SET7/9甲基化转录因子GLI并调控Hedgehog信号通路的机制及功能研究

SET7/9 is one of the most studied lysine methyltransferases (KMTs). Although SET7/9 was first identified as a histone lysine methyltransferase specifically for histone methylation, accumulating evidence indicates that methylation of non-histone proteins and subsequent modulation of multiple signaling pathways are the major biological functions of this enzyme. Our previous findings indicate that the activation/inhibition of Hedgehog (Hh) signaling pathway, one of the most evolution conserved pathways, is subjected to regulation of multiple post-translation modifications. However, whether protein methylation, especial SET7/9 mediated protein methylation, is involved in this regulation remains elusive. To answer this question, we examined the Hh signaling activity by the Hh luciferase reporter assay and qPCR in NIH-3T3 cells in the condition of SET7/9 over-expression or RNAi. We found that SET7/9 can positively regulate the activity of Hh signaling. Through amino acid sequence alignment, mass spectrometry, in vitro methylation assay and GST pull-down assay, we verified that SET7/9 can specifically bind and methylate GLI, the key transcriptional factor downstream of Hh signaling. According to these findings, we hypothesized that SET7/9 can positively regulate Hh signaling activity by methylating GLI. To test our hypothesis, we will further examine the Set7/9-mediated GLI methylation and Hh signaling activation in additional cell lines, dissect the underlying mechanism of how SET7/9 mediated GLI methylation affects the activity of Hh signaling and evaluate the role of SET7/9 in tumor initiation and progression mediated by Hh signaling. This study will provide us a solid theoretical base to eventually comprehend the transduction and regulation of Hh signaling, and develop novel and effective therapeutic strategies for Hh signaling related diseases.

赖氨酸甲基转移酶SET7/9除甲基化组蛋白外，其更主要的功能是对非组蛋白进行甲基化修饰，并因此调控生物体内多种信号通路。我们实验室之前研究发现，Hh通路作为最保守的信号通路之一，其激活/抑制均受到多种蛋白翻译后修饰调控，但甲基化是否参与其中目前尚不清楚。通过SET7/9过表达和RNA干扰，结合Hh报告基因及实时荧光定量PCR，我们发现SET7/9正向调控Hh通路活性；通过氨基酸序列比对、质谱分析、体外甲基化及GST pull-down实验，我们确定SET7/9能结合并特异甲基化Hh信号通路转录因子GLI，提示SET7/9可能通过甲基化GLI调控Hh通路活性。本项目拟从SET7/9甲基化GLI入手，开展SET7/9调控Hh信号通路的分子机制研究，并进一步阐明SET7/9在Hh通路参与的肿瘤发生、发展中的生物学功能，为最终理解Hh通路信号转导机制及功能调控以及相关疾病的诊治提供坚实的理论基础。

赖氨酸甲基转移酶SET7/9除甲基化组蛋白外，其更主要的功能是对非组蛋白进行甲基化修饰，并因此调控生物体内多种信号通路。我们实验室之前研究发现，Hh通路作为最保守的信号通路之一，其激活/抑制均受到多种蛋白翻译后修饰调控，但甲基化是否参与调控Hh通路，特别是SET7/9是否调控Hh通路活性，目前尚不清楚。我们前期通过SET7/9过表达和RNA干扰，结合Hh报告基因及实时荧光定量PCR，发现SET7/9可正向调控Hh通路活性；通过氨基酸序列比对、质谱分析、体外甲基化及GST pull-down实验，我们确定SET7/9能结合并特异甲基化Hh信号通路转录因子GLI，提示SET7/9可能通过甲基化GLI调控Hh通路活性。在以上前期研究结果基础之上，本项目着重开展了SET7/9调控Hh信号通路的分子机制研究，并进一步阐明SET7/9在Hh通路参与的肿瘤发生、发展中的生物学功能。取得以下重要结果：1）通过甲基化质谱分析、氨基酸序列比对、体外甲基化实验，我们证明SET7/9甲基化修饰GLI3蛋白上的第463和595位赖氨酸；2）通过免疫共沉淀实验、定点突变实验，我们发现SET7/9只能甲基化修饰全长GLI3，却不能修饰GLI3的短片段剪切体；3)通过构建甲基转移功能失活的SET7/9突变体，结合Hh荧光报告素酶系统和qPCR检测，我们发现SET7/9与Hh信号通路的活性呈正相关，同时Hh信号通路对SET7/9的表达起正向调控作用，提示Hh信号通路与SET7/9之间可能存在正调控关系；4）通过定点突变、免疫印迹及染色体免疫共沉淀实验，我们发现SET7/9对463和595位赖氨酸上的甲基化修饰能分别促进GLI3全长的蛋白稳点性和增强GLI3全长蛋白结合DNA的能力；5）最后我们证明SET7/9能通过调控Hh信号通路，促进非小细胞肺癌的生长及转移。我们的研究首次揭示了SET7/9对Hh信号通路的调控作用，并提示SET7/9可通过调控Hh信号通路促进肿瘤发生发展。为Hh信号通路依赖型肿瘤治疗提供了新的靶点。