| DNA methylation is an important epigenetic modification in eukaryotes, which can change the state of chromatin spatial conformation and thus enrich specific transcription factors to switch gene transcription, MBD (methyl-CpG-binding domain) protein family play an important role between DNA methylation and gene expression. As a member of MBD family, MBD1was reported to regulate gene expression through combination with low-density methylated CpG.In this study, firstly, the expression of MBDl gene were detected by RT-PCR with specific primers designed according to mRNA sequences of bovine MBDl gene, and the DNA methylation status of12CpG sites upstreaming MBD1gene regulatory region were detected by bisulfite-sequencing PCR in five bovine adult tissues. Secondly, the expression of the MBD1gene and the DNA methylation status of its regulatory region were detected in in vitro cultured oocytes (GV stage,8h,12h,18h,24h) and in in vitro fertilized embryos (2-cell,4-cell,8-cell, blastocysts). Finally, chromatin immunoprecipitation (ChIP) technology was utilized to examine the combination of MBD1protein with pluripotent regulatory factors, such as Nanog, Oct4, and maternal genes including Hlfoo and ZAR1in GV and MⅡ stage oocytes and in8-16cell stage embryos.The results showed that MBD1gene transcription levels were changed among bovine tissues, and gradually increased from heart, kidney, liver, oocyte to testis. The DNA methylation status of12CpG sites in MBDl gene upstream regulatory region were different in five adult tissues. The methylation ratio of heart, kidney, liver, oocyte, testis were49.17%,30%,87.5%,81.67%, and79.17%, respectively. In heart and kidney with lower transcription level of MBDl and also had lower level of DNA methylation ratio than that of liver, ovarian and testis with higher transcription level.Results from oocyte and embryos showed that gene expression of MBD1has significant change during oocyte maturation, the methylation ratio of CpG sites were exactly the same, all of which were83.3%, the-443bp and-696bp CpG sites upstreaming ATG were kept non-methylated while the other CpG sites maintained methylated status. In preimplantation embryos, expression of MBDl were specifically detectable in2-cell, and not detectable from4-cell to blastocyst stage, but the CpG site methylation status and ratio were exactly the same as oocytes.The ChIP results showed that in GV stage oocytes, MBDl protein binded with the gene regulatory region of Nanog, OCT4, and Hlfoo but not ZAR1; in MⅡ stage oocytes, MBD1protein binded with Nanog, H1foo and ZAR1but not OCT4; at8-16cell stage embryos, MBD1protein binded with H1foo but not Nanog, OCT4and ZAR1. In summary, transcriptional expression of the MBD1gene was different in bovine tissues, as well as the DNA methylation status and pattern, which implied that DNA methylation might related to the tissue specific expression of MBD1gene.MBD1gene expression was maintained at a relatively stable level during oocyte maturation, but not detected after2-cell embryos, while the methylation status of CpG sites kept the same pattern from oocyte stage to embryo stage, which suggested that MBD1accumulated during oocyte maturation and function in preimplantation embryo, and DNA methylation co-operated with other regulatory mechanisms regulate the MBD1gene expression.The dynamically binding or dissociation of MBD1with Nanog, Oct4, H1foo, ZAR1gene analyzed by ChIP assay in oocytes or early embryos indicated that MBD1selected binding with the regulatory region of related genes during embryo development, to switch the suppression or activation of gene transcription in an spatial-temporal pattern. |
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