Mechanistic Analysis of How a Plant DNA Methylase Acts on Chromatin

该项目由分子和细胞生物科学部的遗传机制组和生物科学局综合组织系统部的植物基因组研究计划共同资助。细胞的特性是通过一组基因的选择性表达来确定和维持的。该项目将有助于揭示新的分子机制，揭示遗传基因组的大片区域如何关闭或未被阅读，从而在植物等有机体中形成不同类型的细胞。要做到这一点，必须创建和操纵称为异染色质的结构。在异染色质内部是称为核小体的结构，核小体是包裹着DNA的蛋白质包，有助于组织DNA链。玉米(玉米)中的一种重要蛋白质的机制将被研究，它可能会降低DNA对负责读取DNA的蛋白质相互作用的可用性。这个程序将试图找到两个问题的答案：(1)这种蛋白质如何在化学上改变位于核小体中的DNA，以及(2)这种蛋白质如何改变核小体堆积，从而创造出可以关闭或沉默某些基因的结构。将以多种生物物理方法以及遗传方法相结合的形式对参与的学生进行多学科培训。通过与首席调查员实验室和旧金山州立大学硕士项目的合作，以及女研究生的带头参与，积极参与代表不足的少数族裔，将鼓励这些学生在团队环境中将生物调控概念应用于实际问题。这项研究的结果也将有助于更好地理解基于异染色质的沉默机制是如何干扰具有抗虫害和增强生长和营养含量的作物的形成的。在植物和哺乳动物中，异染色质是由两个翻译后修饰，组蛋白H3赖氨酸9甲基化(H3K9me)和DNA胞嘧啶甲基化以及识别这些标记的特定蛋白质因子定义的。在这两种情况下，异染色质的两个特性被认为是其功能所必需的：(I)从起始点扩散到邻近基因组区域的能力，这使得能够在大范围的基因组中发挥作用；(Ii)能够浓缩潜在的染色质，这被认为是抑制DNA访问并使染色质难以转录。然而，DNA和组蛋白甲基化如何使抑制染色质结构成为可能的生化基础还知之甚少。来自拟南芥的主要胞嘧啶DNA甲基转移酶CMT3及其玉米同源物ZMET2为解决这一根本问题提供了机会。CMT3和ZMET2有两个识别H3K9甲基标记的辅助结构域，这种识别是体内DNA甲基化所必需的。该项目将检验以下假设：(I)ZMET2桥接H3K9me核小体并压缩潜在的染色质；(Ii)ZMET2的活性受确定的染色质结构控制。该项目将机械酶学与多角度光散射、FRET和尖端电子冷冻显微镜等方法相结合。从ZMET2工作中得出的模型将在体内以拟南芥中相关的CMT3酶为背景进行测试。这里的机械解剖有望：(I)阐明DNA甲基转移酶如何与组蛋白甲基化合作产生异染色质的生物物理基础；(Ii)揭示异染色质形成中新的可调控步骤。

This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences and the Plant Genome Research Program in the Division of Integrative Organismal Systems in the Directorate for Biological Sciences.Cell identity is determined and maintained by selective expression of a group of genes. This project will help uncover new molecular mechanisms for how large regions of the inherited genome are turned off or left unread, forming different types of cells in organisms like plants. To do this, structures called heterochromatin must be created and manipulated. Inside heterochromatin are structures called nucleosomes, which are packages of DNA wrapped proteins that help organize the DNA strands. The mechanism of an important protein in maize (corn), which may reduce the availability of DNA to protein interactions responsible for reading DNA will be investigated. This program will attempt to find answers to two questions: (1) how this protein chemically changes the DNA located in nucleosomes, and (2) how this protein alters nucleosome packing to create structures that can turn off or silence certain genes. Multi-disciplinary training will be given to the participating students in the form of combining a variety of biophysical approaches, as well as genetic approaches. Active participation of under-represented minorities through a partnership with the principle investigator's laboratory and the San Francisco State University's Masters Program, and leading participation by women graduate students in this program will encourage these students to apply bio-regulatory concepts to real problems in a team setting. The results from this research will also build towards a better understanding of how heterochromatin based silencing mechanisms interfere in the formation of crops with pest-resistance and enhanced growth and nutritional content.In plants and mammals heterochromatin is defined by the presence of two post-translational modifications, histone H3 lysine 9 methylation (H3K9me) and DNA cytosine methylation as well as specific protein factors that recognize these marks. In both contexts two properties of heterochromatin are considered to be essential for its function: (i) the ability to spread to adjacent genomic regions from the sites of initiation, which enables action across large stretches of the genome and (ii) the ability to condense the underlying chromatin, which is thought to inhibit DNA access and make the chromatin refractory to transcription. However the biochemical basis for how DNA and histone methylation enable repressive chromatin structures is poorly understood. The major cytosine DNA methyltransferase, CMT3 from Arabidopsis thaliana and its maize homolog ZMET2, provide an opportunity to tackle this fundamental question. CMT3 and ZMET2 have two accessory domains that recognize the H3K9 methyl mark and this recognition is essential for DNA methylation in vivo. This project will test the following hypotheses: (i) ZMET2 bridges H3K9me nucleosomes and compacts the underlying chromatin and (ii) ZMET2 activity is controlled by defined chromatin architectures. The project combines mechanistic enzymology with methods such as multi-angle light scattering, FRET and cutting edge electron cryo-microscopy. The models derived from the work on ZMET2 will be tested in vivo in the context of the related CMT3 enzyme in Arabidopsis thaliana. The mechanistic dissection here is expected to: (i) elucidate the biophysical basis for how DNA methyltransferases collaborate with histone methylation to generate heterochromatin and (ii) uncover new regulatable steps in heterochromatin formation.