The Regulatory Effects Of TFAP2C And LncT On Epigenetic Modifications During Early Development Of Pig Embryos And Their Mechanisms

The organ size and physiological functions of pigs are close to those of humans.Research using pigs as animal models for human diseases involves diabetes,cardiovascular diseases,neurodegenerative diseases,genetic diseases,tumors and other fields.These models are important for revealing the mechanisms of disease occurrence,finding drug targets,conducting drug screening and efficacy evaluation,and finding ways to treat and diagnose diseases.Assisted reproductive technologies such as in vitro fertilization and somatic cell nuclear transfer have provided effective tools for animal production.The quality of early embryonic development directly affects the success rate after implantation,so studies on early embryonic development are important for the successful production of animals.Each division during early embryonic development is accompanied by significant gene silencing and activation,and it is critical to find the key factors that can regulate gene expression during this process.Transcription factors and non-coding RNAs are abundantly present in the genome and have the function of regulating gene expression,while studies in early embryo development are still scarce.In this study,we screened transcription factors and lncRNAs that may have regulatory functions in embryonic development by comprehensive analysis of transcriptome data from in vivo fertilized(IVV)embryos.And we investigated the effects of transcription factor TFAP2 C and non-coding RNA lnc T on embryonic development by injection of interfering RNA in in vitro fertilized(IVF)embryos,the mechanism of their effects on embryo development was indicated by RNAseq,q PCR,immunofluorescence staining,and Ch IP etc..The aim is to lay a foundation for elucidating the regulation mechanism of early embryonic development and improving the efficiency of animal production.The main research results are as follows:1.Differentially expressed transcription factors and lncRNAs were screemed by analyzing gene expression patterns in the transcriptome of IVV embryos,it was found that(1)The expression pattern of transcription factors in embryos highly coincided with the timing of gene activation with potential regulatory effect in gene expression;(2)Lnc RNAs show dynamic expression patterns during embryonic development and are highly consistent with the progression of embryonic development.Lnc RNAs in IVV embryos were not changed significantly from the oocyte fertilization to the 2-cell embryos,and a large number of lncRNAs changed from the 4-cell embryos;(3)37major genes were screened by target gene prediction and co-expression analysis of lncRNAs.Based on the prediction of target genes,lnc T(named for its predicted target gene TFAP2C)was found to have potential regulation of embryonic development and further research was conducted.The results showed that this fraction of lncRNAs was highly consistent with gene activation at all stages of embryonic development,with large-scale gene activation at both the 4-cell and 8-cell stages,indicating that this fraction of genes may have the potential to regulate embryonic development.2.The effect of transcription factor TFAP2 C on the development of porcine embryos was studied by microinjection of interfering RNA in IVF embryos,and the mechanism of its effect on embryo development was investigated by RNAseq,q PCR,immunofluorescence staining,Ch IP,and dual luciferase reporter.The results showed that(1)Interfering with TFAP2 C significantly inhibited the embryonic development process,and the blastocyst development rate was significantly lower(7.76±1.86%)after interfering with TFAP2 C compared with the control group(22.92±1.97%),while there was no difference between the NC group(injection of meaningless interfering RNA fragments group)(20.4±0.95%)and the control group;(2)RNAseq results showed that after interfering with TFAP2 C there were 792 genes expression was upregulated and 1208 genes expression was down-regulated after interference with TFAP2 C,GSEA Biological process GO analysis revealed that the upregulated genes were mainly related to apoptosis intrinsic signaling pathway regulation,leukemia suppressor response,translation and other processes,while the downregulated genes were mainly related to the regulation of protein localization to the cell periphery,motor behavior;(3)Interference with TFAP2 C disrupted the expression of several epitopemodifying enzyme genes,with SETD2,KDM5 B,EP300 expression significantly upregulated and EZH2,KAT2 A,CARM1 expression significantly downregulated;(4)Immunofluorescence results showed that the modification levels of H3K4me3 and H3K4me2 were significantly reduced 4-cell embryos after TFAP2 C interference,and H3K36me3 modification levels were significantly increased.The level of 5m C modification was increased,and DNMT1 entry to nuclei after TFAP2 C interference;(5)Transcription factor binding site prediction showed that the binding motif of TFAP2 C binds to a variety of epitope modifying enzymes,and Ch IP results and dual luciferase reporter assay results showed that TFAP2 C can bind to the promoter region of SETD2 to regulate its expression.The results indicate that interference with TFAP2 C disrupts gene expression patterns in the embryo,disrupting histone modifications and DNA methylation modifications,thereby affecting embryonic development.TFAP2 C is able to bind to the SETD2 promoter region to affect its expression.3.Based on the screened lncRNA,the gene presence was verified by PCR,and the interfering RNA was designed to study the effect of this lncRNA on embryonic development by microinjection in IVF embryos,combined with RNAseq,q PCR,and immunofluorescence staining to investigate the mechanism of its effect on embryonic development.The results showed that(1)The expression of lnc T started to be elevated at the 4-cell stage.Compared with the control group(22.92±1.97%),the blastocyst development rate was significantly reduced after lnc T interference(13.65±0.82%),while there was no difference between the NC group and the control group(20.4±0.95%);(2)RNAseq results showed that 1564 genes expression was upregulated and 636 genes expression was downregulated after lnc T interference.The pathway enrichment analysis of these genes showed that the upregulated genes were mainly enriched in translation,cellular nitrogen compound metabolic processes,gene expression and cellular biosynthesis processes,while the downregulated genes were mainly enriched in actin cytoskeleton organization,regulation of actin filament organization,regulation of anatomical structures and regulation of cellular components;(3)The interference of lnc T disturbed the epigenetic modifying enzyme expression,compared with the control group,DNMT3 B,PRMT1,HDAC3,CARM1 expression was downregulated and KDM5 C,ESCO1,SETD2,KDM5 B expression was upregulated in the lnc T interference group;(4)Immunofluorescence results showed that the modification of H3K4me3 and H3K4me2 were significantly decreased at 4-cell embryos,and the modification levels 5m C were significantly elevated at 4-cell embryos and blastocyst;(5)SIN3A was one of the genes with significant changes in gene expression after lnc T interference,and the blastocyst rate in SIN3A-KD group was significantly lower than the blastocyst rate in the control group;(6)5m C was similarly observed to be elevated at the 4-cell embryos in the SIN3A-interfered group.DNMT1 was more in the nucleus in the SIN3A-KD and lnc T-KD groups compared to the control group,while DNMT1 was mostly expressed in the cytoplasm in the control group;(7)Interference with both lnc T and SIN3 A was able to reduce the expression of the pluripotency gene NANOG in embryos.The cells were induced to apoptosis.The results showed that interfering with lnc T disrupted gene expression patterns,histone modifications and DNA methylation modifications in embryos,thus affecting embryonic development.As one of the downstream genes of lnc T,SIN3 A is involved in the regulation of lnc T on early embryonic development,affects the nucleation of DNMT1 and thus affects DNA methylation modification,reduces the expression of pluripotent genes and induces cell apoptosis.