Abstract：Epigenetic mechanisms preside over genetic information to enable development from the fertilized, totipotent oocyte to the adult body. Methylation of histones at specific residues is one of the most important epigenetic modifications and has an essential role in both transcriptional repression and activation during embryonic development, including lineage specification and cell differentiation. The UTX (ubiquitously transcribed tetratricopeptide repeat X chromosome) and JMJD3 (jumonji domain containing 3) are identified as a histone H3 lysine 27 trimethylation (H3K27me3) demethylase, which promote muscle development, regulates metabolism and other important physiological functions. The principle of in vitro transcription system is based on DNA template by RNA polymerase in vitro synthesis of RNA. In this study, 3 pairs of primer were designed based on the mRNA sequence of JMJD3 and UTX in mouse (Musculus mus), and the in vitro transcription vectors pMD19T-T7UTX and pMD19T-T7JMJD3 were constructed, respectively. The in vitro transcription of mRNA was injected into the mouse oocytes, and the H3K27me3 was detected by immunofluorescence assay. The results showed that the in vitro transcription of UTX and JMJD3 was detected by electrophoresis, and the bands were clear and consistent with the expected size. The electrophoresis of the correct size of UTX and JMJD3 in vitro transcription products were gel recovery and purified. The use of microinjection technology taking UTX and JMJD3 gel product was injected into mouse oocytes. H3K27me3 expression was detected by laser confocal immunofluorescence technology. Result showed that the expression of H3K27me3 after injection of oocytes was significantly decreased. In this study, in vitro transcription of UTX and JMJD3 vectors were successfully constructed, respectively, using the piecewise PCR method, and proved to had very good enzyme activity at the cell level. This study successfully constructed pMD19T-T7UTX and pMD19T- T7JMJD3 vectors. At the cellular level, pMD19T-T7UTX and pMD19T- T7JMJD3 vectors could be transcribed and translated into active proteins. These vectors can be used to study the role of UTX and JMJD3 in the development and related diseases.
Agger, K., Cloos, P.A., Christensen, J., et al., 2007. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature 449, 731-734.Dalgliesh, G.L., Furge, K., Greenman, C., et al., 2010. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 463, 360-363.Gomez-Sanchez, J.A., Gomis-Coloma, C., Morenilla-Palao, C., et al., 2013. Epigenetic induction of the Ink4a/Arf locus prevents Schwann cell overproliferation during nerve regeneration and after tumorigenic challenge. Brain : a journal of neurology 136, 2262-2278.Greenfield, A., Carrel, L., Pennisi, D., et al., 1998. The UTX gene escapes X inactivation in mice and humans. Human molecular genetics 7, 737-742.Gui, Y., Guo, G., Huang, Y., et al., 2011. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nature genetics 43, 875-878.Jankowska, A.M., Makishima, H., Tiu, R.V., et al., 2011. Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A. Blood 118, 3932-3941.Lan, F., Bayliss, P.E., Rinn, J.L., et al., 2007. A histone H3 lysine 27 demethylase regulates animal posterior development. Nature 449, 689-694.Lee, M.G., Villa, R., Trojer, P., et al., 2007. Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination. Science 318, 447-450.Lee, S., Lee, J.W., Lee, S.K., 2012. UTX, a histone H3-lysine 27 demethylase, acts as a critical switch to activate the cardiac developmental program. Developmental cell 22, 25-37.Miller, S.A., Mohn, S.E., Weinmann, A.S., 2010. Jmjd3 and UTX play a demethylase-independent role in chromatin remodeling to regulate T-box family member-dependent gene expression. Molecular cell 40, 594-605.Miyake, N., Mizuno, S., Okamoto, N., et al., 2013. KDM6A point mutations cause Kabuki syndrome. Human mutation 34, 108-110.Morin, R.D., Johnson, N.A., Severson, T.M., et al., 2010. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nature genetics 42, 181-185.Pan, D., Huang, L., Zhu, L.J., et al., 2015. Jmjd3-Mediated H3K27me3 Dynamics Orchestrate Brown Fat Development and Regulate White Fat Plasticity. Developmental cell 35, 568-583.Salminen, A., Kaarniranta, K., Hiltunen, M., et al., 2014. Histone demethylase Jumonji D3 (JMJD3/KDM6B) at the nexus of epigenetic regulation of inflammation and the aging process. Journal of molecular medicine 92, 1035-1043.Seenundun, S., Rampalli, S., Liu, Q.C., et al., 2010. UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis. The EMBO journal 29, 1401-1411.Shaw, T., Martin, P., 2009. Epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair genes. EMBO reports 10, 881-886.Shpargel, K.B., Sengoku, T., Yokoyama, S., et al., 2012. UTX and UTY demonstrate histone demethylase-independent function in mouse embryonic development. PLoS genetics 8, e1002964.Staller, P., 2010. Genetic heterogeneity and chromatin modifiers in renal clear cell carcinoma. Future oncology 6, 897-900.Thieme, S., Gyarfas, T., Richter, C., et al., 2013. The histone demethylase UTX regulates stem cell migration and hematopoiesis. Blood 121, 2462-2473.van Haaften, G., Dalgliesh, G.L., Davies, H., et al., 2009. Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer. Nature genetics 41, 521-523.Wang, A.H., Zare, H., Mousavi, K., et al., 2013. The histone chaperone Spt6 coordinates histone H3K27 demethylation and myogenesis. The EMBO journal 32, 1075-1086.Wang, C., Lee, J.E., Cho, Y.W., et al., 2012. UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity. Proceedings of the National Academy of Sciences of the United States of America 109, 15324-15329.Wartman, L.D., Larson, D.E., Xiang, Z., et al., 2011. Sequencing a mouse acute promyelocytic leukemia genome reveals genetic events relevant for disease progression. The Journal of clinical investigation 121, 1445-1455.Welstead, G.G., Creyghton, M.P., Bilodeau, S., et al., 2012. X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex-specific manner. Proceedings of the National Academy of Sciences of the United States of America 109, 13004-13009.
