多能性相关lncRNA Osclr8调控细胞重编程的机制研究

The terminally differentiated-somatic cells can be reprogrammed into “induced pluripotent stem” (iPS) cells. The technology has been honored with the 2012 Nobel Prize for its breakthrough discovery in stem cell research. Nonetheless, this reprogramming process is extremely inefficient and time consuming, thus significantly impeding its highly anticipated clinical application. Recent studies suggest that long noncoding RNAs (lncRNAs) may play a key role in determining the fate of stem cells. However, whether lncRNAs are able to overcome epigenetic bottleneck to promote pluripotency remains poorly defined..In studying the mechanism underlying cell reprogramming, we have recently identified Osclr8, a novel lncRNA that interacts with the promoters of multiple pluripotency-associated genes. Knockdown of Osclr8 causes loss of intrachromosomal looping and induces the exit of iPS cells from pluripotency. In the present study, we propose to utilize several technologies established in our lab to explore the role and mechanisms of this lncRNA in cell reprogramming, including “Cas9-induced chromosomal looping”, “RNA in situ reverse transcription-associated trap sequencing” (RAT-Seq), “chromosome conformation capture” (3C), “RNA-guided chromosome conformation capture” (R3C), and “chromosomal loop screening” approaches. We will examine how this lncRNA controls pluripotency by orchestrating intrachromosomal looping and altering epigenetic modifications. Finally, we will test if Osclr8 is able to promote cell reprograming. ..The overall objective of this study is aimed to revolutionize the existing approaches to robustly induce iPS reprograming for regenerative medicine.

细胞重编程机理不清、效率低、耗时，是诱导多能干细胞（iPS）至今未能用于临床治疗的最大路障。近期研究提示lncRNA在决定干细胞命运中起到关键作用,但在重编程中lncRNA如何与多能基因互作，如何调控干细胞多能性，能否促进iPS细胞诱导，这些研究尚属空白。 .本课题组发现一条命名为Osclr8的全新lncRNA，在重编程中呈差异表达并能结合到多能基因启动子上，参与染色质内环结构形成；若将其敲除，干细胞将走向分化。提示该lncRNA与干细胞命运决定关系密切。.本研究拟采用人工诱导DNA成环、RNA逆转录结合捕获测序、染色质构象捕获、体内重编程等技术，研究Osclr8 如何通过招募转录因子、参与多元高级转录复合结构形成、促进细胞重编程，并最终通过操纵以lncRNA为媒介的转录复合结构，达到提高干细胞诱导效率的目的。本研究将为诱导细胞重编程开辟新方向，对促进iPS产品临床应用具有重要意义。

本项目在前期研究中，发现了一条命名为Osclr8（现改称为Platr10）的lncRNA，具有调控干细胞多能性和促进细胞重编程的作用。基于课题组前期已建立的研究方法，拟采用人工诱导DNA成环、RNA逆转录结合捕获测序、染色质构象捕获、体内重编程等技术，研究Osclr8对招募转录因子、参与多元高级转录复合结构形成、促进细胞重编程中的作用机制，以达到提高干细胞诱导效率的目的。.在项目开展中，我们应用CRIST-qPCR方法发现了Osclr8与Sox2和Oct4启动子的结合，应用RNA逆转录相关捕获测序（RAT-seq）进一步明确了与Osclr8相结合的靶基因，以及与Osclr8结合的Oct4的靶区定位。我们发现Osclr8可通过基因覆盖的方式结合于Oct4和Sox2的启动子上，通过调控染色质空间构象，拉近Oct4启动子和增强子的空间距离。而将Osclr8用shRNA敲低后使染色质内环破坏，细胞多能性退出。同时，Osclr8通过招募DNA去甲基化酶Tet1促进Oct4启动子DNA去甲基化，而增强Oct4的转录，促进细胞多能性维持。在细胞重编程过程中过表达Osclr8，可促进重编程克隆的产生。说明Osclr8具有促进细胞重编程效率的潜在应用价值。.除Osclr8外，项目组还围绕lncRNA对干细胞多能性的调控开展了拓展性研究，发现了多个对多能性具有关键调控作用的lncRNA分子，并且各具不同的调控方式和调控靶点，包括：1）lncRNA Oslr9可与Oct4和Sox2启动子，参与调控Oct4基因启动子和增强子多个环状结构形成而促进多能基因Oct4的表达；2）lncRNA Oplr16可募集SMC1以维持Oct4的染色质内环结构，并通过招募去甲基化酶Tet2而促进Oct4启动子去甲基化；3）lncRNA Oeblr20可促进Oct4增强子eRNA的转录而促进细胞多能性维持。.本项目发表论文9篇，培养博士生6人。