We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved arginine residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved arginine residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.
Keywords: A-tract; Arabidopsis thaliana; CArG-box; MADS-box; SELEX-seq.