This award supports the generation of mutations in genes that control gene expression at the level of chromatin. The overall goal of the project is to identify and functionally analyze most, if not all, of the several hundred genes in maize (corn) and Arabidopsis that contribute to chromatin-level control of gene expression. Maize is the most important agricultural crop in the US, as well as the premier model system for addressing fundamental questions in monocots, particularly cereals. Chromatin is the proteinaceous material that together with DNA comprises chromosomes. A key requirement for the expression of genes in chromosomes is that chromatin be remodeled (i.e., "opened") in such a way that transcriptional activator proteins and RNA polymerases can have access to the DNA, permitting the assembly of a transcription complex which then transcribes the gene into messenger RNA. The approach exploits conserved chromatin genes identified in the human, yeast, worm, and fly genome projects to: (1) identify similar genes in the complete Arabidopsis genome sequence and (2) isolate related genes from maize. Identification of genes in Arabidopsis greatly facilitates the isolation of genes from maize. Certain tests of chromatin gene function require dominant mutations, so dominant negative mutations will be made for each target chromatin gene. Most importantly, all mutations will be characterized to determine their effects on genetic transmission, plant growth and development, and a comprehensive battery of biochemical and epigenetic tests. These tests include histone acetylation, DNA methylation, the processes of epimutation and paramutation, reactivation of silenced transgenes and transposons, the efficiency of Agrobacterium T-DNA integration, and nucleolar dominance. Also, fusions of chromatin gene products to the GAL4 DNA binding domain will be tested for effects on a reporter transgene possessing a GAL4 DNA binding site to determine the ability of candidate genes to reverse or promote the formation of repressive chromatin. These lines will be valuable for isolation of additional mutations that suppress activity of chromatin genes. This "forward" genetic approach will be important to identify the many interesting regulatory components in chromatin that are not highly conserved or are plant specific. This award will result in the generation and classification of a large set of useful mutations that will facilitate investigations of gene regulation in plants, leading to deeper understanding of the complex mechanisms by which plants control the expression of their genes. Equally important, a chromatin database and web site will be created that will facilitate communication among scientists and dissemination of information on chromatin level control in plants and other organisms. Understanding how plants control gene expression is essential for understanding how plants grow and develop and how they respond and adapt to the environment. Quality and yield improvements in crops continue to depend on applying new genetic insights and tools like those to be gained from this program. The research here will have a direct impact on genetic improvement of maize, and will also be applicable to many other important crop plants.
Deliverables:
T-DNA insertion mutations of Arabidopsis genes encoding chromatin proteins: Arabidopsis Biological Resource Center
RNAi mutants of Arabidopsis genes encoding chromatin proteins: Arabidopsis Biological Resource Center
RNAi mutants of maize genes encoding chromatin proteins: Maize Genetic Stock Center RT-PCR and cDNA sequences corresponding to transcripts from chromatin genes: Genbank Curated information regarding Arabidopsis, maize and rice genes encoding chromatin proteins: www.chromdb.org
