Abstract
DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding of gene regulation in any organism of choice.
Highlights
DNA-binding proteins (DBPs), such as transcription factors, polymerases, methyl-transferases or histones, play pivotal roles in the regulation of chromatin structure and the control of gene expression
We developed a new algorithm that was optimized for double-stranded DNA (dsDNA) probe design of a variable library region
We could show the quick and economic identification of DNA-binding motifs by DPI-ELISA screen that is potentially applicable to any given DBPs irrespective of the DNA-binding domain architecture
Summary
DNA-binding proteins (DBPs), such as transcription factors, polymerases, methyl-transferases or histones, play pivotal roles in the regulation of chromatin structure and the control of gene expression. Sequencing of eukaryote genomes disclosed that about 10% of all genes encode potential DBPs. every higher plant or vertebrate genome harbors over 2000 of these DBP genes [1,2,3,4]. Every higher plant or vertebrate genome harbors over 2000 of these DBP genes [1,2,3,4] Despite their importance in many fundamental processes, e.g. during stress or disease, throughout development and in controlling yield or growth, our knowledge on this tremendous number of putative DBPs and their interaction with DNA is limited [1,2]. As many classes of DBPs are not (yet) in the focus of investigations, only for approximately 7% of all DBP family members encoded in a eukaryote genome a DNA-binding motif has been described [2]
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