The Arabidopsis Transcription Factor ORFeome and downstream genomic application

University Of California, San Diego, La Jolla CA

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Abstract

DESCRIPTION (provided by applicant): Transcription factors (TF) have a crucial role in controlling gene expression, and exploring their molecular targets, binding partners and mode of regulation is essential to understand any plant biological process. Arabidopsis offers some unique advantages for the development of large-scale genomic approaches for the study of TFs function such as the ease and low cost to generate large transgenic collections, or the propensity of most gene-regulatory regions to be circumscribed to a short region upstream the transcription start site. The potential of these types of strategies is greatly exemplified in a recent report from our laboratory where, by using a fraction of the full TF collection, a novel clock component was identified. For these reasons, we started gathering all available Arabidopsis TFs from the different ORFeome resources (Salk, Pekin-Yale, REGIA, TIGR and RIKEN) to generate a complete TF collection. However, our findings from resequencing the different clones revealed a large overlap between the collections and several hundred mislabeled or missing ones, resulting in a final coverage close to 75% of all the Arabidopsis transcription factors and regulators. Here, we propose to generate an homogenous gold standard GATEWAYTM compatible collection containing every Arabidopsis TF. The corresponding coding sequences will be cloned in the same vector using the available ORFeomes as the template resource when possible. The remaining 25% missing ones will be generated by following different complementary amplification protocols and subsequently cloned in the same vector backbone. In addition, we propose to create and distribute to the community, nine application-ready genomic collections containing each TF in fusion with different tags for a multitude of applications. These nine collections will allow (1) overexpression screens in plants of wild type as well as EAR or VP64 translational fusions, (2) protein-protein interaction screens, (3) protein-DNA interaction screens, (4) subcellular localization and, (5) bacterial recombinant protein expression. In addition, in collaboration with Dr. Joe Ecker at the Salk Institute, we propose to test and compare the efficiency of different protein epitope-tags suitable to perform ChIP-seq experiment. A collection of TF tagged with the selected epitope will be generated. Finally, we propose to devise a simple protocol to perform yeast one-hybrid screens with full TF collections at a reasonable cost. We strongly believe these resources have the potential to greatly enhance research in Arabidopsis and other crop species. As the knowledge gap is being filled, the study of transcriptional networks in Arabidopsis will ultimately help us understand the biochemical complexity of multicellular organisms and positively impact the biomedical research community.