Matthias Schleiden Institute/Genetics, Friedrich Schiller University Jena, Philosophenweg 12, 07743, Jena, Germany.
Department of Computer Science, University of Helsinki, Pietari Kalmin katu 5, 00014, Helsinki, Finland.
Plant Mol Biol. 2021 Mar;105(4-5):543-557. doi: 10.1007/s11103-020-01108-6. Epub 2021 Jan 24.
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.
我们通过 SELEX-seq 研究了 MADS 结构域转录因子 SEPALLATA3 和突变变体的 DNA 结合谱。SEPALLATA3 突变蛋白的 DNA 结合特性使我们提出了一种新的 DNA 结合模式。MIKC 型 MADS 结构域蛋白作为植物发育中必不可少的转录因子，以二聚体的形式结合到一个称为 CArG 盒的富含 10 个碱基对的 AT 基序上。然而，这个共识基序并不能完全解释开花植物中丰富的家族成员如何以特定的方式结合不同的靶基因。本研究旨在更好地理解 MADS 结构域转录因子的 DNA 结合特异性。此外，我们还想了解高度保守的精氨酸残基在 MADS 结构域转录因子家族的结合特异性中的作用。在这里，我们通过进行 SELEX followed by high-throughput sequencing (SELEX-seq) 来研究花同源 MADS 结构域蛋白 SEPALLATA3 的 DNA 结合谱。我们发现了一组多样化的结合序列，并能够估计 SEPALLATA3 对大量不同序列的体外结合亲和力。我们发现了富含 AT 基序作为侧翼序列的偏好证据。虽然不同的 CArG 盒可以作为 SEPALLATA3 的结合位点，但我们的研究结果表明，首选的侧翼基序几乎总是相同的，因此主要不依赖于中央 CArG 盒基序的身份。对在 DNA 结合 MADS 结构域第 3 位具有单个氨基酸取代的 SEPALLATA3 蛋白的分析进一步表明，保守的精氨酸残基，已被证明参与形状读出机制，对于识别 CArG 盒基序的第 3 和 8 位的核苷酸特别重要。这使我们提出了一种新的 SEPALLATA3 DNA 结合模式，与已知的其他 MADS 结构域蛋白不同。
