Understanding the contribution of a chromatin remodeler to regulation of tissue-specific gene expression in Arabidopsis plants

了解染色质重塑剂对拟南芥植物组织特异性基因表达调节的贡献

• 批准号: 1413183
• 负责人: Joseph Ogas
• 金额: $ 75万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1413183&HistoricalAwards=false
• 关键词: Understanding; contribution; chromatin; remodeler; regulation

The long-term goal of this research is to enable scientists to manipulate the identity of specific tissues in plants to increase their agricultural potential. For example, this research may identify a factor that can be inhibited to enable leaf tissue to begin producing root proteins, and thereby form roots. Unshackling the constraints imposed by a fixed tissue identity could have a substantial practical impact by enabling modification of plants to enhance their utility for food, fiber, and fuel. All organisms contain the blueprint of how tissues are formed and what proteins are produced in genes in a highly organized dynamic complex of proteins and nucleic acids referred to as chromatin. Factors play a critical role in regulating production of tissue-specific genes that determine the developmental identity of specific tissues such as leaf and root. How genes are expressed in different tissues is of particular interest in plants because changes in gene expression during development are extremely malleable in this kingdom. Although some of the factors have been identified that control tissue-specific genes, very little is known about the regulatory mechanisms and machinery that enable genes to transition from one state or the other. The protein will study one of those factors to decipher the mechanism by which it can control gene regulation in plants. The proposed research is also aligned with the education mission of NSF. The Principal Investigator (PI) strongly supports undergraduate research and has considerable record of engaging undergraduates in research projects that provide them with a transformative scientific experience. These types of research experiences often play a critical role in prompting students to pursue a Science, Technology, Engineering and Math (STEM) career. In addition, graduate students will undertake the bulk of the proposed research, which will provide them with a solid foundation in science associated with research involving genomics, biochemistry, and genetics. The PI has an extensive experience in teaching undergraduate and graduate students and strongly believes in motivating topics in the classroom based on current efforts in the lab. Lessons learned by working with students on the proposed research will provide the bases by which the PI will continue to transform the educational experience of students as head advisor in the Department of Biochemistry.The fundamental organizational subunit of chromatin is the nucleosome, which consists of DNA wrapped around an octamer of histone proteins. Factors that control chromatin structure, referred to as chromatin remodelers, play a critical role in regulating expression of tissue-specific genes that determine the developmental identity of specific tissues such as leaf and root. The protein that will be studied in this work, the chromatin remodeler PICKLE (PKL), is thought to contribute to both regulation of tissue-specific genes by serving as a component of a chromatin-based switch that enables both negative and positive regulation of these genes. Thus deciphering the mechanism of PKL action at these loci will illuminate for the first time how the machinery of plants enables fluid determination of gene expression and developmental identity. The proposed experiments will elucidate a fundamental chromatin-based pathway that restricts developmental identity in plants and thus, are likely to identify novel targets by which developmental identity can be manipulated in recalcitrant species. This proposal will examine the role of the chromatin remodeling protein PKL in determination of developmental identity in Arabidopsis thaliana, a plant that is widely used for laboratory research. PKL alters the structure of nucleosomes and is necessary for proper expression of genes that specify developmental identity. Many of these tissue-specific genes are regulated by the repressive epigenetic mark H3K27me3 (trimethylation of lysine 27 of histone H3 ? a specific modification of a histone protein), which is used to prevent expression of tissue-specific genes in inappropriate tissues (for example to keep root genes off in leaf tissue). PKL is necessary for both repression and activation of H3K27me3-regulated genes, indicating that it acts as both a repressor and an activator of gene expression. This proposal will address the hypothesis that PKL is a component of a chromatin-based switch that enables both negative and positive regulation of developmental genes. This proposal will characterize how PKL contributes to chromatin structure and expression from genes that are regulated by H3K27me3. Specifically, the project will analyze the chromatin of the entire genome to distinguish between different models of PKL action. It will examine how PKL activity is regulated by identifying the modification state of nucleosomes that interact with PKL as well as test the hypothesis that nucleosomes remodeled by PKL are a better substrate for the complex that promotes the H3K27me3 modification. Genetic and biochemical screens will be used to identify new factors and pathways that enable PKL-dependent processes.

