APETALA1 and SEPALLATA3 interact with SEUSS to mediate transcription repression during flower development

The transcriptional repression of key regulatory genes is crucial for plant and animal development. Previously, we identified and isolated two Arabidopsis transcription co-repressors LEUNIG (LUG) and SEUSS (SEU) that function together in a putative co-repressor complex to prevent ectopic AGAMOUS (AG) transcription in flowers. Because neither LUG nor SEU possesses a recognizable DNA-binding motif, how they are tethered to specific target promoters remains unknown. Using the yeast two-hybrid assay and a co-immunoprecipitation assay, we showed that APETALA1 (AP1) and SEPALLATA3 (SEP3), both MADS box DNA-binding proteins, interacted with SEU. The AP1-SEU protein-protein interaction was supported by synergistic genetic interactions between ap1 and seu mutations. The role of SEU proteins in bridging the interaction between AP1/SEP3 and LUG to repress target gene transcription was further demonstrated in yeast and plant cells, providing important mechanistic insights into co-repressor function in plants. Furthermore, a direct in vivo association of SEU proteins with the AG cis-regulatory element was shown by chromatin immunoprecipitation. Accordingly, a reporter gene driven by the AG cis-element was able to respond to AP1- and SEP3-mediated transcriptional repression in a transient plant cell system when supplied with SEU and LUG. These results suggest that AP1 and SEP3 may serve as the DNA-binding partners of SEU/LUG. Our demonstration of the direct physical interaction between SEU and the C-terminal domain of SEP3 and AP1 suggests that AP1 and SEP3 MADS box proteins may interact with positive, as well as negative, regulatory proteins via their C-terminal domains, to either stimulate or repress their regulatory targets.