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.

基因表达的转录调节代表了许多细胞事件以及整个生物体的正确发育的中心要求之一。传统上，转录激活在基因调节的研究中受到了最大的关注，而转录抑制的基本作用直到最近才获得认可。然而，尽管对动物系统中转录基因沉默的分子机制进行了深入的研究，但我们对植物中这些过程的理解非常稀疏，而且因为植物可能利用独特的策略来建立和维持染色质状态，但只能在非植物系统中提供有关表观修饰的信息的有限使用。为了帮助填补这一空白，启动了类似类似的拟南芥组蛋白赖氨酸脱甲基酶（LSD1）的共抑制配合物的全面研究。 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和ATCZ被证明可以阻止Planta中报告基因的表达。 （iii）使用反向遗传学，ATSWP1，ATSWP2和ATCZS基因敲除突变体可产生几乎相同的表型（苯尾部）和染色质修饰，这与这些基因产物可能在单个共抑制器复合物中起作用的想法一致。这些发现将用作寻求两个具体目标的基础：I。纯化重组ATSWP1/2和ATCZ的生化特征。将检查ATSWP1/2的预测生化活性：组蛋白去甲基酶，多胺氧化酶和FAD结合。 ATCZ将分析其作为HMT的功能。将确定通过ATSWP1/2和/或ATCZ（即特定核心组蛋白甲基化标记）对组蛋白修饰的身份。 ii。蛋白质相互作用网络的识别和初始表征，用于ATSWP/ATCZS共抑制剂复合物的活性。基于我们的初步数据，ATSWP1/2和ATCZ并不是共抑制器复合物的唯一组成部分。该复合物的其他成员将通过TAP-TAG纯化确定。这些成分之间的蛋白质 - 蛋白质相互作用将通过双分子荧光互补（BIFC）在植物中表征。这项研究的工作假设将植物LSD1样共抑制剂复合物视为植物特异性和一般功能组件和建筑的组合。因此，这个项目很可能会发现染色质修改的共抑制络合物的新颖方面，这些复合物在其他模型系统中无法解决。拟议的研究活动将纳入初级教育水平的科学教学。具体来说，除了博士后和研究生，本科生和高中生（其中大多数是少数族裔）外，实验室还将培训他们将学习科学思维和现代生物的经验。