Epigenetic Control of DNA Replication in Arabidopsis

智力价值：DNA的忠实复制是所有生物体遗传的基础。DNA复制是一个受到严格控制的过程，但人们对它的理解仍不完全透彻。例如，人们对DNA复制和细胞中包装DNA的组蛋白的化学修饰等过程之间的关系知之甚少。许多不同的组蛋白修饰是已知的，但它们的功能尚不清楚。该项目的长期目标是了解模式植物拟南芥（Arabidopsis thaliana）基因组中DNA复制与组蛋白修饰之间的关系。这将通过详细探索最近观察到的DNA复制与特定组蛋白修饰之间的联系来实现，该修饰的功能是阻止拟南芥基因组某些区域的基因表达。两个名为ATXR5和ATXR6的蛋白（ATXR5/6）冗余作用维持拟南芥基因组中位于重复元件的基因表达的抑制。最近发现，在atxr5/6突变体中，基因组的这种沉默区域在每个细胞周期内以一种称为“再复制”的异常DNA复制形式复制不止一次。使用新一代测序方法绘制了基因组的重复复制区域，并发现与以组蛋白修饰为特征的与基因表达抑制相关的区域密切对应。本项目旨在利用拟南芥的遗传和基因组资源，研究组蛋白修饰和DNA复制的基本生物学过程之间的相互作用。新的基因组测序技术将用于探索DNA复制和修复途径与局部染色质环境相互作用的方式。此外，多种生化和遗传学方法将被用于扩大对ATXR5和ATXR6途径机制的理解。更广泛的影响：该项目将产生大量的基因组数据集，这为本科生参与最先进的基因组技术和数据分析提供了一个独特的机会。最初，三名本科生将参与这项研究。每个人都将接受项目所需的标准分子生物学技术的广泛培训。此外，这些学生将接受数据分析技术的培训，从高通量测序读数的初始处理和映射到下游更高水平的分析。这将有助于培养具有分子生物学和计算生物学界面技能的新一代学生。目标是将这种跨学科培训扩大到更多的加州大学洛杉矶分校本科生，作为新的生物医学研究辅修项目的一部分，该项目由分子细胞和发育生物学的首席研究员部门和MIMG部门牵头。将开发一门课程，通过新的跨部门教学实验课程来教授，在这门课程中，更多的学生可以使用来自该项目的真实世界数据集来学习计算生物学技术，并将其应用于假设检验。了解组蛋白修饰与DNA复制之间的关系不仅对植物研究，而且对其他真核生物的DNA复制研究具有广泛的影响。此外，该项目的数据将通过公共浏览器和网站广泛传播，并将广泛应用于对发育，应激反应，基因表达和植物基因组学感兴趣的植物生物学家。

Intellectual merit: The faithful replication of DNA is the basis of inheritance in all organisms. DNA replication is a carefully regulated process, but it is still incompletely understood. For example, little is known about the relationship between DNA replication, and processes such as the chemical modification of histone proteins that package the DNA in the cell. Many different modifications of histones are known, but their function is unclear. The long-term objective of this project is to understand the relationship between DNA replication and such histone modifications in the genome of the model plant, Arabidopsis thaliana. This will be approached through detailed exploration of a recently observed link between DNA replication and a particular histone modification whose function is to prevent gene expression within certain regions of the Arabidopsis thaliana genome. Two proteins named ATXR5 and ATXR6 (ATXR5/6) act redundantly to maintain repression of gene expression of genes located within repetitive elements in the Arabidopsis genome. It has been recently discovered that in atxr5/6 mutants, such silenced regions of the genome replicate more than once per cell cycle in a form of aberrant DNA replication termed "re-replication". Re-replicating regions of the genome have been mapped using next-generation sequencing approaches, and found to closely correspond to regions that are characterized by histone modifications associated with repression of gene expression. This research project aims to use the genetic and genomic resources for Arabidopsis to investigate the interplay between the fundamental biological processes of histone modifications and DNA replication. Novel genome sequencing techniques will be used to explore the ways in which DNA replication and repair pathways interact with local chromatin environments. Furthermore, multiple biochemical and genetics approaches will be used to expand the understanding of ATXR5 and ATXR6 pathway mechanisms. Broader impacts: This project will generate a significant number of genomic datasets, which provides a unique opportunity to involve undergraduate students in state-of-the-art genomic techniques and data analysis. Initially, three undergraduates will be involved in the research. Each will be extensively trained in standard molecular biology techniques required for the project. In addition, these students will be trained in data analysis techniques, from the initial processing and mapping of high throughput sequencing reads to downstream higher-level analysis. This will facilitate the training of a new class of students with skills that lie at the interface of molecular biology and computational biology. A goal will be to expand such cross disciplinary training to a larger pool of UCLA undergraduate students as part of the new Biomedical Research minor program which is being spearheaded by the Principal Investigator's department of Molecular Cell and Developmental Biology, together with the MIMG department. A course will be developed that will be taught through the new interdepartmental instructional laboratory curriculum, in which larger groups of students can use real world data sets from this project to learn computational biology techniques and apply them to hypothesis testing. An understanding of the relationship between histone modifications and DNA replication will have a broad impact not only on plant research, but also on DNA replication research in other eukaryotes. In addition, the data from this project will be widely disseminated through public browsers and web sites, and will be of wide utility to a broad set of plant biologists interested in development, stress responses, gene expression, and plant genomics.