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

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。

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.