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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 factors—generally involved in floral organ identity and phase transition—we 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. [ABSTRACT FROM AUTHOR]

Abstract: Krox-20, originally identified as a member of “immediate-early” genes, plays a crucial role in the formation of two specific segments in the hindbrain during early development of the vertebrate nervous system. Here we cloned a genomic sequence of Xenopus Krox-20 (XKrox-20) and studied functions of a promoter element in the flanking sequence and associated transcription factors, which function in early Xenopus embryos. Using the luciferase reporter assay system, we showed that the 5′ flanking sequence was sufficient to induce luciferase activities when the reporter construct was injected into embryos at the eight-cell stage. Deletion and mutagenesis analyses of the 5′ flanking sequence revealed a minimal promoter element that included two known subelements, a CArG-box and cAMP response element (CRE) within a stretch of 22 bp nucleotide sequence (−72 to −51 from the transcription initiation site), both of which were essential for the promoter activity. The gel mobility shift assay indicated that these two subelements bound to some components in whole cell extracts prepared from stage 20 Xenopus embryos. Antibody supershift and competition experiments revealed that these components in cell extracts were serum response factor (SRF) and a member of CRE binding protein (CREB) family proteins that bound the CArG-box and CRE, respectively. They appeared to assemble on the minimal promoter element to produce a novel ternary complex. When we injected mRNA of a dominant-negative version of Xenopus SRF (XSRFΔC) into animal pole blastomeres at the eight-cell stage, expression of XKrox-20 in the nervous system as well as the minimal promoter activity was strongly suppressed. Suppression by XSRFΔC was counteracted by coexpressed wild-type XSRF. These results indicate that XSRF functions as an endogenous activator of XKrox-20 by forming a ternary complex with CREB on the minimal promoter element. [Copyright &y& Elsevier]