磷酸化修饰热休克转录因子4对晶状体发育的调控

Hsf4 has been found to be a key regulator of lens development at the postnatal age. Defective Hsf4 causes congenital cataracts in both human and animals. Our preliminary data demonstrate that Hsf4 transcriptional activity is regulated by post-translational phosphorylation during lens development. FGF2-MAP kinase signal pathways participate in the regulation of Hsf4 protein stability and transcription activity (Hu, MCB 2006,26:3282;Hu,et.al Int J Biochem Cell Biol 2012,45(2):317). The results of alanine-mutation scanning demonstrated that the phosphorylation of Hsf4/T222 and S299shows different regulatory effects on Hsf4 transcription activity. Hsf4 transcription activity can be significantly downregulated by Hsf4／T222A mutant but activated by the mutation of Hsf4/S299A. Therefore, we hypothesize accordingly that the dynamic activation and inactivation of Hsf4, which are regulated by differential phosphorylation of T222 and S299 by the different lens developmental signal pathways, is important in modulating lens development.In this grant application, we will study:1)constructing the transgenic zeberfish model(Zeberfish/Hsf4/T222A, T222D, Hsf4/S299A and Hsf4/S299D) with CRISPR-Cas 9 to study the roles of phosphorylation of Hsf4/T222 and S299 on zeberfish lens deveopment; 2)investigating the protein kinases and their associated upreseam signal pathways that regulate the phosphorylation of Hsf4/T222 and S299 by usingour hsf4-/- mouse epithelial cells.The aim of this grant application is to determine how the phosphorylation signals control Hsf4’s transcription activity and their potential roles in lens development. The results will be significant for early diagnosis of Hsf4-related cataract.

Hsf4功能障碍能诱发先天遗传性白内障，但调控Hsf4转录活性信号通路不清楚。我们发现Hsf4转录活性受磷酸化调控，突变T222A能抑制Hsf4的转录活性，而突变S299A则作用相反。因此推测不同信号通路通过对T222和S299的动态磷酸化来调控Hsf4的转录活性，进而调控晶状体发育。该课题利用斑马鱼模式生物模型，结合hsf4-/-晶状体细胞系和分子生物学技术探讨：①磷酸化T222和S299位点的上游激酶和信号通路（主要在MAPkinases,AKT1,Aurora, Cdc2,ATM和CDK5)；②应用CRISPR-Cas-9 DNA重组技术,构建携带Hsf4/T222A/D和Hsf4/S299A/D基因的斑马鱼模式生物模型，研究它们对晶状体发育调控，以及诱发白内障的分子机制。③磷酸化修饰Hsf4转录活性与老年白内障发生的相关性。该课题为Hsf4功能障碍诱发白内障的早期诊断提供理论基础。

热休克转录因子4（HSF4）是热休克转录因子家族成员之一，通过特异结合基因启动子中的HSE，调控下游基因（如热休克蛋白）的转录。HSF4的转录作用对晶状体发育至关重要，HSF4基因突变患者或敲除HSF4基因的小鼠和斑马鱼都发生先天性白内障。但是调控HSF4的转录活性还不完全清楚。我们前期研究发现基因突变S299A可上调HSF4对HSP25基因的转录作用，但是，该位点在晶状体组织内是否受磷酸化修饰调控还需研究。利用磷酸化-S299多肽，我们生产了特异识别phospho-S299的多克隆抗体。应用该抗体，我们首次在体内证明，HSF4/S299位点被磷酸化修饰调控。EGF-MEK1-ERK1/2 通路参与磷酸化修饰S299，并抑制HSF4控制的alpha B-crystallin 的表达。在晶状体发育的早期，HSF4转录活性最强，同时， HSF4/S299位点的磷酸化水平也增加。由此推论，S299位点磷酸化负责下调HSF4的转录活性，使HSF4不能过度的激活。另外，我们应用酵母双杂交技术，发现HSF4与DEAD/H-box Helicase UAP56结合，并受UAP56 调控，这一研究揭示了一个转录调控新机制，转录因子可以将RNA-polymerase II控制的mRNA 转录与转录后修饰联系在一起。而且，参与mRAN 剪辑的UAP56 还能调控转录因子的转录调控作用。.上述研究结果对理解HSF4在晶状体组织内的转录调控机制提供了新的理论依据。