解析Nr5a2-L3mbtl2蛋白相互作用及其在多能性干细胞及癌细胞中的调控功能

The grand breaking study by Dr Yamanaka has identified four transcription factors (Oct4, Sox2 Klf4 and c-Myc) that can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Our previous study further identified a novel reprogramming factor Nr5a2, which can replace Oct4 to induce iPSCs from mouse fibroblasts. Nr5a2 is highly expressed in early embryos as well as in mouse embryonic stem cells (ESCs), but depletion of Nr5a2 neither abolish pluripotency in vivo nor in vitro. Studies have also revealed extensive regulatory connections among Nr5a2 and other pluripotency factors, which show that Nr5a2 plays an important role in pluripotency regulation, but cannot fully explain its key role in promoting iPSC induction. On the other hand, Nr5a2 is also expressed in adult tissues, and its abnormal high expression is associated with several types of cancer. This may reveal a connection or similarity between molecular regulatory networks in pluripotent stem cells and cancer; meanwhile, it may provide a new angle to understand Nr5a2 function in promoting reprogramming. .The activity of Nr5a2 is modulated by co-repressors through protein-protein interaction. Hence, to fully understand the biological function of Nr5a2 and its regulations in the pluripotency or cancer network, it is crucial to identify proteins that interact with Nr5a2 and to deconvolute the protein-protein interactions within multi-protein complexes. Through a campaign to search for Nr5a2 interacting proteins, we identified L3mbtl2 as a new Nr5a2 interactor. L3mbtl2 is a transcriptional repressor, and it is known to be involved in regulating mouse ESC proliferation, and associated with cancer occurence. Our finding has uncovered a novel connection between these two factors; therefore, further studies on the interaction between Nr5a2 and L3mbtl2, as well as their functions and molecular mechanisms in pluripotent stem cells and cancer, are necessary and promising..In this project, we will perform a thorough investigation into the interaction nature as well as the assemblies of protein complexes containing Nr5a2 and L3mbtl2. The obtained data will then be combined with the whole transcriptome analysis, integrative bioinformatics analysis, and systematic reporter assays to illustrate the functional roles of Nr5a2-L3mbtl2 protein complexes in the pluripotency network. In these works, Nr5a2-/- mouse ESCs generated with the cutting-edge CRISPR/Cas9 technology will be employed to compare with normal wild type mouse ESCs; meanwhile, by implementing iPSC generation technology, we will examine the functional roles of L3mbtl2 in modulating the Nr5a2-based reprograming process. Furthermore, we will employ previous reported cancer cell lines, to investigate the functional role, as well as interaction and cooperation between Nr5a2-L3mbtl2 in human cancers. .These research objectives will be tackled with multidisciplinary approaches combining proteomics, bioinformatics, biochemistry, molecular and stem cell technologies. The results obtained will generate a wealth of information on the physiological properties of Nr5a2 and L3mbtl2, as well as on the molecular networks in which they are involved to regulate the unique pluripotency property and cancer cell survival.

大量工作显示多能干细胞与癌症细胞在分子调控方面存在着很多相似性。我们在以往研究中发现核受体蛋白Nr5a2可以取代Oct4诱导小鼠体细胞重编程，但其关键调控机制并不清楚。最近，我们发现Nr5a2与另一多能性相关蛋白L3mbtl2相互作用、并在重编程过程中呈现相关性。 值得关注的是，在成体组织中，Nr5a2和L3mbtl的异常高表达都被发现与多种癌症的发生密切相关，但二者之间相互作用尚无报道。因此，本项目中，我们拟采用蛋白组学、生物信息学、分子和细胞生物学等多种实验方法在干细胞及癌症细胞进行综合分析，全面地解析Nr5a2和L3mbtl2相互的分子基础，探讨其相互作用模式、及对下游靶基因的调控功能和机理，研究揭示二者在干细胞多能性分子调控、重编程、及癌症发生过程中的生物学功能及相互影响。这将为理解其在干细胞多能性调控中的功能提供新的视角，并可能揭示干细胞与癌症发生的相关性、及癌症发生的分子基础。

我们在以往研究中发现核受体蛋白Nr5a2可以取代Oct4诱导小鼠体细胞重编程，但其关键调控机制并不清楚。最近，我们发现Nr5a2与另一多能性相关蛋白L3mbtl2相互作用、并在重编程过程中呈现相关性。在成体组织中，Nr5a2和L3mbtl的异常高表达都被发现与多种癌症的发生密切相关，因此，本项目中，我们拟采用蛋白组学、生物信息学、分子和细胞生物学等多种实验方法在干细胞及癌症细胞进行综合分析Nr5a2和L3mbtl2相互的分子基础，研究揭示二者在干细胞多能性分子调控、重编程、及癌症发生过程中的生物学功能。.Nr5a2(nuclear receptor subfamily 5, group A, member 2)基因编码一个核受体蛋白，能够促进小鼠成纤维细胞的重编程，并可以取代 Oct4 诱导iPSCs [1];也被发现能够促进外胚层干细胞（EpiSCs）向胚胎干细胞（ESCs）转变 [2]。 据此，我们建立了dox-Nr5a2介导的小鼠多能干细胞从primed态向naïve态转变的高效诱导系统。此外，在之前的iPSC工作基础上，课题组采用诱导型多順反子表达技术建立了携带可由Doxycycline诱导表达的转录因子（Nr5a2、Sox2、Klf4、cMyc）的小鼠iPSC细胞株，建立了携带Nr5a2的稳定的iPSC 二次诱导系统。.在Nr5a2 与 L3mbtl2相互结合的探讨方面，我们进一步研究探讨L3mbtl2-Nr5a2蛋白之间的相互作用机制，课题组构建了多个Nr5a2与L3mbtl2缺陷质粒，通过免疫共沉淀技术对二者可能相互结合的位点进行了多方面的分析。另外， 为了解析L3mbtl2-Nr5a2对小鼠 ESCs 多能性的调控， 我们通过CRISPR技术建立了Nr5a2敲除细胞，并进行了系统分析。　.与此同时，为进一步推进功能性研究，课题组探索了由CRISPR/Cas9介导的非同源DNA敲入技术在多拷贝基因敲除中的应用。 之前的研究成果表明，由CRISPR/Cas9诱导的、 经非同源末端连接路径(non-homology end joining, NHEJ pathway)介导的DNA修复，可以用于大片段DNA（或基因）在基因组中的定点敲入 [3]。 该方法的敲入效率远远高于传统的、由同源依赖性DNA修复路径( homology-dependent repair