Abstract
Key messageWe 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.
Highlights
Used large-scale methods to study transcription factor (TF) binding specificity are nowadays ChIP-seq, protein-binding microarray (PBM) and1 3 Vol.:(0123456789)Plant Molecular Biology (2021) 105:543–557SELEX-seq (Systematic Evolution of Ligands by Exponential Enrichment followed by high-throughput sequencing) (Orenstein and Shamir 2014; Slattery et al 2014; Stormo and Zhao 2010)
In a previous sitedirected mutagenesis study we have shown that the arginine 3 of SEP3 is important for the recognition of A-tract elements within the CArG-box and for minor groove shape readout (Käppel et al 2018)
We further demonstrate that the highly conserved arginine 3 residue in the MADS-domain of SEP3 is critical for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif
Summary
Used large-scale methods to study transcription factor (TF) binding specificity are nowadays ChIP-seq (chromatin immunoprecipitation followed by high-throughput sequencing), protein-binding microarray (PBM) and. SELEX-seq (Systematic Evolution of Ligands by Exponential Enrichment followed by high-throughput sequencing) (Orenstein and Shamir 2014; Slattery et al 2014; Stormo and Zhao 2010). It enables the study of up to 107 or more selected DNA molecules after only one or two selection rounds (Riley et al 2014; Zykovich et al 2009). Relative affinities for selected sequences can be obtained by comparing the sequence composition of later rounds to that of the unselected DNA library (Riley et al 2014)
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