冷激蛋白PpCSP1通过活性氧途径调控小立碗藓重编程的机制研究

Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. The moss Physcomitrella patens, the first sequenced bryophyte plant, has been commonly used as an ideal genetic material to study reprogramming at cellular level. Previously, we identified a common factor PpCSP1 (Physcomitrella patens Cold-Shock Domain Protein 1), that share conserved domain with the iPSC (induced Pluripotent Stem Cell) factor Lin28 in mammals, could regulate reprogramming of differentiated leaf cells to chloronema apical stem cells in Physcomitrella patens. To further investigate the function of PpCSP1 in the reprogramming, transcriptome analysis indicated that genes involved in oxidative response pathway were upregulated in PpCSP1 overexpression line. This result suggests ROS (Reactive Oxygen Species) is activated in the PpCSP1-mediated reprogramming. Consistently, ROS level were altered in the PpCSP1 overexpression line and ppcsp quadruple deletion mutant upon H2O2 (one major ROS in plants) and KI (one common ROS scavenging reagent) treatments. Zeocin, the most used DNA damage reagent, could induce reprogramming without wounding, whereas PpCSP1 overexpression line and ppcsp quadruple deletion mutant exhibited distinct response in Zeocin induced reprogramming, implying that DNA damage is involved in PpCSP1 pathway. In this study, the molecular interactions of PpCSP1, ROS and DNA damage function in the reprogramming will be investigated. CLIP-seq (Cross-Linking ImmunoPrecipitation sequencing) will be performed to identify the target mRNAs of RNA binding protein PpCSP1, and the identified targets will be studied to elucidate whether they are involved in ROS response. In addition, comet assay will be employed to analyze DNA damage level in PpCSP1 overexpression line and ppcsp quadruple deletion mutant. The fulfillment of this proposal will further reveal the molecular function of PpCSP1 in modulating ROS signaling during reprogramming in Physcomitrella patens. Additionally, the knowledge obtained from this study will advance our understanding of reprogramming mechanisms in land plants.

陆生植物和后生动物均有使已分化细胞重编程为干细胞的能力，但对其重编程分子机制的研究还十分有限。小立碗藓进化地位特殊，重编程能力强，是研究细胞重编程分子机制的理想材料。我们前期工作发现了一个小立碗藓重编程的正向调控因子PpCSP1，其与哺乳动物里iPSC因子Lin28是同源蛋白。为了进一步研究PpCSP1在小立碗藓重编程的分子机理，我们分析了PpCSP1相关材料的转录谱并发现其可能参与活性氧（ROS）途径，而ROS水平的变化可能通过DNA损伤途径来调控重编程。本项目将围绕PpCSP1、ROS、DNA损伤这三者在调控重编程的关系展开，拟外源添加ROS及DNA损伤试剂Zeocin处理并观察重编程表型、ROS定量、CLIP-seq分析PpCSP1结合的mRNAs、彗星电泳检测DNA损伤程度等实验，阐明PpCSP1调控小立碗藓重编程的分子机制，为探究陆生植物重编程的分子调控机理提供范例。

本项目针对模式植物小立碗藓（Physcomitrella patens）的重编程体系来研究RNA结合蛋白PpCSP1的分子功能。小立碗藓PpCSP1蛋白与其哺乳动物同源蛋白Lin28有相似的功能，是小立碗藓中重编程的正调控因子。我们的前期发现，PpCSP1可能通过ROS途径来调控重编程。本项目的主要研究内容即通过研究PpCSP1的分子功能来阐述其如何通过调节ROS的水平来调控重编程。PpCSP1作为一个RNA结合蛋白，其最本质的分子功能即为PpCSP1到底能结合哪些mRNA，以及结合其靶mRNA后，如何调控其靶mRNA的命运。因而，本项目主要围绕PpCSP1的分子功能开展了以下主要工作：.1.PpCSP1靶mRNA的鉴定：首先，我们通过gel shift assay的实验方法，证实了PpCSP1在体外可能结合RNA。通过RIP-seq实验来研究PpCSP1的靶mRNA。初步的分析结果表明PpCSP1可以普遍结合mRNA，并没有发现PpCSP1可以富集特异的mRNA。这提示PpCSP1可能在基因组水平作为一个mRNA的缓冲剂来动态调控其靶mRNA的命运。.2.PpCSP1互做蛋白的鉴定：我们通过寻找PpCSP1的互做蛋白来找寻其分子机制的线索。我们采用了iTRAQ的方法来研究PpCSP1的互做蛋白。结果表明，poly-A binding protein（PABP）与PpCSP1可能互做。共定位结果表明PABP组成性的存在于细胞质中，并且与PpCSP1存在共定位。.3.PpCSP1在基因组水平调控翻译的初步探索：PABP的主要功能是翻译调控，提示PpCSP1在结合mRNA后可能调控其翻译。我们在小立碗藓中成功建立ribosome footprint的技术来研究PpCSP1如何参与翻译调控的分子机制。下一步，我们正在比较野生型，PpCSP1过表达，ppcsp四重突变体这三个样品之间ribosome footprint的变化，期望得到PpCSP1在基因组水平调控翻译的分子机制。.通过目前的研究结果，我们阐述了PpCSP1在转录后水平调控其靶mRNA翻译来影响ROS水平，进而调控小立碗藓重编程的可能机制。主要科学意义在于从分子机制上提示了动植物细胞在进化水平上重编程调控的统一性，广泛存在于动植物的ROS可能是重编程调控的共有机制之一。