Intellectual Merit: The long-term objective of this project is to understand how the CHD3 class of ATP-dependent chromatin remodeling factors contributes to regulation of gene expression and developmental identity. In animal model systems, CHD3 proteins have been shown to act through a multisubunit complex (Mi-2/NuRD) in combination with histone deacetylase to repress transcription of genes that play a critical role in differentiation and development. In the plant model system Arabidopsis thaliana, the CHD3 gene, PICKLE, also represses expression of genes and plays an important role in developmental transitions. However, there are two key differences between PICKLE function and CHD3 function in animals. First, PICKLE does not appear to function in the plant equivalent of Mi-2/NuRD. Second, PICKLE appears to operate through formation of the repressive chromatin mark, trimethylation of histone H3 lysine 27 (H3K27me3). This histone modification is known to play a critical role in gene repression in both animals and plants, but is not directly regulated by CHD3 proteins in animals. To understand how PICKLE regulates gene expression, this project will take advantage of powerful genetic, biochemical, and developmental tools to explore how CHD3 proteins contribute to chromatin structure and gene expression. Specifically, experiments will be aimed at characterizing the remodeling activity of recombinant PICKLE protein and examining the contribution of various conserved protein domains to PICKLE function in vitro and in vivo. The results will shed light on new mechanisms of action for CHD3 remodelers that are anticipated to be generally applicable to all eukaryotes.
Broader Impacts: The planned research aligns well with the education mission of NSF. The project will build on a long tradition of engaging students in research by providing both undergraduates and graduate students with opportunities to carry out research. This will provide the students with a solid foundation in science associated with chromatin biology, model systems, and genome-based approaches. Finally, the PI will continue to synergize his experiences in the lab with those in the classroom and thereby provide a more meaningful educational experience to students.
The goal of our research is to understand how a chromatin remodeling factor contributes to gene expression and developmental identity in plants. The genomic DNA of plants and animals exists in the nucleus of the cell in a nucleoprotein complex referred to as chromatin. Chromatin remodeling factors dynamically alter the composition of chromatin, thus affecting genome architecture and gene expression. Gene expression, in turn, plays a key role in determining developmental identity – whether a plant organ is a leaf or a root for example. We work with a chromatin remodeling factor named PICKLE that belongs to a family of remodeler proteins referred to as CHD3 remodelers that are found in plants and animals. In animals, CHD3 remodelers turn off genes that determine developmental identity by helping to remove a modification (acetylation) from the histone protein H3 (a key component of chromatin). We have previously shown that PICKLE, like animal CHD3 proteins, represses expression of developmental identity genes in plants. As a result of the research supported by the National Science Foundation, we have now shown that PICKLE proteins turn off these genes by a different strategy than animal proteins. PICKLE promotes a different modification (methylation) of a different region of histone H3. It turns out that both plants and animals use this modification to turn off developmental identity genes. Thus our work links a conserved chromatin remodeling pathway (CHD3 proteins) with a conserved chromatin modification pathway (methylation of lysine 27 of histone H3). We are now following up this finding by trying to understand how PICKLE promotes this modification in plants. We also undertook biochemical characterization of the PICKLE protein. PICKLE has been classified as a CHD3 protein based on the amino acid sequence of the protein – the assumption being that if it looks like a CHD3 chromatin remodeler, it must therefore act like a CHD3 chromatin remodeler. However, proteins do not always behave as expected based on sequence conservation. We carried out a series of biochemical assays with the goal of testing the hypothesis that PICKLE protein exhibits chromatin remodeling activity. We isolated highly purified recombinant PICKLE protein. Using this recombinant protein, we found that PICKLE does exhibit chromatin remodeling activity – including the ability to reposition a nucleosome (a fundamental unit of chromatin) on a piece of DNA. Furthermore, the remodeling activity of PICKLE is similar to that of animal CHD3 proteins. Thus we have demonstrated that we have generated biochemically active recombinant protein. It is worth noting that we are only the second laboratory to report generation of an active chromatin remodeler from plants. This capability will put us in a unique position to use biochemistry to test hypotheses of how PICKLE acts to modify chromatin structure. The research program funded by the National Science Foundation has contributed to broader impacts beyond the specific findings listed above. The long-term goal of these and other research efforts in our lab is to enable external manipulation of PICKLE-dependent processes such that scientists will be better able to manipulate plant developmental identity to promote agronomic traits of interest – such as propagating plants as cuttings or promoting seed oil production in roots. In addition, given that PICKLE is related to animal CHD3 proteins and that the repressive modification PICKLE promotes is found in animals as well as plants, it is possible that our research may provide insight into regulation of expression of developmental identity genes in animals as well as plants. In addition, this project contributed substantively to development of human resources and made possible the training of 1 postdoc, 2 graduate students, and 11 undergraduates. Furthermore, the primary investigator of this project is also the head advisor for the undergraduate program in the Department of Biochemistry at Purdue. The hands-on experience of training and supervising the undergraduates as they carried out research in the laboratory has directly informed efforts by the primary investigator to revise and update the research experience for the entire undergraduate population of the Department.
