Screening for Protein-DNA Interactions by Automatable DNA-Protein Interaction ELISA

<div><p>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 <i>de novo</i> 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 <u>D</u>NA-<u>p</u>rotein-<u>i</u>nteraction (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, <i>At</i>WRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of <i>in vitro</i> binding preferences. In addition, protein extracts of <i>At</i>bZIP63 and the DNA-binding domain of <i>At</i>WRKY33 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, <i>At</i>TIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for <i>At</i>TIFY1 binding. This specific interaction between <i>At</i>TIFY1 and its DNA-binding motif was confirmed by <i>in vivo</i> plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for <i>de novo</i> 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.</p>