PGC7及其互作蛋白在Dlk1-Dio3印记区的印记维持与保护机制研究

Placental dysplasia caused by imprinting disorders is the main cause of failures of somatic cell nuclear transfer (SCNT). PGC7 is a critical maternal factor for zygotic imprinting maintenance and protection. Our previous work showed that PGC7 was an intrinsically disordered protein, and we have established the protein interaction networks of PGC7 which contains 291 proteins. Here we propose to investigate the molecular mechanism of PGC7-mediated imprinting maintenance through analyzing the PGC7 interacting protein network. In this study, we use embryonic stem cells as the model cell, treatment of cells with Vc or transfected with TET3 as the stress condition of DNA demethylation, and Dlk1-Dio3 imprint region as the target site of imprinting regulation. By studying the interaction regulations between PGC7 and UHRF1, DMAP1 or HP1BP3, we want to reveal the molecular mechanism of PGC7 and its interaction proteins in recruiting DNMT1 or regulating chromatin structure at Dlk1-Dio3 imprint region. At the same time, this project is intended to reveal the factors affecting the maintenance of imprinting in cloned embryos by observing the differences of PGC7 interactions between bovine IVF embryos and cloned embryos, and to provide new ideas and methods for improving the efficiency of SCNT.

印记紊乱引起的胎盘发育异常是体细胞核移植技术失败的主要原因。PGC7是合子期维持和保护印记的关键母源因子。我们发现PGC7是一个天然无序蛋白，并建立了包含291个蛋白的PGC7互作蛋白网络。本项目拟以胚胎干细胞为实验材料，以维生素C处理或转染TET3作为DNA去甲基化的胁迫模型，以Dlk1-Dio3印记区作为印记调控靶标位点，从PGC7的互作蛋白网络入手，研究PGC7在印记维持和保护方面的功能机制。通过研究PGC7与UHRF1、DMAP1及HP1BP3之间的互作调控关系，揭示PGC7及其互作蛋白在Dlk1-Dio3印记区招募维持DNA甲基化酶DNMT1和调节染色质结构等方面的分子机制，阐明PGC7印记维持和保护功能的分子作用机理。同时本项目拟通过观察牛IVF胚胎与克隆胚胎中PGC7及其互作蛋白的互作调控差异，揭示克隆胚胎中影响印记维持或保护的因素，为提高体细胞克隆效率提供新的思路和方法。

我们前期发现PGC7是一个天然非折叠蛋白。这类蛋白由于缺乏有序的三级结构，通常具有两个功能特点：一是主要通过与其它蛋白的相互作用而发挥功能；二是与多种蛋白互作而具有多种功能。这也是本项目研究思路的基础。通过研究发现：PGC7与多种蛋白互作，参与调控细胞内多种生物学过程。目前普遍认为PGC7主要参与调控DNA甲基化（包括印记区的甲基化），在本项目的资助下，我们揭示了PGC7与UHRF1和HP1BP3互作对Dlk1-Dio3印记区DNA甲基化的调控机制。除了这些跟踪研究之外，我们还发现PGC7与Prdm14及YY1互作，参与调控印记区和染色质的H3K27的甲基化和乙酰化，进一步揭示了PGC7在DNA甲基化和组蛋白修饰之间的协同调控关系及其在哺乳动物早期胚胎发育中的调控作用。.此外，我们还发现PGC7可以影响ERK或AKT激酶活性参与细胞内信号转导过程，进而调控DNA甲基化和卵母细胞蛋白质翻译激活。总之，在本项目的资助下，我们对母源效应因子PGC7的认识发生了从追踪前人到原创发现的变化。