| The growth arrest and DNA damage gene family plays a key role in the regulation of cell cycle and apoptosis. Gadd45a is one member of this family. The function of Gadd45a is maintaining the genomic stability. Gadd45a inhibits cell growth at G2/M checkpoint, lacking of Gadd45a result in abnormal G2/M checkpoint. Gadd45a induces the decline of cytoskeleton stability followed by apoptosis. In addition, Gadd45a plays an important role in active DNA demethylation. Over expression of Gadd45a can promote global DNA demethylation, depressing the expression of Gadd45a can lead to gene silence.The expression and regulation of Gadd45a occur in transcription, post transcription and translation. There are no data related to the epigenetic regulation of this gene. RT-PCR, bisulfite-sequencing PCR, chromatin immuno-precipitation assay were used to detect the dynamic change of DNA methylation and histone modification in the regulation region of Gadd45a gene. The DNA methylation status in IVF embryos, parthenogenetic embryos, and nuclear transfer embryos were also analyzed. Results are as followed:1. the expression of Gadd45a geneRT-PCR was used to detect the expression of Gadd45a gene in bovine heart, kidney, liver and testis. Expression of Gadd45a mRNA were not detected in heart but were found in the other three tissues with different levels.2. DNA methylation status of Gadd45a geneDNA methylation is one of the most frequent chemical modifications in the eukaryotic genome. It is associated with inactive chromatin state and, therefore, negative regulation of transcription. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues.Seven CpG sites in the 3' regulation region of Gadd45a were chosen to examine their DNA methylation status by the bisulfite-sequencing PCR. The result showed that the methylation level in heart was obviously higher than in the other three tissues, and the expression of bovine Gadd45a is related to DNA methylation.3. Gadd45a gene histone modification statusHistone modifications affect the structural dynamics of the nucleosome that regulates chromatin function through the recruitment of specific interacting proteins that recognize a single or conformational set of modifications. H3 acetylation was considered to activate gene, and H3K9 methylation was related to inhibit gene transcription.Chromatin immuno-precipitation (ChIP) was used to examined histone modification in bovine heart, kidney, liver and testis. The result showed that the acetylation level of H3 in heart and kidney were higher than in liver and testis. The methylation level of H3K9 in heart and kidney were higher than that of the other two tissues. These results showed that H3K9 methylation affects the Gadd45a tissue-specific expression, while H3 acetylation may play a limited role in thetissue-specific expression of Gadd45a gene.4. DNA methylation in embryos of Gadd45a geneEpigenetic modification of the genome through DNA methylation is the key to the maintenance of the differentiated state of the cell, and it must be reset during somatic cell nuclear transfer.In this study DNA methylation of Gadd45a gene were detected in vitro fertilization embryos, parthenogenenic embryos and nuclear transfer embryos. Results showed that the methylation level of nuclear transfer embryo is higher than that of the others. These results demonstrate that the DNA methylation status of Gadd45a gene is abnormal in nuclear transfer embryos.In conclusion, DNA methylation and histone modification synergistically regulate the tissue-spicific expression of Gadd45a gene. The results showed that the DNA methylation and H3K9 methylation in regulation region of Gadd45a gene are associated with gene supression. The difference of DNA methylation status among embryos from differrent sources indicated that abnormal reprogramming of a single gene occurred during nuclear transfer embyo. This abnormal may affect the embryo development.
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