转录因子SOX2与长链非编码RNA在基因组调控中的相互作用机制研究

SOX2 is a member of SOX (SRY-related high-mobility-group box) transcription factor family. It is a vital player in diverse processes, including early embryonic development, neural differentiation, and carcinogenesis. It was recently found that long noncoding RNAs (lncRNAs) play key roles in genome regulation and stem cell fate determination in a SOX2-dependent way. However, the cooperation mechanism between them are still unrevealed. Our data obtained through electrophoretic mobility shift assays (EMSA) show that SOX2 directly binds to RNA via its DNA-binding domain- HMG box, leading to influences on its DNA binding property. In this research, we will first test whether RNAs are bound by SOX2 in a sequence-specific or structure-specific manner through EMSA and fluorescence resonance energy transfer (FRET); next, in combine with crystallization trail and isothermal titration calorimetry (ITC), we will construct the model of interaction between SOX2 and RNA; then, SOX2-interacted lncRNAs will be ascertained in human umbilical vein endothelial cells (HUVECs) and their effects on DNA binding by SOX2 will be identified through EMSA and FRET. This work will contribute to understand the relations between other SOX transcription factors and lncRNAs and mechanism of cell fate determination, and support a new direction for related diseases treatments.

SOX2是SOX转录因子家族的成员，它在早期胚胎发育、神经分化以及癌症发生等过程中起到关键作用。最近研究发现长链非编码RNA以SOX2依赖的方式调控基因组进而决定干细胞命运，但它们在该过程中的相互作用机制至今仍不清楚。本课题组通过凝胶迁移实验发现SOX2可通过其DNA结合结构域－HMG结构域直接结合RNA进而影响其对DNA的结合。本项目拟利用凝胶迁移实验等技术检测SOX2的RNA结合特性；结合蛋白结晶和等温滴定量热法等技术构建SOX2的RNA结合模型；从人脐静脉内皮细胞中鉴定与SOX2结合的长链非编码RNA并通过荧光能量共振转移实验等手段检测它们对SOX2结合DNA序列的影响，从而阐明SOX2与长链非编码RNA在基因组调控中的相互作用机制。本研究将有助于理解其他SOX转录因子和长链非编码RNA的关系，深化认识细胞命运的决定机制，为相关疾病的治疗提供新的方向。

在人类细胞中约8.1%的DNA结合蛋白也可以结合RNA，其中相当比例的为转录因子。Sox2是SOX转录因子家族的成员，它在早期胚胎发育、神经分化以及癌症发生等过程中起到关键作用。最近研究发现长链非编码RNA以Sox2依赖的方式调控基因组进而决定干细胞命运，但它们在该过程中的相互作用机制至今仍不清楚。本项研究重点研究了Sox2与RNA和DNA的相互作用机制以及对细胞重编程过程的影响。我们体外表达了Sox2蛋白，并通过紫外交联免疫共沉淀（CLIP）技术指数富集的配体系统进化（SELEX）技术从体内和体外两个角度验证了Sox2的RNA结合活性，同时发现其偏好结合富含G/C的RNA底物；我们构建了一系列的Sox2缺失突变体蛋白，利用凝胶迁移实验找到了一个长为60个氨基酸的RNA结合基序（RBM）；设计了RNA fishing实验，并且证明了Sox2可以通过HMG结构域和RBM同时结合DNA和RNA；另外我们还检测到Sox2不仅可以和RNA及DNA结合，还与剪切复合体存在相互作用；随后的转录组测序（RNA seq）和RNA免疫共沉淀（RIP）的实验证明Sox2可以通过RNA结合活性影响其调控转录的与细胞多能性有关的基因转录产物的可变剪切，并进而影响细胞的重编程过程。本研究首次发现了具有DNA/RNA双结合活性的Yamanaka因子，找到了转录因子调控细胞发育的新的方式，深化认识细胞命运的决定机制，为基于可诱导多功能干细胞的细胞治疗提供新的方向。