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

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，以提高它们的农业潜力。例如，这项研究可能确定一种可以被抑制的因子，使叶组织开始产生根蛋白，从而形成根。通过对植物进行改造以增强它们对食物、纤维和燃料的效用，解开固定组织身份施加的限制可能会产生重大的实际影响。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高度组织的蛋白质和核酸的动态复合体中，基因中产生了什么蛋白质。因子在调节组织特异性基因的产生方面发挥着关键作用，这些基因决定了特定组织的发育特性，如叶和根。基因如何在不同的组织中表达是植物特别感兴趣的问题，因为在这个王国里，发育过程中基因表达的变化是非常可塑性的。尽管已经确定了一些控制组织特异性基因的因素，但人们对使基因从一种或另一种状态过渡的调控机制和机制知之甚少。该蛋白质将研究其中一个因素，以破译它控制植物基因调控的机制。这项拟议的研究也与国家科学基金会的教育使命保持一致。首席调查员(PI)大力支持本科生的研究，并有相当多的记录让本科生参与研究项目，为他们提供变革性的科学经验。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，这将为他们提供与涉及基因组学、生物化学和遗传学的研究相关的坚实的科学基础。PI在教授本科生和研究生方面有丰富的经验，并坚信基于实验室目前的努力，课堂上的激励话题。通过与学生在拟议的研究中合作所获得的经验将为PI继续改变学生作为生物化学系首席顾问的教育经验提供基础。染色质的基本组织亚单位是核小体，它由包裹在组蛋白八聚体周围的DNA组成。控制染色质结构的因子，称为染色质重构体，在调节组织特异性基因的表达方面发挥关键作用，这些基因决定了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，染色质重塑泡菜(PKL)，被认为对组织特异性基因的调节都有贡献，因为它是基于染色质的开关的一个组成部分，使这些基因既能进行负调控，也能进行正调控。因此，破译PKL在这些基因座上的作用机制将首次阐明植物的机械如何能够流动地决定基因的表达和发育特性。拟议中的实验将阐明一种基于染色质的基本途径，该途径限制植物的发育同一性，从而可能识别新的靶点，通过这些靶标可以操纵顽固物种的发育同一性。这项建议将研究染色质重塑蛋白PKL在确定拟南芥发育特性方面的作用，拟南芥是一种广泛用于实验室研究的植物。PKL改变了核小体的结构，对于特定发育特性的基因的正确表达是必要的。这些组织特异性基因中的许多都受抑制性表观遗传标记H3K27me3(组蛋白H3的赖氨酸27的三甲基化？组蛋白的特定修饰)，用于防止组织特定基因在不适当的组织中表达(例如，使叶组织中的根基因远离)。PKL对H3K27me3调控基因的抑制和激活都是必需的，表明它既是基因表达的抑制者，也是基因表达的激活者。这项提议将解决一种假设，即PKL是基于染色质的开关的一个组成部分，该开关能够实现对发育基因的负向和正向调节。这项提案将表征PKL如何对染色质结构和受H3K27me3调控的基因的表达做出贡献。具体地说，该项目将分析整个基因组的染色质，以区分不同的PKL作用模式。它将通过确定与PKL相互作用的核小体的修饰状态来研究PKL活性是如何调节的，并检验由PKL重塑的核小体是促进H3K27me3修饰的复合体的更好底物的假设。基因和生化筛选将被用来识别新的因素和途径，使PKL依赖的过程成为可能。

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.