Study On Regulation Of Nuclear Transfer Reprogramming During Goat Zygotic Genome Activation By Long Noncoding RNA

In mammals,their proper development during the early cleavage stages strongly relies on the gene products newly transcribed by zygotic genome activation?ZGA?.The ZGA process is abnormal during the development of early embryo generated by somatic cell nuclear transfer?SCNT?.It was reported that long noncoding RNA?lncRNA?plays vital roles during the zygotic genome activation.However,the ZGA stage in goat embryo is not clear and lncRNAs were not well conserved.Moreover,there are few reports regarding the regulation of nuclear transfer reprogramming by IncRNAs.Thus,the aim of this study is to investigate the regulation of nuclear transfer reprogramming by lncRNA.To achieve this,four experiments have been conducted in this study:1.Study of differentially expressed mRNA and lncRNA during goat zygotic genome activationThe aim of this study is to determine the ZGA stage in goat and to investigate the mRNAs and lncRNAs that regulate the ZGA process.Firstly,the ZGA was determined by in vitro-cultured,alpha-amanitin treatment and Immunofluorescence staining.Secondly,expression of mRNA and IncRNA during goat ZGA were investigated by using single-cell RNA sequencing.Finally,functions of three potential lncRNAs were investigated by using microinjection of siRNAs against them.Our data reveal that?1?early development was arrested at 8-cell stage in in vitro-cultured and alpha-amanitin treatment embryos and the RNAP ? was strongly stained in 8-cell embryos.?2?Expression of the mRNA and lncRNAs were dynamically changed during goat ZGA process.There are 800 mRNAs and 250 lncRNAs that differentially expressed during ZGA.?3?The differentially expressed mRNA Kdm4d was highly expressed in 8-cell embryos and the level of H3K9me3 was gradually removed during ZGA,reaching its lowest level in 8-cell embryos,However,H3K9me3 was highly stained in developmentally arrested 8-cell embryos.?4?The role of three functional lncRNAs,lnc₁178,lnc₁37,and lnc₃263,were investigated.The percentage of embryos develop to the blastocyst stage was significantly reduced?19.4±0.44%Vs.75±2.89%,P<0.01?in lnc-137 knocked down embryos.Moreover,the expression level of the zygotic genes,CXCL6,EIF-1A,and HSP70.1,was compromised in the lnc₁37 knocked down embryos?P<0.01?.Taken together,the ZGA occurs at the 8-cell stage in goat early development and lncRNAs were dynamically changed during the ZGA process.Importantly,the lncRNA lnc₁37 is crucial for goat zygotic genes expression and the maternal genes degradation,thus affect the development of the embryos.Our results are consistent with the notion that lncRNAs play vital roles during ZGA,and the data presented here provide an excellent source for further ZGA lncRNA studies.2.Study of differentially expressed mRNA during ZGA of SCNT-generated embryosThe ZGA process of SCNT-generated embryos was aberrant,and the histone modification-related mRNAs play vital roles during the nuclear transfer reprogramming.Therefore,in the present study,we first investigated the transcriptome of the SCNT-generated embryos during ZGA.Secondly,the expression level of the histone methylation modifiers and the histone modification H3K4me3 was analyzed during ZGA.Finally,goat Kdm5b mRNA was synthesized and injected by micro injection to investigate its influence on nuclear transfer reprogramming.Data in this study reveal that?1?ZGA was abnormal in SCNT-generated embryos and there are 813 mRNAs up-regulated and 235 mRNAs down-regulated in the 8-cell stage SCNT-generated embryos compared to the Intracytoplasmic sperm injection?ICSI?-generated embryos,respectively.?2?The expression level of the histone methylation modifiers,Kdm5a and Kdm5b was significantly down-regulated in SCNT-generated 8-cell embryos.?3?Histone modification H3K4me3 was gradually erased during ZGA of the ICSI-generated embryos and was barely observed at the ICSI-generated 8-cell embryos.However,at the SCNT-generated 8-cell embryos,H3K4me3 was strongly stained.?4?Injection of goat Kdm5b mRNA effectually removed the H3K4me3 at the SCNT-generated 8-cell embryos.Importantly,the ratio of the embryos developed to blastocysts was significantly increased.However,the higher level of H3K4me3 was maintained in SCNT-generated 8-cell embryos when Kdm5b was knocked down,and no embryos developed to blastocyst.Our data suggest that the ZGA process was abberrant in goat SCNT-generated embryos and the H3K4me3 was abnormal during ZGA of goat SCNT-generated embryos.Thus,H3K4mc3 was a barrier during goat nuclear transfer reprogramming,and Kdm5b enhanced the embryonic development following nuclear transfer in goat.Thus,the present study provides a theoretical reference for exploring the mechanism of nuclear transfer reprogramming and improving the in vitro development of SCNT-generated embryos.3.Study of regulation of nuclear transfer reprogramming during goat ZGA by IncRNALncRNAs play vital roles during ZGA.However,whether IncRNA is associated with the nuclear transfer reprogramming during ZGA is seldom investigated.The present study,therefore,investigates functional IncRNA during ZGA in goat