The START domain is a highly conserved protein motif associated with a number of diverse proteins, including key regulators of plant development. START domains are well known in animal systems where they regulate protein function through binding of specific lipids. The function of this domain in plant proteins is not well understood. Genetic analysis has shown that this motif is critical for function of the HD-ZIPIII proteins, which are required for normal embryonic, vegetative and floral development. The goal of this project is to elucidate the mechanism via which the START lipid-binding domain regulates activity of the HD-ZIPIII proteins. Genetic and biochemical approaches will be used to determine the effects of START domain mutations on the localization of HD-ZIPIII proteins within the cell, their ability to bind protein partners or DNA, and their ability to induce expression of target genes. A second aspect of this project is to identify lipids that bind the START domain to modulate HD-ZIPIII activity. A large library of distinct chemical compounds will be screened for substances that bind the HD-ZIPIII START domain and affect its ability to modulate target gene expression. Resolving the role of the START domain in HD-ZIPIII function will have wide-ranging implications for the large number of START domain-containing proteins in plants that contribute to diverse developmental processes, disease resistance and the regulation of flowering time. Likewise, identification of agonists / antagonists of the START domain will provide an entirely new toolkit to research START domain proteins and can reveal signals important for the fundamental developmental processes regulated by START domain factors. This project will also make significant educational contributions through the training of a Postdoctoral Fellow who is a member of a group underrepresented in science, and the training of undergraduate and high school students. In addition, participants in this project will lecture in the NSF-funded CSHL Plant Science Course and will teach in a summer training program for high school teachers at the CSHL Dolan DNA learning center.
The goal of this project was to elucidate the role of the START lipid-binding domain in the regulation of highly conserved family of plant transcription factors, the HD-ZIPIII proteins. The HD-ZIPIII factors are required for the normal progression of a number of fundamental developmental processes in plants, including embryogenesis, shoot growth, vascular development, and patterning of leaves and floral organs. Reflective of their prominent roles in plant development, HD-ZIPIII activity is under exquisite control. Interestingly, this class of transcription factors contains a so-called START domain. This protein structure is found in all species from yeast to humans, and is required for the transfer of lipids and sterols (including cholesterol) within the cell. Coupling of a START domain to proteins that regulate gene expression, as is the case for the HD-ZIPIII proteins, is however a unique feature seen only in plants. The function of the START domain in this context remains unknown, however, preliminary observations indicated that the START domain regulates HD-ZIPIII activity. This presents the hypothesis that binding of a lipid or sterol to the START domain serves as a switch that turns on the HD-ZIPIII proteins and allows them to activate downstream genes with functions in the above-mentioned key developmental processes. The first Aim of this project was to resolve the mechanism via which lipid/sterol binding to the START domain regulates HD-ZIPIII activity. To this end, we created small changes in the START domain and studied their effects on plant development. In addition, we determined whether these START domain variants affected the site in the cell where the HD-ZIPIII proteins accumulate, or changed their ability to interact with DNA or protein partners. We showed that the START domain is indeed essential for HD-ZIPIII function. Mutations that alter the "lining" of the lipid/sterol-binding pocket render these proteins inactive. Experiments to discern how lipid/sterol binding switches on HD-ZIPIII activity indicate that binding of a lipid/sterol promotes the interaction of HD-ZIPIII proteins with protein partners that activate expression of downstream genes. The second Aim was to gain insight into the nature of the lipid or sterol bound by the HD-ZIPIII START domain. For this, a set of experiments was developed that allow the efficient screening of a wide range of chemical compounds for their ability to interact with and modulate the activity of the HD-ZIPIII START domain. In addition, with outcomes from computer modeling and biochemical binding studies, this showed that the HD-ZIPIII START domain binds a long-chain fatty acid with a small head group, rather than a sterol based compound. Tools generated in this project are currently being used to identify the endogenous START domain-bound lipid. Plant development is largely determined by mobile signals that instruct a cell of their precise position within the developing plant. The identification of such instructive signals is one of the main challenges in plant biology. The work completed through this project uncovers a new mechanism via which mobile signals affect development; binding of a lipid to the START domain facilitates binding of the HD-ZIPIII proteins to protein partners, this switches on activity and induces downstream genes. This finding has wide-ranging implications for the large number of START domain-containing proteins in plants that contribute to diverse processes, e.g. disease resistance and the regulation of flowering time. Besides advancing our basic knowledge of plant development, these findings have other exciting applications. The identification of compounds that bind the START domain provide an entirely new toolkit to control HD-ZIPIII activity and possibly modulate aspects of plant development. Last but certainly not least, this projected provide opportunities for training and development to a minority postdoctoral fellow, one female visiting graduate student, and two female undergraduates.
