Regulatory role of the START lipid/sterol binding domain in homeodomain transcription factors from plants

Doctor of PhilosophyBiochemistry and Molecular Biophysics Interdepartmental ProgramKathrin SchrickClass IV homeodomain leucine-zipper transcription factors (HD-Zip TFs) are master regulators of cell-type differentiation in the plant epidermis. These transcription factors contain a putative START (STeroidogenic Acute Regulatory (StAR)-related lipid Transfer) lipid/sterolbinding domain that is hypothesized to link metabolism to gene expression in plant development. This study is focused on two class IV family members that serve as models in many of the experiments: GLABRA2 (GL2) is a key regulator of differentiation in hair cells called trichomes as well as other epidermal cell types in various plant tissues. The second member addressed in this study is PROTODERMAL FACTOR2 (PDF2), which plays a crucial role in epidermal cell specification in shoots. A leading hypothesis is that the START domain, by binding a ligand, controls transcription factor function, analogously to nuclear receptors from mammals. Domain swap experiments indicated that the START domain from both plants and mammals is a conserved ligand-binding motif that is required for transcription factor activity. To further address its function in ligand binding, mutational analysis of the START domain of GL2 was performed. Several of the mutations remove charged residues in the predicted ligand-binding pocket and resulted in loss-of-function phenotypes, suggesting that ligand binding is critical for HD-Zip TF activity. Chromatin immunoprecipitation–based sequencing (ChIP-seq) revealed that the START domain is dispensable for transcription factor binding to DNA. Using a high throughput thermal shift assay to screen a library of pure natural compounds, specific secondary metabolites were identified as putative START domain ligands for PDF2. Experiments in both yeast and N. benthamiana demonstrated that the START domain is required for homodimerization of GL2 through its Zip domain. It was also found that the START domains physically interact with RHAMNOSE SYNTHASE I (RHM1). Further, this work provided evidence for a previously elusive redundancy between GL2 and another class IV HD-Zip TF, and unveils a positive feedback loop in the maintenance of the GL2 activity during trichome differentiation. Taken together, these findings support the premise that START domains are central players in metabolic regulatory networks that can modulate transcription factor activity by binding ligands and mediating protein-protein interactions

Shared functions of plant and mammalian StAR-related lipid transfer (START) domains in modulating transcription factor activity

Abstract Background Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity. Results Our results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein–metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts. Conclusions The START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein’s nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity

Shared functions of plant and mammalian StAR-related lipid transfer (START) domains in modulating transcription factor activity

Background: Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity. Results: Our results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein–metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts. Conclusions: The START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein’s nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity

A series of dual‐reporter vectors for ratiometric analysis of protein abundance in plants

Ratiometric reporter systems enable comparisons of the abundance of a protein of interest, or "target," relative to a reference protein. Both proteins are encoded on a single transcript but are separated during translation. This arrangement bypasses the potential for discordant expression that can arise when the target and reference proteins are encoded by separate genes. We generated a set of 18 Gateway-compatible vectors termed pRATIO that combine a variety of promoters, fluorescent, and bioluminescent reporters, and 2A "self-cleaving" peptides. These constructs are easily modified to produce additional combinations or introduce new reporter proteins. We found that mScarlet-I provides the best signal-to-noise ratio among several fluorescent reporter proteins during transient expression experiments in Nicotiana benthamiana. Firefly and Gaussia luciferase also produce high signal-to-noise in N. benthamiana. As proof of concept, we used this system to investigate whether degradation of the receptor KAI2 after karrikin treatment is influenced by its subcellular localization. KAI2 is normally found in the cytoplasm and the nucleus of plant cells. In N. benthamiana, karrikin-induced degradation of KAI2 was only observed when it was retained in the nucleus. These vectors are tools to easily monitor in vivo the abundance of a protein that is transiently expressed in plants, and will be particularly useful for investigating protein turnover in response to different stimuli