GSE95730
Differences in DNA-binding specificity of floral homeotic protein complexes predict organ-specific target genes
Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors (TFs). How these factors achieve their regulatory specificities is however still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS-domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high throughput DNA sequencing (SELEX-seq) on several floral MADS-domain protein homo- and heterodimers to measure their DNA-binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 (SEP3) and AGAMOUS (AG). Binding specificity is further modulated by different binding site (BS) spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows to differentiate between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA-binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA-binding specificity of floral MADS-domain proteins. DNA-binding specificity of individual dimers, as well as DNA-binding preferences of higher-order complexes differ between floral homeotic protein complexes. