Dissecting the Function and Regulation of Stress-Induced SUMOylation

描述（由申请人提供）：在真核生物中，应激快速触发小泛素相关修饰物（SUMO）与细胞内靶蛋白的缀合，以改变其活性 并采取防御措施应激诱导的SUMO化（SIS）缺陷与许多人类疾病相关，包括由遗传毒性应激诱导的急性和慢性肝病以及癌症。此外，几种致病菌（单核细胞增生李斯特菌，肺炎链球菌）和病毒（人腺病毒，流感病毒）改变SIS，这对绕过防御和促进感染很重要。像后生动物一样，一些植物病原体劫持SUMO化途径使宿主防御无效，这表明该途径在防御生物攻击中起保守作用。因此，我们必须精确地确定SUMO化如何触发变化，以最大限度地减少应激损伤，抑制发病机制，并促进生存。特别是，本研究的一个长期目标是确定SIS反应如何介导导致应激保护的下游效应。开花植物拟南芥非常适合理解SUMO化在胁迫过程中的作用。它已经表征了影响驱动SIS的SIZ 1连接酶的突变体，这些突变体对许多胁迫高度敏感并且不能引起SIS响应;从而允许在胁迫期间解剖SUMO化。此外，植物和后生动物中SUMO靶标之间的大量重叠表明了一种保守的反应，该反应收敛于对转录调控、RNA代谢和染色质修饰重要的因子，表明SIS反应普遍影响基因表达和染色质可及性。为了鉴定与SIS反应相关的转录和染色质改变，将在热应激之前和之后进行野生型和siz 1突变体植物的比较。具体而言，下一代测序将用于跟踪SUMO 1/2沿染色质沿着结合占据率的变化（ChIP-Seq）和mRNA丰度/剪接的改变（RNA-Seq）。发生在野生型植物中的变化是不存在的siz 1突变体将定义如何SUMO化重新布线的基因组在应对压力，并在这样做，有可能确定新的基因参与胁迫耐受性。一些SUMO靶标在应激期间也被泛素特异性修饰，其动力学表明它们是降解的靶向。该建议的第二个焦点旨在使用新的蛋白质组学方法鉴定双重修饰的靶标。这些目标将有助于鉴定催化该反应的候选SUMO靶向泛素连接酶。热应力前后的功能丧失分析将确定这种双重修饰在不利条件下的重要性和功能。总的来说，这些实验的目的是提供一个全面的了解SIS响应的输出和调节。从广义上讲，这项工作将为理解生物体如何快速应对环境压力和病原体入侵提供一个机制基础。

DESCRIPTION (provided by applicant): In eukaryotes, stress rapidly triggers the conjugation of small ubiquitin-related modifier (SUMO) to intracellular target proteins to alter their activity and mount a defense response. Defects in stress-induced SUMOylation (SIS) are associated with a number of human diseases including acute and chronic liver disease and cancer induced by genotoxic stress. In addition, several pathogenic bacteria (Listeria monocytogenes, Streptococcus pneumoniae) and viruses (human adenovirus, influenza virus) alter SIS, and this is important for bypassing defenses and promoting infection. Like metazoans, several plant pathogens hijack the SUMOylation pathway to render host defense ineffectual, suggesting that this pathway serves a conserved role in defense against biotic attack. Thus, it is essential that we determine precisely how SUMOylation triggers changes that minimize stress damage, inhibit pathogenesis, and promote survival. In particular, a long-term objective of this study is to determine how the SIS response mediates downstream effects that lead to stress protection. The flowering plant Arabidopsis thaliana is well suited for understanding the role of SUMOylation during stress. It has characterized mutants affecting the SIZ1 ligase that drives SIS, which are hypersensitive to many stresses and fail to elicit the SIS response; thereby permitting the dissection of SUMOylation during stress. In addition, substantial overlap between SUMO targets in plants and metazoans suggests a conserved response that converges on factors important for transcriptional regulation, RNA metabolism, and chromatin modification, indicating that the SIS response universally affects gene expression and chromatin accessibility. To identify the transcriptional and chromatin alterations associated with the SIS response, comparisons of wild type and siz1 mutant plants will be performed before and after heat stress. Specifically, next-generation sequencing will be used to track changes in SUMO1/2 binding occupancy along chromatin (ChIP-Seq) and the alterations to mRNA abundance/splicing (RNA-Seq). Changes occurring in wild type plants that are absent in siz1 mutants will define how SUMOylation rewires the genome in response to stress and, in doing so, has the potential to identify novel genes involved in stress tolerance. Some SUMO targets are also modified with ubiquitin specifically during stress with their dynamics suggesting that they are targeted for degradation. A second focus of this proposal aims to identify dual- modified targets using a novel proteomics approach. These targets will assist in identifying candidate SUMO- targeted ubiquitin ligases that catalyze this reaction. Loss-of-function analyses before and after heat stress will establish the importance and function of this dual modification during adverse conditions. Collectively, these experiments aim to provide a comprehensive understanding of the outputs and regulation of the SIS response. Broadly, this work will provide a mechanistic basis for understanding how organisms can rapidly respond and defend themselves from environmental stress and pathogen invasion.