Klf4在神经干细胞染色质高级结构变化和细胞分化过程的作用

The cell fate control of neural stem cells (NSCs) during brain development is tightly regulated. Misregulation of NSC differentiation leads to the developmental neurological diseases. Epigentic regulation is critical for NSC differentiation. While much is known about DNA methylation or histone modification, very little is known on high-order chromatin structure during this process. Mutations that cause aberrant chromatin structure leads to ageing and developmental disorders. We have discovered that Klf4 protein plays an important role in regulating chromatin structure in somatic cell reprogramming. In addition, our preliminary data suggest that Klf4 gene deletion in NSCs leads to differentiation of NSC in vitro. Therefore, we hypothesize that Klf4 may play crucial roles in higher-order chromatin structure in NSCs and thus in regulating NSC differentiation. To test the hypothesis, we will perform the following experiments: analyze the conditional Klf4 knockout mice to determine the roles of Klf4 in the cortical neuronal differentiation; perform RNA-seq on wide-type and Klf4-knockout NSCs to identify the genes regulated by Klf4; perform the CHIA-PET，CLIP-seq and CHIP-seq to investigate the Klf4-mediated chromatin interaction, RNA interaction and the Klf4-binding site in genome; Correlate these data with RNA-seq data respectively to determine the role of Klf4-mediated higher-order chromatin structure in the neural differentiation and the Klf4-regulated genes. This project will not only highlights the higher-order chromatin structure in NSC, but also provides novel mechanisms for neural differentiation.

神经干细胞（NSC）分化异常是神经系统疾病的主要原因。染色质高级结构在维持干细胞“干”性和体细胞重编程过程中起关键作用。Klf4介导Oct4增强子与基因组中多个位点相互作用，调节重编程过程。前期结果表明Klf4敲除促进NSC分化和相关基因表达。因此，我们推测Klf4在长非编码RNA辅助下，介导抑制子与启动子相互作用，形成独特染色质高级结构，调节基因表达和NSC分化。为证明假说，我们检测Klf4小鼠脑中NSC分化；利用RNA测序，寻找Klf4调控的基因；利用CHIA-PET、CLIP-seq和CHIP-seq等检测Klf4介导的DNA相互作用以及Klf4与RNA和与DNA的结合位点；把这些信息分别与Klf4调控基因信息进行关联，确定Klf4介导的染色质高级结构对NSC分化和基因表达的调节作用。本研究从染色质高级结构这一全新视角揭示NSC中基因转录调控机制和NSC分化机制。

神经干细胞的增殖分化与神经系统发育和神经系统疾病密切相关。染色质高级结构及相关的转录调控元件在干细胞的增殖与分化中发挥重要作用。本项目通过研究发现染色质高级结构相关蛋白Klf4通过与RNA结合蛋白Staufen1以及RNA解旋酶Ddx5/17相互作用维持神经祖细胞的自我更新，进而调控神经发生过程，同时Klf4通过结合长非编码RNA5430416N02Rik并维持其基因座与Mid1基因之间的相互作用，调控Mid1基因的表达来影响干细胞增殖。另外我们也发现Smek1/2与Ryk-ICD共同调控靶基因表达并参与神经干细胞分化过程。本研究发现包括Klf4在内的多个在神经干细胞多能性维持及分化过程中重要的调控元件和基因并阐明了神经干细胞、胚胎干细胞干性维持和分化的新机制。