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.
