AKT激酶对猪植入前胚胎DNA甲基化印记调控机制的研究

Imprinted genes play quite important role in the development of embryos. During the stage of embryo cleavage, genomic imprints could survive the extensive reprogramming and maintain DNA methylation at imprinted loci, during which DNMT1 play an important role. It has been confirmed that in cell lines, AKT could stabilizes DNMT1 by phosphorylation. However, in the porcine preimplantation embryos, whether AKT maintain DNA methylation imprints through stabilizing DNMT1 need further confirmation. To answer this question, we use CRISPR/Cas9 genome editing, Laser scanning confocal microscope and Sulfite sequencing technology to study the expression level and intracellular localization of AKT and DNMT1 as well as methylation dynamic change in the DMRs region, making sure if there is an interaction between AKT and DNMT1, interpreting the mechanism how does this interaction regulate DNA imprints. Not only does this project provide new directions of the role in which AKT may play in the preimplantation embryos, but also it broaden horizons in the study of the mechanism how imprinted genes are regulated in the stage of embryo cleavage.

印记基因对维持胚胎的正常发育非常关键。卵裂期基因印记通过抵御基因组范围内的重编程而得以精确保留，其中甲基转移酶DNMT1在维持DNA甲基化印记过程中扮演着重要的角色。研究证实，AKT激酶对DNMT1的磷酸化修饰能使其在细胞中处于稳定状态。然而，在植入前胚胎中AKT激酶是否通过稳定DNMT1蛋白而维持DNA甲基化印记尚有待于进一步研究。本项目拟通过CRISPR/Cas9基因修饰、激光共聚焦、亚硫酸盐测序等技术，研究AKT激酶与DNMT1在猪植入前胚胎的表达水平、胞内定位及相关印记基因差异甲基化区域(DMRs)甲基化动态变化，从而明确猪植入前胚胎中AKT激酶与DNMT1之间的相互作用，并解析二者在维持DNA甲基化印记调控机制中的作用。该研究不仅为探索AKT激酶在植入前胚胎中的功能提供新的研究思路，同时也为揭示卵裂期印记基因的调控机制提供理论依据。