The Mechanism Of Key Histone Modification Regulations During Porcine Early Embryos

Early embryonic development begins with the fusion of sperm and oocyte in mammals.In this process,chromatin undergoes extensive reprogramming,which converts terminally differentiated cells into totipotent embryos.As an important epigenetic modification,histone modification underwent distinct reprogramming processes during early embryonic development.Previous studies have mostly focused on model animals,and about the reprogramming dynamics of histone modifications in porcine embryos were not clear.As an important agricultural livestock and potential biomedical model,it is of great significance to study the histone modification dynamics in porcine early embryos.Furthermore,comparative analysis between pig,mouse and human would highlight the conservation and specificity of epigenetic modifications in the reprogramming process of mammalian embryos.In this study,by using immunofluorescence staining and ultra-low-input native chromatin immunoprecipitation and sequencing(ULI-NCh IP-seq),we profiled key histone modifications,namely H3K4me3,H3K27me3 and H3K27 ac in porcine oocytes and in vitro fertilized embryos for the first time.In addition,we compared with the histone modifications dynamics in somatic cell nuclear transfer(SCNT)embryos,and found that there was an abnormal enrichment of histone modifications in cloned embryos.The main research results are as follows:(1)In porcine oocytes,H3K4me3 formed as broad peaks in the distal partially methylated domains(PMD)regions,and were maintained until the zygotic genome activation(ZGA),and then reshaped into sharp peaks.In addition,on the proximal promoter of the ZGA genes,H3K4me3 in oocytes formed in sharp peaks,which will become broader after fertilization,and then reshaped into sharp peaks during ZGA.This observation also exists in human early embryos,but not in mouse early embryos.(2)It has been proposed that broad H3K4me3 domains inhibit the activations of ZGA genes,but this proposal was not supported by experimental evidences.To test the biological functions of broad H3K4me3 domains in pre-ZGA embryos.We knocked down KDM5 B and KDM5C(demethylase of H3K4me3)in porcine zygotes.Early embryonic development was significantly disrupted after the knockdown.RNA-seq analysis at the 4-cell stage showed that the overall transcriptional levels were reduced,and ZGA-related genes was suppressed.Ch IP-seq analysis revealed that abnormal H3K4me3 established in promoters of ZGA genes.These results indicate that KDM5 B and KDM5 C safeguard the entry of ZGA by erasing the broad enrichment of H3K4me3 in porcine early embryos,thereby ensuring subsequent embryonic development.(3)Strong H3K27me3 enrichment exists in porcine oocytes,and it is highly co-localized with H3K4me3 in the PMD regions.After fertilization,H3K27me3 underwent global removal,and began to re-establishment at the morula stage.On the promoter of the ZGA genes,the enrichment of H3K27me3 at the morula stage shows up as bivalent marks with H3K4me3.By inhibiting H3K27me3 reconstruction in post-ZGA embryos,we found that embryonic development was impaired and the ZGA genes were abnormally highly expressed at the blastocyst stage.It is speculated that promoter bivalency inhibits the continuous activation of the ZGA genes and contributes to facilitating ZGA exit.In addition,the reprogramming process also exists in human early embryos,but not in mouse early embryos.(4)There is no H3K27 ac enrichment in porcine oocytes,but it began to re-establishment in the genomic regions erased by H3K27me3 after fertilization,indicating that there is a conversion relationship between the two histones.By detecting the expression patterns of H3K27me3 and H3K27 ac modification-related regulatory factors,it is found that the conversion may be regulated by the PRC2 complex.(5)By analyzing the enrichment of H3K4me3 and H3K27me3 at four-cell and blastocyst of SCNT embryos,we found abnormal enrichment of these two histone modifications in the promoter and PMD regions in the SCNT four-cell embryos.It is speculated that it inhibited the expression of the ZGA genes in cloned embryos,thereby affecting the development of the cloned embryos.At the blastocyst stage,it was found that the two histone modifications are not significantly different between cloned embryos and fertilized embryos,indicating that the abnormal enrichment of histone modifications during ZGA is the main factor affecting the development of cloned embryos.In summary,this is the first time to describe the dynamics of H3K4me3,H3K27me3 and H3K27ac modifications during porcine early embryonic development.At the same time,two novel mechanisms of epigenetic modification regulating ZGA initiation were elucidated: one is that the broad H3K4me3 peaks in ZGA gene promoter after fertilization inhibits the premature activation of ZGA,and the other is that H3K27me3 peaks re-establishment on the promoters will inhibit the expression of the ZGA genes to promote the exit of ZGA at morula stage.These observations also exist in human early embryos,indicating the conservation of reprogramming between pig and human.Additionally,abnormal histone modifications are likely served as an important epigenetic barrier in pig cloned embryos.This study is beneficial to deepen the biological functions of histone modification during early embryonic development in future studies.