Chromatin Charting: Organization and Dynamics of Plant Nuclear DNA in situ

Rutgers University New Brunswick, New Brunswick NJ

Investigators

Abstract

The nucleus is the subcellular organelle in which the bulk of the genomic information within an eukaryotic cell is organized. From studies using hybridization technologies and microscopy work with serial or optical sections of fixed cells, a picture of an organized subnuclear structure has emerged. More recently, the application of the Green Fluorescent Protein (GFP) as an in vivo tag of genomic DNA has allowed the visualization of chromatin in live cells of animals and fungi. Based on studies using 3-D fluorescence microscopy, the chromosomes within an interphase nuclei are perceived to have an ordered arrangement that is relatively static except for slow motions that can be attributed to Brownian movement. However, it is also clear that during other phenomena that are known to occur within a nucleus, such as transvection and recombination, that relatively large and long-range movement of chromatin must be possible. The goal in this project is to contribute to the general understanding of subnuclear architecture by charting the relative physical position and movement of sequences for each of the chromosomes in cells of living plants. To achieve the objective of visualizing and charting the sequences for all the chromosomes of Arabidopsis, GFP and two different color variants of this protein will be deployed as in vivo tags for about 1,000 dispersed sites within the 5 chromosomes of Arabidopsis. Comparative analyses of the relative positions between defined regions of the genome in space and time will provide novel information about the organization principles that control the structure and dynamics of chromatin. Concurrent with optical studies to track the relative subnuclear location and movement for distinct regions of the genome, the effects of genome location on transcription potential of a reporter gene will be quanitified. Together, these studies should provide the first comprehensive 3-D physical and transcription activity maps for a genome and should contribute significantly to understanding the roles that subnuclear location may play in controlling gene expression. This study should generate more than 1,000 mapped insertion lines of Arabidopsis with 3 distinct and optically tractable GFP-tags at defined locations within the genome. These materials should be invaluable for the characterization of chromatin-related mutations that affect gene expression and development. The number of such mutations are likely to rapidly increase due to the efforts of several genome projects that have been funded by the NSF in the past two years. Molecular tools generated from this project will also be applied to an important crop plant such as maize. The fusion of cutting edge imaging technology with the wealth of classical and modern cytogenetics in maize should provide new perspectives on global control of genetic information as well as epigenetic phenomena such as paramutation. These new insights will facilitate understanding of how genomic information is organized in plants and how gene expression can be regulated at a global scale. As such, the tools and knowledge generated by this proposed work should benefit future efforts to improve the quality and yield of crop plants. Deliverables: 1. About 5,000 lac-operator-tagged (beacon) Arabidopsis thaliana lines. 2. 3-D coordinate maps of 1,000 selected beacon insertions with maximal dispersion across the whole genome. 3. A global gene expression map that shows the 3-D coordinates and the luciferase marker expression levels of the beacons. These materials and information will be available at http://aesop.rutgers.edu/~lamlab/ccharting.html