This project links two important cellular processes that were previously thought to be unrelated in plant systems: epigenetic regulation of gene expression and lipid signaling. The studies follow up on the recent discovery that the Arabidopsis ATX1, encoding a protein involved in chromatin modifications, specifically binds the lipid phosphoinositide 5-phosphate (PtdIns5P) in vitro and the two control a shared set of target genes in vivo. The long-term goal of this project is to fully characterize this novel ATX-PtdIns5P signaling pathway. The studies are aimed at gaining insights into the mechanics of the signaling mechanism, into its functioning in vivo, and at identifying other participants of the pathway. A major focus will be on studies of two Arabidopsis proteins from the myotubularin family, which, by virtue of their biochemical activity can produce the ligand, PtdInd5P. Although myotubularins are highly conserved in the evolution of eukaryotes and play important roles in animal systems, nothing is known about myotubularin activities in plants. Plant cells provide an attractive and convenient model system to study myotubularin function at the molecular, cellular, and organismal levels.
The project activities will also positively impact human resource development in Nebraska through the direct training of young scientists at the undergraduate, graduate and postgraduate level. Several small colleges in rural Nebraska supply our classes and laboratories with enthusiastic and motivated undergraduate students. In addition to UNL students, two undergraduate students from Doane College will be trained every summer. The recruitment of students from underrepresented groups will be emphasized and these students will be encouraged to consider a career in biology. Additional impacts include the development and instruction of a graduate level Developmental Genetics/Epigenetics course (BIOS910) designed to survey and to critically synthesize current topics in epigenetics.
Myotubularin (MTM) and myotubularin related (MTMR) proteins are evolutionary conserved enzymes in eukaryotes that produce a lipid molecule, Phosphatidylinositol-5 phosphate (PtdIns5P). This lipid functions as a signaling molecule that is present at trace amounts under regular cellular conditions but rapidly increase under certain stress conditions. Defects in the MTM functions leading to PtdIns5P deficiency result in severe diseases in humans, like muscle dystrophy, neuronal diseases and leukemia. Plants also contain genes encoding MTM-related proteins that are highly similar to the mammalian (human) proteins. Given the strong disease phenotypes caused by misregulated PtdIns5P levels, it has been unclear what roles plant MTMs play and why they were conserved in the eukaryotic evolution. Our studies provided first insights into the roles of the myotubularin proteins in plants. We found that the plant proteins, like their animal counterparts, have enzyme activity that converts the substrate (PtdIns3,5)P2 into the signaling molecule, PtdIns5P, and that its cellular levels were very low under homeostatic conditions but rapidly increased under dehydration stress. These results suggested that PtdIns5P may function as a signaling molecule in plant dehydration stress response pathways. Although well known that plants respond to biotic and abiotic stresses by altering expression of specific genes, how environmental signals are translated into altered gene expression is less clear. We have proposed that PtdIns5P regulates the activity of the chromatin modifier, ATX1 which, in turn, influences the expression of ATX1-regulated genes. As the Arabidopsis counterpart of the Trithorax factor from the TrxG complex regulating expression from many genes involved in developmental processes as well as in responses to a variety of environmental stresses, ATX1 belongs to the category of the epigenetic regulators. Thereby, the finding that signaling molecules (like PtdIns5P altering their levels in response to environmental changes) can modulate the activity of ATX1 affecting the expression of the numerous ATX1-regulated genes, linked lipid-signaling with epigenetic regulation. The majority of our efforts, accordingly, were focused on developing molecular, cellular, organismal and genomic approaches to test this hypothesis. Among the most important results from these studies are: 1). Establishing the origin of the two myotubularin genes in Arabidopsis (AtMTM1 and AtMTM2) in the context of the evolution of the myotubularin gene family in animal and plant lineages; 2. Revealing the specific and distinct roles of the two, MTM1 and MTM2, genes in Arabidopsis. In collaboration with the Laboratory for Phosphoinositide Signaling at the Patterson Institute for Cancer Research (University of Manchester, Great Britain) we found that only AtMTM1 is involved in elevating the cellular level of phosphatidylinositol 5-phosphate (PtdIns5P) in response to dehydration stress, that the two myotubularins differentially affect the Arabidopsis dehydration stress-responding transcriptome, and that AtMTM1 and AtMTM2 display different localization patterns in the cell consistent with the idea that they associate with different membranes to perform specific functions; 3) A single amino acid mutation in AtMTM2 (L250W) results in a dramatic loss of subcellular localization. Mutations in this region are linked to disease conditions in humans; 4) By a functional genomics approach, we showed that ATX1 and AtMTM1 participate in overlapping drought-response pathways. The shared set of genes, found in whole-genome transcriptome analyses provided insights into the relationship of the epigenetic factor and the lipid phosphatase from the other end of the response pathway; 5) During the period of these studies, we trained 6 postdoctoral fellows, one graduate student and 6 undergraduate student researchers, all of whom contributed significantly to the results. Numerous constructs, mutant and transgenic lines generated during this period have been distributed among several interested labs worldwide. A graduate student in Bonn, Germany, continues research in this area largely using materials and knowledge accumulated in the course of our work on this project and I serve as a co-advisor for her PhD thesis. In summary, our results indicated that despite the remarkable structural conservation, plant and animal myotubularins have significantly diverged in their functions. While loss of myotubularin function causes severe disease phenotypes in humans it is not essential for the cellular homeostasis under normal conditions in Arabidopsis thaliana. Instead, myotubularin deficiency is associated with altered tolerance to dehydration stress. The two Arabidopsis genes AtMTM1 and AtMTM2 originating from a segmental chromosomal duplication and encoding catalytically active enzymes have significantly diverged in function. The shared gene sets co-regulated by ATX1, AtMTM1 and PtdIns5P established an unknown cross talk between lipid-signaling and chromatin-regulated gene expression contributing to our understanding of possible mechanisms converting environmental stress signals into altered expression of the response genes. These results have been published in several papers.
