Chromatin-modifying Co-repressor Complexes in Plants

植物中染色质修饰辅阻遏物复合物

• 批准号: 0743974
• 负责人: Vitaly Citovsky
• 金额: $ 45万
• 依托单位: SUNY at Stony Brook
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-15 至 2012-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0743974&HistoricalAwards=false
• 关键词: Chromatin; modifying; Co; repressor; Complexes

Transcriptional regulation of gene expression represents one of the central requirements for many cellular events and for the correct development of the entire organism. Traditionally, transcriptional activation has received the most attention in studies of gene regulation whereas the fundamental role of transcriptional repression has gained recognition only recently. However, while the molecular mechanisms of transcriptional gene silencing in animal systems are being intensively studied, our understanding of these processes in plants is very sparse and, because plants likely utilize unique strategies to establish and maintain chromatin state, only limited use can be made of information available on epigenetic modifications in non-plant systems. To help fill this gap, a comprehensive study of Arabidopsis histone lysine demethylase (LSD1)-like co-repressor complexes was initiated. Three major lines of data were developed: (i) Three components of an LSD1-like co-repressor complex in Arabidopsis were identified: LSD1-like AtSWP1 and AtSWP2 proteins, and a direct interactor of AtSWP1 (but not of AtSWP2), AtCZS, a C2H2 zinc finger histone methyltransferase (HMT). (ii) AtSWP1, AtSWP2, and AtCZS were shown to block expression of a reporter gene in planta. (iii) Using reverse genetics, AtSWP1, AtSWP2, and AtCZS knockout mutants were shown to produce almost identical phenotypes (phenocopies) and chromatin modifications, consistent with the idea that these gene products may function in a single co-repressor complex. These findings will be used as a foundation to seek two specific goals: I. Biochemical characterization of purified recombinant AtSWP1/2, and AtCZS. AtSWP1/2 will be examined for their predicted biochemical activities: histone demethylase, polyamine oxidase, and FAD binding. AtCZS will be analyzed for its function as a HMT. The identity of histone modifications by AtSWP1/2 and/or AtCZS, i.e., specific core histone methylation marks, will be determined. II. Identification and initial characterization of a protein interaction network for activity of the AtSWP/AtCZS co-repressor complexes. Based on our preliminary data, AtSWP1/2 and AtCZS are not the only components of the co-repressor complex. Additional members of this complex will be identified by TAP-tag purification. Protein-protein interactions between these components will be characterized in planta by bimolecular fluorescence complementation (BiFC). The working hypothesis of this research, regards the plant LSD1-like co-repressor complex as a combination of plant-specific and general functional components and architecture. Thus, this project most likely will uncover novel aspects of chromatin-modifying co-repressor complexes that are not possible to address in other model systems. The proposed research activities will be integrated into the teaching of science at junior educational levels. Specifically, the laboratory will train, in addition to postdocs and graduate students, undergraduates and high school students (most of whom are minorities) who will learn scientific thinking and modern bioexperimentation.

基因表达的转录调控是许多细胞事件和整个生物体正确发育的核心要求之一。传统上，转录激活在基因调控的研究中受到最多的关注，而转录抑制的基本作用直到最近才得到承认。然而，虽然在动物系统中的转录基因沉默的分子机制正在深入研究，我们的理解这些过程中的植物是非常稀疏，因为植物可能利用独特的策略来建立和维持染色质状态，只有有限的使用可以在非植物系统中的表观遗传修饰的信息。为了帮助填补这一空白，拟南芥组蛋白赖氨酸去甲基化酶（LSD 1）样共阻遏复合物的全面研究开始。（i）在拟南芥中鉴定了LSD 1样共阻遏物复合物的三种组分：LSD 1样AtSWP 1和AtSWP 2蛋白，以及AtSWP 1（但不是AtSWP 2）的直接相互作用物AtCZS，C2 H2锌指组蛋白甲基转移酶（HMT）。(ii)AtSWP 1、AtSWP 2和AtCZS显示阻断植物中报告基因的表达。(iii)使用反向遗传学，AtSWP 1，AtSWP 2和AtCZS敲除突变体显示出产生几乎相同的表型（表型）和染色质修饰，这与这些基因产物可能在单个辅阻遏物复合物中起作用的想法一致。这些发现将被用作寻求两个具体目标的基础：I。纯化的重组AtSWP 1/2和AtCZS的生物化学表征。将检查AtSWP 1/2的预测生化活性：组蛋白脱甲基酶、多胺氧化酶和FAD结合。将分析AtCZS作为HMT的功能。AtSWP 1/2和/或AtCZS对组蛋白修饰的鉴别，即，将确定特定的核心组蛋白甲基化标记。二. AtSWP/AtCZS共阻遏物复合物活性的蛋白质相互作用网络的鉴定和初步表征。基于我们的初步数据，AtSWP 1/2和AtCZS不是辅阻遏物复合物的唯一组分。该复合物的其他成员将通过TAP-标签纯化来鉴定。这些组分之间的蛋白质-蛋白质相互作用将在植物中通过双分子荧光互补（BiFC）来表征。本研究的工作假设，认为植物LSD 1样辅阻遏物复合物是植物特异性和一般功能成分和结构的组合。因此，该项目很可能会发现染色质修饰的辅阻遏物复合物的新方面，这是不可能解决在其他模型系统。拟议的研究活动将纳入初中科学教学。具体来说，除了博士后和研究生外，该实验室还将培养本科生和高中生（其中大多数是少数民族），他们将学习科学思维和现代生物实验。