Chromatin-modifying Co-repressor Complexes in Plants

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中心要求之一。传统上，转录激活在基因调控研究中受到最多的关注，而转录抑制的基础作用直到最近才得到承认。然而，虽然动物系统中转录基因沉默的分子机制正在被深入研究，但我们对植物中的这些过程的了解非常少，而且由于植物可能利用独特的策略来建立和维持染色质状态，因此在非植物系统中关于表观遗传修饰的信息只能被有限地利用。为了帮助填补这一空白，一项全面的拟南芥组蛋白赖氨酸去甲基酶(LSD1)类共抑制物复合体的研究被启动。(1)在拟南芥中鉴定了LSD1类共抑制蛋白复合体的三个组分：类LSD1的AtSWP1和AtSWP2蛋白，以及与AtSWP1(而不是AtSWP2)直接相互作用的AtCZS，即C2H2锌指蛋白甲基转移酶(HMT)。(Ii)AtSWP1、AtSWP2和AtCZS可阻断报告基因在植物中的表达。(Iii)利用反向遗传学，AtSWP1、AtSWP2和AtCZS基因敲除突变体被证明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共抑制物复合体中发挥作用的想法一致。这些发现将作为寻求两个特定目标的基础：一、纯化重组AtSWP1/2和AtCZS的生化特性。AtSWP1/2将被检测其预测的生化活性：组蛋白去甲基酶、多胺氧化酶和FAD结合。将分析AtCZS作为HMT的功能。将确定AtSWP1/2和/或AtCZS对组蛋白修饰的同一性，即特定的核心组蛋白甲基化标记。II.AtSWP/AtCZS共抑制物活性的蛋白质相互作用网络的鉴定和初步表征。根据我们的初步数据，AtSWP1/2和AtCZS并不是辅助抑制物复合体的唯一成分。这个复合体的更多成员将通过TAP-Tag纯化来鉴定。这些组分之间的蛋白质相互作用将通过双分子荧光互补(BIFC)来表征。本研究的工作假说认为，植物LSD1样共抑制物复合体是植物特有的和一般的功能成分和结构的组合。因此，这个项目很可能会发现染色质修饰的共抑制物复合体的新方面，这些方面在其他模型系统中是不可能解决的。拟议的研究活动将纳入初中阶段的科学教学。具体地说，该实验室将培养博士后和研究生、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。

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.