Tet家族在小鼠成纤维细胞诱导为肝干细胞的谱系重编程过程中的作用及相关机制研究

The "lineage reprogramming" is referred to that differentiated somatic cells could be directly converted into other somatic cells or progenitors with defined transcription factors. Both the breaking of the epigenetic molecular barriers of the somatic cell and the establishment of the epigenetic molecular landscape of the stem cells are the essential steps for lineage reprogramming and iPSCs formation. DNA methylation pattern, as one of the molecular barriers of reprogramming, has a profound impact on genome stability, transcription and development; it is even regarded as the identity of somatic cells and stem cells. In most recent years, ten-eleven translocation (TET) family enzymes, including Tet1, Tet2 and Tet3, are evidenced as critical enzymes in regulating the DNA methylation pattern, which can oxidize 5-methylcytosine to 5-hydroxymethylcytosine, an intermediate nexus in demethylation. Many recent reports showed that Tet family enzymes play a vital role in ES maintenance and iPSCs formation. However, the function and the underlying mechanism of Tet family involving in lineage reprogramming have remained unclear. We succeeded in reprogramming MEF to induced Hepatic Stem Cells (iHepSCs) with Hnf1b and Foxa3 in 2013, as a successful model in the field of lineage reprogramming，which gives us a unique advantage to explore the basis of Tet family in lineage reprogramming. Recently we found that the expression of Tet1, Tet2 and Tet3 were activated in iHepSCs and kept high expression in vitro culture. Our recent results exhibited that knockdown of Tet1 ,Tet2 and Tet3 at the same time, resulted in a morphological abnormality of iHepSCs and a dramatic decrease of the hepatic stem cell-specific gene Dlk and Sox9. In addition, we discovered that the three Tet-shRNAs could interfere with the formation of iHepSCs clone when they were co-transfected into MEF with Hnf1b and Foxa3, which suggests the function of Tet members in lineage programming. Basing on these above and other observations, and our experience in transcriptomics and CpG methylation profiling, we design to reveal the function and mechanism of Tet family in iHepSCs maintenance and iHepSCs formation by interfering with the expression of Tet family in this proposal. The ultimate destination is to optimize the reprogramming strategy with Tet family enzymes and finally improve the efficiency of reprogramming and quality of iHepSCs.

"谱系重编程"是指成熟体细胞可以通过外源转录因子的导入直接重编程为其它类型的体细胞或干（祖）细胞。体细胞基因组DNA甲基化修饰是谱系重编程首先需要突破的表观遗传分子屏障之一。研究表明Tet家族能将5mC氧化成5hmC来实现DNA的去甲基化从而在ES细胞干性维持和iPSCs形成过程中发挥重要作用，但Tet家族在谱系重编程过程中的作用尚未见报道。我们实验室2013年在Cell Stem Cell上发表了将MEF重编程为肝干细胞iHepSCs的研究成果，为深入研究Tet家族的作用提供了理想平台。本课题将在前期Tet相关实验结果的基础上，通过干扰Tet的表达，结合微量细胞转录组分析技术，研究Tet家族在外源基因诱导MEF重编程为iHepSCs过程中的作用、iHepSCs干性维持中的作用以及相关机制，以完善对Tet家族在调控细胞基因组表观特性方面的功能的认识，并提出提高谱系重编程效率和质量的新思路

Tet家族能将5mC氧化成5hmC来实现DNA的去甲基化从而在ES细胞干性维持和iPSCs形成过程中发挥重要作用，但Tet家族在谱系重编程过程中的作用尚未见报道。我们实验室2013年在Cell Stem Cell上发表了将MEF重编程为肝干细胞iHepSCs的研究成果，为深入研究Tet家族的作用提供了理想平台。本课题将在前期Tet相关实验结果的基础上，通过干扰Tet的表达，结合微量细胞转录组分析技术，研究Tet家族在外源基因诱导MEF重编程为iHepSCs过程中的作用、iHepSCs干性维持中的作用以及相关机制。.本课题计划主要研究内容包括① Tets在诱导性肝脏干细胞干性维持中的作用及相关机制；② Tets在谱系重编程过程中的作用及相关机制；③ 如何利用Tets来提高重编程效率。.项目围绕研究内容展开，取得了如下研究结果.1. 对Tet1与iHepSCs干性维持的关系进行了较深入的研究，发现Tet1表达的抑制会造成自我更新能力的改变；证明Tet1与CTCF结合区域结合并造成CpG的去甲基化或羟甲基化是CTCF结合的前提，即Tet1通过与CTCF结合序列或临近区域结合并改变CTCF结合序列的CpG甲基化状态从而改变了CTCF与结合序列的结合，最终造成c-Myc基因表达的改变.2.对Tet1敲低和敲除情况下，iHepSCs细胞的生物学特性的变化进行了研究，同时利用全基因组水平的转录组和甲基化组测序筛选出了受Tet1调控的候选基因，并对其在iHepCS细胞自我更新中的作用进行了验证。发现Wnt通路相关基因的表达与其相关调控区段和机制。.3.进行了Tet1与iHepSCs重编程相关性的研究，并对重编程过程的相关分子机制做了探讨，发现Tet1的激活是发生在重编程的早期事件，是重编程发生的必要条件。.4. 构建了永生化小鼠胚胎成纤维细胞系SV40-MEF，并对其生物学特性进行了检测，证明通过感染双因子可以将其转分化为iHepSCs样的上皮细胞，并对其生物学特性做了鉴定。本细胞系的构建为转分化机制的研究提供了适合的起始细胞系，大大提高了工作效率.本课题利用肝脏谱系重编程的技术体系，研究TET蛋白家族在谱系重编程过程中的作用及相关机制，完善对于Tet基因生物学功能的认识，并为进一步优化肝脏谱系重编程体系开辟新的思路。