal H3K9me2 was further increased following the combined inhibition of ERK1/2 and Tet3.(5)After inhibition of zygotic ERK1/2,the embryos showed a decreased percentage of blastocysts in both 50 μM and 100 μM GDC-0994 treatment groups(P<0.01).Moreover,the transcription of Oct4,Nanog,Sox2,and Klf4 were all highly expressed in ERK1/2-inhibited blastocysts(P<0.05).These results suggest that the level of ERK1/2 in the zygotic stage affects the reprogramming of 5mC and 5hmC,and high levels of zygotic ERK1/2 activity are contributing to the developmental potential of preimplantation embryos and cell lineage differentiation in mice.3.Effect of hypoxic culture on the transcriptome in embryos during major ZGAIn vitro,embryonic developmental outcomes in hypoxia were generally better than those in normoxia.The present study,therefore,investigates the effect of oxygen concentration on the development of preimplantation embryos and further studies the mechanism that regulation of embryonic development in vitro by oxygen concentration.Firstly,the phenotypic changes of embryonic development were determined between hypoxic and normoxic conditions.Secondly,transcriptome sequencing of mouse 2-cell embryos cultured with normoxia or hypoxia was performed to identify changes in gene expression due to oxygen concentration.Finally,the transcriptome remodeling of major ZGA due to oxygen concentration was determined by taxonomic analysis of transcripts in Maternal-to-zygotic transition(MZT).Data in this study reveal that(1)Compared to the 5%O2-treated group,the 21%O2-treated embryos showed significantly more 2cell and 4-cell arrest during development.The blastocyst cavity appeared in 5%O2treated embryos at the 75hpf stage while the 21%O2-treated embryos were still in the compaction stage.(2)GO functions were annotated on DEGs and the number of genes in various GO subsets was counted,and we found that the effects of hypoxic culture on major ZGA mainly involved biological processes,regulation of transcription(DNA template-dependent),and positive regulation of transcription by RNAPII.(3)In all DEGs of normoxic and hypoxic embryos,core DEGs were significantly enriched in ribosome structural components and RNA binding.Furthermore,the DEGs subnetwork was significantly enriched in ribosome structural components,RNA binding,RNAPII-specific DNA binding,and maternal mRNA degradation.(4)Compared with embryos cultured with 5%O2,more Decayed mRNAs and maternal mRNAs were retained in the 21%O2-treated 2-cell embryos.(5)More 2-cell transient transcripts,major ZGA,and Midpreimplantation gene activation(MGA)genes were expressed in 5%O2-treated embryos.Meanwhile,the signal intensity of 5-EU was significantly increased in 5%O2-treated 2-cell embryos(P<0.01).These results indicate that the oxygen concentration in the culture environment is crucial for the embryonic development of mice before implantation,and the physiologically hypoxic environment is conducive to the decay of maternal mRNA,promotes the transcription initiation of major ZGA,and the establishment of developmental potential before embryonic implantation in mice.4.Effects of hypoxic culture on H3K4me3 and H3K27me3 during major ZGAH3K4me3 deposition is associated with the expression of major ZGA genes,and H3K27me3 deposition regulates the pre-implantation development of embryos.The present study,therefore,investigates the effect of oxygen concentration in the deposition of H3K4me3 and H3K27me3 in major ZGA.Firstly,the protein levels of H3K4me3 and H3K27me3 in 21%O2-treated and 5%O2-treated 2-cell embryos were detected by immunofluorescence and Western blotting,and whether oxygen concentrations had an effect on them was determined.Secondly,the distribution characteristics of genome-wide H3K4me3 and H3K27me3 deposition in 21%O2treated and 5%O2-treated 2-cell embryos were analyzed by CUT&Tag-seq.Finally,the effect of the H3K4me3 redistribution on the alteration of gene expression level caused by oxygen concentration was determined by the combined analysis of CUT&Tag-seq and mRNA-seq.Data in this study reveal that(1)The results of immunofluorescence and Western blotting showed that H3K4me3 deposition was significantly reduced while H3K27me3 levels showed an increasing trend in 5%O2-treated 2-cell embryos.(2)CUT&Tag-seq results showed that the genome-wide H3K4me3 broad domains(bdH3K4me3)decreased and the H3K4me3 domain at TSSs increased,while the H3K27me3 domain did not change significantly in 5%O2-treated 2-cell embryos.(3)The combined analysis of CUT&Tag-seq and ATAC-seq showed that the accumulation of H3K4me3 in the open chromatin was increased during major ZGA,and more H3K4me3 was accumulated at the same following hypoxia culture.(4)The combined analysis of Cut&tag-seq and mRNA-seq showed that expression levels of core DEGs Rpsll,Rps29,Rps4l,Rpl9,Rpl36al,Rpl14,Mrps12,and Mrpl57 were significantly higher in the 5%O2-treated group(P<0.01),and the genome browser view showed that H3K4me3 domains in the promoter region of these core genes were accumulated significantly following hypoxia culture(P<0.05).These results indicate that more H3K4me3 enrichment was obtained in the promoter region following hypoxia culture during major ZGA,and the oxygen concentration regulated the expression of ribosome genes during major ZGA by influencing the distribution of H3K4me3.In summary,ERK1/2 activity during minor ZGA is involved in impeding the deposition of heterochromatin marker H3K9me2,and promoting the de novo 5mC and its oxidation following ERK1/2 gradient inactivation.Physiological hypoxia during Major ZGA contributes to the ordered remodeling of zygotic transcriptomes and the reprogramming of H3K4me3.High levels of ERK1/2 and physiological hypoxia provide an excellent intracellular and extracellular environment for zygotic genome activation.The relevant research results provide great theoretical significance to analyzing the mechanism of livestock embryonic development and improve the in vitro development ability of livestock embryos.