The role of PIAS3 in retinal development

描述(由申请人提供)：我们的团队已经确定依赖于PIAS3的SUMO化在调节视杆和视锥感光细胞分化中发挥核心作用。然而，关于PIAS3在光感受器中的功能和蛋白质SUMO化的几个关键问题仍然没有解决。虽然蛋白质SUMO化对光感受器的活性是必不可少的，但我们之前的工作并没有确定PIAS3是否是分化的光感受器成熟、维持或存活所必需的。为了解决这个问题，我们将产生并鉴定选择性缺乏光感受器PIAS3表达的突变小鼠。其次，目前尚不清楚依赖PIAS3的SUMO化如何在与锥体特异基因的启动子结合时诱导NR2E3作为转录抑制因子，但当它与杆状特异基因的启动子结合时不会这样做。我们将测试TFIIH相关蛋白ERCC2和CDK7是否通过阻止PIAS3依赖的SUMO化NR2E3与杆特定基因的启动子结合来介导这一效应。最后，利用蛋白质芯片获得的相扑连接酶特异性的初步研究表明，PIAS3和TOPORS，另一种对光感受器功能至关重要的E3相扑连接酶，可能调节核外功能的多个光感受器特异蛋白的活性。我们发现BBS4是被PIAS3 SUMO化的，并建议研究这一过程如何调节BBS4在体内的组装和视紫红质的运输。我们还计划研究PIAS3是否也通过位点特异性的SUMO化来调节其他纤毛和中心体蛋白如CEP290的功能，以及我们的初步体外分析表明，PIAS3的活性是否受TOPORS依赖的SUMO化所控制。

DESCRIPTION (provided by applicant): Our group has determined that PIAS3-dependent SUMOylation plays a central role in regulating both rod and cone photoreceptor differentiation. However, several critical questions about the function of PIAS3 and protein SUMOylation in photoreceptors still remain unresolved. While protein SUMOylation is essential for photoreceptor viability, our previous work did not determine whether PIAS3 is required for the maturation, maintenance or survival of differentiated photoreceptors. To address this question, we will generate and characterize mutant mice that selectively lack PIAS3 expression in photoreceptors. Second, it is not known how PIAS3-dependent SUMOylation induces NR2E3 to act as a transcriptional repressor when bound to the promoters of cone-specific genes, but does not do so when it bound to the promoters of rod-specific genes. We will test whether the TFIIH-associated proteins ERCC2 and CDK7 might mediate this effect by blocking PIAS3-dependent SUMOylation of NR2E3 when it is bound to the promoters of rod-specific genes. Finally, preliminary studies of SUMO ligase specificity obtained using protein microarrays have suggested that PIAS3 and TOPORS, another E3 SUMO ligase critical for photoreceptor function, may regulate the activity of multiple photoreceptor-specific proteins that function outside the nucleus. We have found that BBS4 is SUMOylated by PIAS3, and propose to study how this process regulates BBSome assembly and rhodopsin transport in vivo. We also plan to investigate whether PIAS3 also regulates the function of other ciliary and centrosomal proteins such as CEP290 through site-specific SUMOylation, and will whether the activity of PIAS3 is controlled by TOPORS-dependent SUMOylation, as suggested by our preliminary in vitro analysis.