| Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) have demonstrated that the terminal differentiated adult cells can be reprogrammed into that of embryonic cells. However, this two processes are highly inefficient, especially for the terminal differentiation state cells. Previous studies have demonstrated that this inefficient process can be facilitated by the inclusion of additional factors.Histone methyltransferase contains lots of epigenetic enzymes, which had been demonstrated that played an important role in regulating embryo development, stem cell pluripotency maintenance and differentiation and cell reprogramming. SMYD3 (SET and MYND domain-containing protein 3) is a histone H3 lysine 4 (H3K4) di-and tri-mathyltransferase that forms a transcriptional complex with RNA Polymerase Ⅱ and activates the transcription of oncogenes and cell cycle genes in human cancer cells. Previous report have demonstrated that enhanced expression of SMYD3 is essential for the growth of human cancer cell, whereas the suppression of SMYD3 expression leads to apoptosis and the inhibition of cell growth, migration and invasion. However, the role of SMYD3 in mammalian early embryonic development has not yet been addressed.In this study, we first investigated the expression pattern of SMYD3 in bovine preimplantation embryos and the effects of RNA interference (RNAi)-mediated SMYD3 repression on the development of bovine embryos. Next we detected the cell proliferation and reprogramming relatived genes expression after SMYD3 was factitious over-expressed in bEF, and used traditional induced stem cell method to induce our SMYD3 overexpressed bEF for estimating the reprogramming efficiency. The details are showed as follows:1. The effect of SMYD3 on bovine oocyte maturation and early embryonic developmentIn this study, we first investigated the expression pattern of SMYD3 in bovine preimplantation embryos. The results showed that SMYD3 mRNA was maintained at an appropriate level from oocytes maturation to the 8 cell stage of preimplantation embryos, where the expression had a fluctuation, but the difference was not significant (p>0.05). After which, the SMYD3 mRNA level peaked at the morula stage, and decreased at blastocyst stage (p<0.01). Moreover, immune-fluorescent staining result showed that in M Ⅱ stage, SMYD3 mainly stained in chromosome. During preimplantation embryonic development stages, SMYD3 stained both in nuclei and cytoplasm, and the cytoplasm was stained more intense than the nuclei. Microinjection of SMYD3 siRNA into oocytes at germinal vesicle stage down-regulated the SMYD3 mRNA during the oocytes maturation and also decreased the transcription level of NANOG in the MII stage oocytes, finally blocked the development of in vitro fertilization embryos at 8 cell stage. Conversely, Microinjection of SMYD3 siRNA at pronuclear stage did not affect early embryonic development. Our findings suggest that SMYD3 accumulated in the oocytes can regulate the expression of NANOG, and plays an essential role in bovine early embryonic development.2. SMYD3 functions in the bEF growth and gene expressionA number of factors have been shown to affect reprogramming efficiency through cell-cycle-dependent or-independent mechanisms. The cell-cycle-dependent manner affectes the reprogramming efficiency and accelerates the reprogramming dynamics by affecting cell proliferation; whereas, the cell-cycle-independent manner enhances reprogramming presumably through an epigenetic mechanism without changing the proliferation status. This study will detected the bEF reprogramming mediated by SMYD3 according to the above two aspects.To detect the effect of the SMYD3 on the bEF, the over-expression vector, pSMD3-IRES2-Zsgreenl and the knockdown vector, pRFP-CB-shLenti-SAYD3 were constructed and introduced into the bEF by lipofectamine or lentivirus, respectively. With overexpression of SMYD3, The proliferation of bEF cells was improved, accompanied by elevated expression of the epigenetic related genes such as DNMT1, DNMT3a and EZH2 and lineage decision genes and cell reprogramming related genes such as NANOG, c-MYC, GATA6, PAX6 and NESTIN, while the overexpression of SMYD3 has no significant effect on cell cycle regulated genes CCNG1 and CDK2, but significantly improve the expression of MAP3K11 (p<0.001). However, when the expression of SMYD3 were knockdown, the expression of the above genes declined significantly (p<0.001), it means that SMYD3 played a role on their expression.Whether in embryos or bEF cells, NANOG expression was regulated by SMYD3. In order to detecte the relationship of SMYD3 and NANOG, Bisulphite PCR (Bis-PCR) and chromatin immunoprecipitation PCR (ChIP-PCR) were used to analyze the NANOG promoter methylation and its combination with SMYD3. The results showed that the DNA methylation of NANOG promoter were increased after SMYD3 was over-expressed, but it did not combine with the SMYD3, which implied that SMYD3 did not affected the expression of NANOG directly.Overexpression of SMYD3 in bEFs can improve them proliferation, and regulate the pluripotent genes, lineage decision genes and epigenetic related genes’ expression. Therefor, SMYD3 may affect the cell reprogramming. So we transducted the mouse oct4, sox2, klf4 and c-myc defined-factors to the SMYD3 transgenic cells to induce them reprogramming. The result showed that SMYD3 can improve the clone formation compared with the control bEF cells. Meanwhile these clones showed typical colony morphology and immunofluorescence stained positively for OCT4, NANOG and SOX2. The results declare that SMYD3 can improve the clones formation and reprogramming process. Whether these clone have other iPSCs characteristics need still study.This study not only identify an important factor for bovine oocyte maturation and embryo development, but also reveal that SMYD3 can accelerates the reprogramming of the bEF by changing the proliferation status and enhancing activation of pluripotent genes and epigetic enzymes in reprogramming. |
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