SCNT-generated embryos and further study the mechanism that regulation of nuclear transfer reprogramming by lncRNAs.Firstly,differentially expressed lncRNAs during ZGA in goat SCNT was characterized using single-cell RNA sequencing.Secondly,functional IncRNA was predicted by using Kdm5b as the target.Finally,the development of the SCNT-generated embryos after knocking down of functional IncRNA was observed and the mechanisms behind this were further investigated.Data in this study reveal that?1?lncRNAs were dynamically changed during ZGA of goat SCNT-generated embryos,and 131 and 28?a total of 159?lncRNAs were up-regulated and down-regulated,respectively.?2?There are 18 lncRNAs which were predicted to target the Kdm5b and lnc₃712 was selected for further study for its expression level was significantly increased after knocking down of Kdm5b.?3?The blastocyst rate of SCNT-generated embryos after lnc₃712 knocking down was greatly improved;?4?There are 700 mRNAs which were up-regulated after knocking down of the lnc₃712 in SCNT-generated 8-cell embryos,incuding the expression level of the zygotic genes,such as BTG3,UBE2M,UBE2S,EIF3G,BTG1,and HSF1 was significantly increased?P<0.05?,and expression level of the transcription factors,such as ABTB1,GATA2,and TEAD4 were significantly increasd?P<0.01?,whereas the maternal genes,WEE2 and GDF9 was significantly decreased?P<0.01?after knocking down of the lnc₃712 in SCNT-generated 8-cell embryos.?5?Knocking down of the lnc₃712 increased the expression level of the Kdm5b,and successfully removed the histone modification H3K4me3 at the SCNT-generated 8-cell embryos,and the H3K27me3 signal was re-occurred in the blastocysts of SCNT-generated embryos.Taken together,our data reveal that lncRNAs were dynamically changed during ZGA of goat SCNT-generated embryos and suggest that the IncRNA lnc₃712 was an epigenetic barrier of nuclear reprogramming by repressing the expression of Kdm5b in goat.This study reveals the regulation network of lncRNAs and histone modifiers that regulating the nuclear transfer reprogramming in goat.The present study not only enriches the research content of IncRNA regulatory nuclear transfer reprogramming,but also provides references for improving the reprogramming efficiency of nuclear transfer.4.Study of Xist promoter methylation in SCNT-generated embryos and cloned goatsLncRNA Xist is crucial for initiating XCI,which achieves dosage equivalence of X-linked genes between XX and XY cells.However,information about Xist in goat genome remains unknown and Xist-related research in SCNT cloned goats was seldom reported.The present study aims to determine the expression level of Xist and the methylation status ofXist promoter in cloned goats and SCNT-generated embryos during ZGA.Firstly,the promoter sequence of the goat Xist was amplified and differentially methylated region?DMR?was identified using MethPrimer.Secondly,the methylation status ofXist promoter was investigated in cloned goats and SCNT-generated embryos during ZGA.Finally,the expression level of Xist was investigated in cloned goats and SCNT-generated embryos during ZGA.Data in this study reveal that?1?the puptative DMR was differentially methylated in oocytes,sperm,female fibroblasts,and male lung tissue,indicating that this DMR is differentially methylated.?2?During the ZGA stage in goat,the Xist promoter was highly methylated in SCNT-generated embryos compared to that of the ICSI-generated embryos.?3?When compared to naturally bred controls,the methylation level of Xist was significantly increased in ear,lung,and brain tissues of 3-day-old female deceased cloned goats but remained unchanged in ear tissue of female live cloned goats and in lung and brain tissues of male deceased cloned goats.?4?The expression level of Xist was not differentially changed in SCNT-generated embryos during ZGA.However,in the ear,brain,and lung tissues of female deceased cloned goats,the expression level of Xist was significantly decreased.Based on these data,we conclude that the methylation of the Xist promoter was abnormal in SCNT-generated XX embryos and in female deceased cloned goats.We further infer that the hypermethylation of Xist was failed to be reprogrammed in SCNT-generated embryos during ZGA,which was retained in the female deceased cloned goats and subsequently led to dysregulation of Xist.Our data presented here will help to improve the efficiency of cloning and provides fundamental data for investigating the effect of Xist in goat nuclear reprogramming.In conclusion,we found that the ZGA occurs at 8-cell stage in goat.During which process,there are 800 mRNA and 250 lncRNA that differentially expressed.One of the lncRNAs,lnc₁37,is crucial for the early development of goat embryos.The ZGA process is abnormal in goat SCNT embryos.Importantly,injection of Kdm5b mRNA or siRNA against lnc₃712 greatly increases the development of goat SCNT embryos.Moreover,lnc₃712 might act as a barrier of reprogramming by targeting the Kdm5b.Our study not only enriches the research content of lncRNA regulatory nuclear transfer reprogramming,but also provides references for improving the reprogramming efficiency of nuclear transfer.