Homotypic Clusters of Transcription Factor Binding Sites in the First Large Intron of AGL24 MADS-Box Transcription Factor Are Recruited in the Enhancement of Floral Expression

The occurrence of homotypic clusters of transcription factor binding sites (HCTs) and their contribution to regulatory evolution is documented in animals, but their manifestation in regulating plant genome expression remained an enigma. To explore the existence and functions of HCTs in highly constrained non-coding sequences of MADS-box transcription factorsgenerally involved in floral organ identity and phase transitionwe employed cis-regulatory cluster finding in silico tools augmented with in vitro assays and site-directed mutagenesis in the STMADS11 superclade of this family. Thousands of transcription factor binding sites (TFBSs) organized into HCTs and multiple-factor binding elements belonging to various families of TFs were identified in the promoter as well as in the intronic region of the selected members of STMADS11 subfamily. A total of 151 HCTs were detectable in the defined promoter in comparison with 144 in the intronic regions. The MADS-domain binding HCTs (overlapping CArG-boxes) constitute a major portion (18%) of these HCTs. A protrusive HCT of 11 TFBSs was identified in the 1st large intron of the AGAMOUS LIKE 24 (AGL24) pertinent to the SVP clade of the STMADS11 subfamily but no such cluster could be detected in the region proximal to the core promoter. These HCTs in the intronic region could bind the SEP4 MADS-domain protein as revealed by in vitro assay. Using HCT cluster swapping of CArG-boxes through the GUS reporter assay, it was possible to empirically validate that a change in the position of intronic MADS-box cluster of AGL24 to the promoter region adjacent to the transcriptional start site (TSS) invoke an enhancement of floral expression. This study unveils the occurrence of HCTs in both the promoter and downstream intronic regions in the selected members of the STMADS11 subfamily of plants. These HCTs play an important role in deciphering the gene regulatory code of MADS-box TFs particularly AGL24.