The Expression Pattern Of H3K27me3 Demethylase UTX And JMJD3 In Mouse Parthenogenetic Embryos

Histone methylate can be removed specifically from Histone 3 lysine 27 t ri-methylation (H3K27me3) by H3K27me3 demethylases UTX (ubiquitously tra nscribed TPR gene on the X chromosome) and JMJD3 (jumonji domain contai ning protein 3), which cause the activation of gene expression. UTX and JMJ D3 play an important role in ESC (embryonic stem cell) self-renewal, cell diff erentiation and the formation of cancers. But, so far, the roles of UTX and J MJD3 in the development of embryos are not clear.First, we designed two and three primers of UTX and JMJD3 based on mRNA sequences in GenBank database of NCBI respectively, using the strategy of sectional type PCR and selecting pMD19-T as a transition vector, then connecting the section of PCR products which sequence are correct. At last, we constructed UTX and JMJD3 expression vectors in vitro transcription pMD19T-T7UTX and pMD 19T-T7JMJD3 which under the control of T7 promoter. After in vitro transcription, the mRNA products of UTX and JMJD3 electrophoretic bands were clear and the size of mRNA products were agree with expectation. The mRNA of UTX and JMJD3 were injected to mouse MⅡ oocytes by microinjection, the immunofluorescent staining result shown that H3K27me3 were significantly decreased. So, the construction of UTX and JMJD3 in vitro transcription vectors is successful and the transcription products in vitro could be translated into active proteins at cell level in this study.Next, we tested the expression of UTX and JMJD3 in parthenogenetic activation (Parthenogenetic activation, PA) embroys by RT-qPCR (Real-time quantitative PCR, RT-qPCR). The result indicated that the expression level of UTX and JMJD3 was highest in MⅡ oocytes. With following embryo development, UTX and JMJD3 began to gradually degradation and reached the lowest level in blastocyst. In order to reveal the function of UTX and JMJD3 in PA embryos further, we knocked down UTX and JMJD3 respectively in MⅡ oocytes using siRNA (Small interfering RNA, siRNA) interference technology through micro-injection.The knock-down efficiency of UTX and JMJD3 were 90% and 70% respectively by RT-qPCR detection. And we also found, when we knocked down one of UTX and JMJD3, another expression would increase. The immunofluorescence staining result of 8-cell and morula suggested that there are no H3K27me3 fluorescence signal when we interfered UTX or JMJD3 alone, while the signal of H3K27me3 were notable when we interfered UTX and JMJD3 all. At last, we also examined the level of UTX and JMJD3 protein in PA 2-cell, the result of western blot were conform to outcome from the RT-qPCR. When we knocked down one, another would increase.The above results shown that the function of UTX and JMJD3 are complementary both in mRNA and protein level. This found is significantly to further study the mechanism of UTX and JMJD3 and in mice provides a new clue for preimplantation embryo development.