Genome-wide binding of SEPALLATA3 and AGAMOUS complexes determined by sequential DNA-affinity purification sequencing

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3–AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3–AG heteromeric complexes, including SEP3(Δtet)-AG, a complex with a SEP3 splice variant, SEP3(Δtet), which is largely dimeric and SEP3–AG tetramer. SEP3 and SEP3–AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3–AG and SEP3(Δtet)-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3(Δtet)-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3–AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.