| After the fusion of mammalian sperm and oocyte, the genome begins to the drastic epigenetic reprogramming. First, the paternal genome is actively demethylated without the DNA replication. Followed, the maternal genome is passively demethylated with DNA replication. The paternal genome rapid active DNA demethylation, which does not depend on DNA replication, needs DNA demethylation catalytic enzymes. These DNA demethylation and de novo methylation during early embryonic development provides a suitable time window to study the influence of DNA methylation on gene expression and the relationship between DNA methylation and pluripotency.There are two methods for DNA methylation removal. One is DNA replication-dependent, while another is catalyzed by the DNA demethylase. At present, there was not an effective DNA demethylase has been identified in mammal, and active DNA demethylation pathway is also controversial. Activation-induced cytidine deaminase (AID, also known as AICDA) was first recognized through its role in the generation of antibody diversity in B lymphocytes, and was later found to be able to deaminate 5mC into thymine (T). In addition, AID was hypothesized to be necessary for the initiation of active DNA demethylation in the promoter of pluripotent genes (such as OCT4 and NANOG). However, the demethylation role of AID remains controversial. The main reason is that the deamination efficiency of AID on mC was much lower than that on C.To further investigate the effect of AID in active demethylation in mammalian cells, this study were designed to examine the expression of AID and the DNA methylation status of its regulation region in bovine tissues, oocytes, fertilized eggs and preimplantation embryos. Meanwhile, gene overexpression, knockdown technologies and DNA demethylation agent 5-Aza-CdR treatment were used to study the role of AID in the reprogramming process and in embryo development.1. The dynamic changes of AID T-DMR during bovine early embryonic developmentAccording to the expression of AID and the DNA methylation status of its promoter region in four kinds of tissues, the tissue-dependent and differentially methylated region (T-DMR) was located at the transcription start site -88 bp～-431 bp. The expression of AID was related to tissue type. The testis was significantly higher than that of heart, kidney and liver, but its DNA methylation status of AID in T-DMR region was lower than the other three kinds of tissues. This suggests that DNA methylation status of AID gene in T-DMR region is closely related to its expression level.Then the expression of AID gene and its methylation status of promoter region T-DMR were detected during oocyte maturation and preimplantation embryo development. AID was gradually accumulated along with the oocyte maturation. At the same time, No.2 and 3 CpG sites in the T-DMR region of AID were obviously demethylated, whereas the other sites are not changed. With the combination of sperm and oocyte, the expression of AID sharply declined until 8-cell which refer to maternal zygotic transition period. After 8-cell stage, AID gene gradually increased again through blastocyst stage. However, in this process, AID T-DMR has maintained the DNA methylation status except for No.2 and 3 CpG sites which were unmethylated. By immunofluorescence detection, AID proteins are uniformly distributed in the nucleus and cytoplasm from the oocyte maturation stage to morula stage. And a large number of AID protein concentrated in the inner cell mass at the blastocyst stage, which may related to the maintenance of pluripotency of the inner cell mass.In summary, AID which are accumulated during oocyte maturation has an effect on fertilization. And the DNA methylation status of AID gene promoter is closely related to its expression. The expression of AID gene, after activation of the embryonic genome, may be associated with embryo development.2. The effect of AID in the bovine fetal fibroblasts and oocytes during early embryonic developmentIn this study, gene overexpression and knockdown technologies were used to change AID expression in bovine fibroblasts and GV oocytes and to detect the role of the AID gene on fertilization and development. The overexpression vector pCDsRed-PbAID was constructed and transfered into bovine fetal fibroblasts. After G418 selective progression, we obtain five clones, which were identified by PCR to use as nuclear donor cells in somatic cell nuclear transfer. In AID-transfected cells, the overexpression of the AID gene did not change the cellular morphology, chromosomal composition, growth curve and global genomic DNA methylation status, but the overexpression of AID changed the DNA methylation status of the OCT4、NANOG、SOX2 and CDX2 promoters, and their transcription. Using AID-overexpressing cells as the nuclear donor cells, the cleavage and blastocyst rates were increased. In addition, in the blastocysts of the AID-overexpression group, the expression of OCT4, NANOG and SOX2 was increased, whereas the DNA methylation levels of their promoters exhibited no significant change. Our data provide new evidence implicating AID in active DNA demethylation in mammalian cells and demonstrate that AID-dependent DNA demethylation act in a loci-specific mode.For the AID knockdown experiments, siRNAs were transferred into GV oocytes. To ensure the high efficiency of AID knockdown, a mixture of three interference fragments was injected into GV oocytes. After 18 h of in vitro maturation, the AID mRNA level in the MII oocytes was found to be markedly reduced (26.7%, respectively) compared with the level obtained with the control siRNA. The reduction of AID did not affect the maturation of oocytes and embryo development, but altered the expression of OCT4, NANOG and SOX2 and their methylation status of promoter region. Then, the changes of 5-mC and 5-hmC were analyzed in pronuclear stage eggs by immunofluorescence after injection of AID-siRNA. There were no obvious changes between 5-mC and 5-hmC, this meant the mechanism of AID and 5-hmC did not share a same channel. In conclusion, this study demonstrated that AID selectively catalyzes gene DNA demethylation and plays a role in the dynamic epigenetic regulation of cell pluripotency and embryo development.3. The effect of 5-Aza-CdR on somatic reprogrammingIn this study, AID overexpressing cells and AID gene knock down cells are used to investigate the effect of 5-Aza-CdR(1、2、3、4、5μM) on cell morphology, cell cycle, related gene expression and genomic DNA methylation status and to analyze the relationship between genomic DNA demethylation and site-specific DNA demethylation. The results showed that 5-Aza-CdR had effect on transgenic donor cells growth in dose-dependent manner. As the 5-Aza-CdR concentration being increased to 4 and 5μM, cellular activity significantly decreased (P<0.01). When treated by 1,2, and 3 μM for 24 h, cells in the GO/G1 and G2/M phase were increased and cells in the S phase were decreased. Karyotype analysis showed that 3 μM treatments resulted in the emergence of a small proportion of aneuploid cells, indicating that high concentrations of 5-Aza-CdR could increase the rate of aneuploidy. The number of cells which expressed red fluorescent protein (DsRed) was significantly increased after 1 μM 5-Aza-CdR treatment, compared to the control group, the expression of AID and SOX2 were also increased, while the promoter methylation levels of SOX2 was significantly decreased. After 24 h treated by 5-Aza-CdR, the expression of the OCT4 and SOX2 genes were increased in AID knockdown cells, but the expression of the NANOG did not change. The above results showed that 5-Aza-CdR treatment effected the cell morphology, cell cycle and reporter gene expression in bovine transgenic cells. In conclusion, the genomic demethylation by 5-Aza-CdR and loci specific demethylation by AID act synergistically in somatic cell reprogramming. |
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