The DNA binding landscape of four seed-specific bZIP transcription factors in bread wheat: use of Bind-n-seq in vitro analysis

Abstract Sequence-specific transcription factor (TF)-DNA binding is a vital step in biology since it directly regulates genetic information inside the organism. However, the binding sites for many TFs are unknown in plants and animals.Using high-throughput sequencing technologies,studies have shown that TFs, including basic leucine zipper (bZIP), bind to a myriad of sequences, many of which are biologically relevant. bZIPs are eukaryote-specific dimeric TFs, and in plants, they are reported to bind to 8–12 nts sequences (with ACGT core) in the promoter region of many genes. In sedentary plants, they are the conduit for converting environmental cues into gene activity. A key question is what determines bZIP DNA binding specificity.This study adopts the Bind-n-seq methodology, which uses a 108 nts oligonucleotides library with an 18 nts random sequence that either incorporates ACGT tetranucleotides core or a completely random sequence to generate in vitro DNA-bZIP interaction maps of four seed-specific bread wheat bZIPs. Subsequently, de novo binding motifs representing the complete binding landscape for each bZIP were determined. Binding analysis shows a high degree of overlap for bZIP binding to G-box and C-box but with distinct flanking bases, suggesting a discrete role of flanking regions in ordaining TF-DNA binding specificity. From this study binding motifs obtained for Hy5 and Embp-1 show similarity with previously reported sequences, suggesting the protocol's robustness.