Chromatin organization in Arabidopsis root epidermal development

In general, all the different types of cells that comprise a complex organism like a plant or an animal have the same genes. An important, but little understood, question is how a single set of genes can be used in different ways to produce these different types of cells. One answer that is now emerging is that the genes are modified in semi-permanent ways - epigenetically - so that some can be switched on and others cannot. Thus different types of cells have the same genes, but different epigenetic states, and use different subsets of genes. Important questions are how these epigenetic states are set up and to what extent and under what circumstances they can be changed. An understanding of this will have far-reaching consequences for biology and medicine. In animals, it is relatively difficult to change epigenetic states, which is probably one reason why many animal cell types, such as most nerve cells, cannot be regenerated. On the other hand, plant cells are much more flexible in their development, which is why, for example, plants can often be regenerated from pieces of tissue (cuttings) or even single cells. We think one reason for this is likely to lie in the way plant genomes, as embodied in chromatin (the complex of the DNA with many different specific proteins), are modified epigenetically. We have begun a detailed study of one gene that is responsible for causing specific cells in the root of a plant to become root hair cells. We have been able to show by advanced microscopy that the region of the DNA containing this gene is in a different physical state in cells that will become root hair cells from cells that will not. Furthermore, we have shown by looking at naturally occurring aberrant cells that this difference in state is reversible and is potentially reset every time the cells divide. In this proposal we want to study the detailed biochemical basis of the physical difference in this gene. This should ultimately allow us to find out how and why the genes are in different states in different cells, and how this state is set and reset. We expect that this will help to explain how plant cells can change their developmental fates more easily than animal cells.

One of the major current questions in biology is how a single genome can be used in different ways to produce the different cell types of a multicellular eukaryotic organism. A current paradigm is that during development, specific genomic regions are modified in semi-permanent ways to express different sets of genes. Thus different cell types have different chromatin states. It is important to understand how these different states are set up and how they can be changed. We have recently shown by fluorescence in situ hybridization that there are different chromatin states in the region of a key regulatory gene (GL2) in trichoblast and atrichoblast root epidermal cell types in Arabidopsis. Furthermore, we have shown that this chromatin state can be changed within a single cell cycle. This proposal is to understand the biochemical differences at the GL2 locus that are responsible for these different chromatin states, using a multidisciplinary imaging, genetic, and biochemical approach. First, we will apply our in situ assay of chromatin at GL2 to survey a number of available mutants lines in order to determine what known factors and processes might be involved. Second, we will purify nuclei from trichoblasts and atrichoblasts by fluorescence activated cell sorting, and determine the differences at the GL2 locus by chromatin immunoprecipitation. Finally, we will tag two factors known to be involved in the regulation and which are thus candidate components of a complex binding to the GL2 locus and effecting the chromatin changes, and purify complexes containing them for proteomic analysis.