这项研究的长期目标是使科学家能够操纵植物中特定组织的身份，以增加其农业潜力。例如，这项研究可能会确定一个因子，可以抑制，使叶组织开始生产根蛋白，从而形成根。 解开固定组织身份所施加的限制可能会产生实质性的实际影响，使植物的改造，以提高其对食物，纤维和燃料的效用。 所有生物体都包含组织如何形成的蓝图，以及在被称为染色质的蛋白质和核酸的高度组织化的动态复合体中基因中产生的蛋白质。因子在调节组织特异性基因的产生中起着关键作用，这些基因决定了特定组织如叶和根的发育特性。 基因如何在不同组织中表达在植物中特别有趣，因为在发育过程中基因表达的变化在这个王国中具有极强的可塑性。虽然已经确定了一些控制组织特异性基因的因素，但对使基因从一种状态或另一种状态转变的调节机制和机制知之甚少。这种蛋白质将研究其中一种因子，以破译它控制植物基因调控的机制。 拟议的研究也符合NSF的教育使命。首席研究员（PI）大力支持本科生的研究，并有相当多的记录从事本科生的研究项目，为他们提供一个变革性的科学经验。这些类型的研究经验通常在促使学生追求科学，技术，工程和数学（STEM）职业方面发挥着关键作用。此外，研究生将承担大部分拟议的研究，这将为他们提供与基因组学，生物化学和遗传学研究相关的科学基础。PI在教授本科生和研究生方面拥有丰富的经验，并坚信根据实验室目前的努力，在课堂上激发主题。通过与学生在拟议的研究工作中吸取的教训将提供基础，PI将继续改变学生作为生物化学系首席顾问的教育经验。染色质的基本组织亚基是核小体，它由DNA包裹在组蛋白八聚体周围组成。控制染色质结构的因子，称为染色质重塑因子，在调节组织特异性基因的表达中起关键作用，所述基因决定特定组织如叶和根的发育特性。 将在这项工作中研究的蛋白质，染色质重塑PICKLE（PKL），被认为有助于作为一个组件的染色质为基础的开关，使这些基因的负和正调控的组织特异性基因的调节。因此，破译PKL在这些位点的作用机制将首次阐明植物的机制如何使基因表达和发育身份的流体决定。拟议的实验将阐明一个基本的染色质为基础的途径，限制在植物中的发育身份，因此，有可能确定新的目标，发育身份可以操纵在植物物种。这项建议将检查的作用，染色质重塑蛋白PKL在确定发育的身份，在拟南芥，植物，被广泛用于实验室研究。PKL改变核小体的结构，并且对于指定发育身份的基因的适当表达是必需的。这些组织特异性基因中的许多基因受抑制性表观遗传标记H3K27me3（组蛋白H3赖氨酸27的三甲基化？组蛋白的特定修饰），其用于防止组织特异性基因在不适当的组织中表达（例如，防止根基因在叶组织中表达）。PKL对于H3K27me3调节的基因的阻遏和激活都是必需的，这表明它既充当基因表达的阻遏物又充当基因表达的激活物。这项建议将解决的假设，PKL是一个基于染色质的开关，使负和正调控的发育基因的组成部分。该提案将描述PKL如何有助于染色质结构和H3K27me3调控的基因表达。具体来说，该项目将分析整个基因组的染色质，以区分PKL作用的不同模型。它将研究如何通过识别与PKL相互作用的核小体的修饰状态来调节PKL活性，并测试由PKL重塑的核小体是促进H3K27me3修饰的复合物的更好底物的假设。遗传和生物化学筛选将用于确定新的因子和途径，使PKL依赖的过程。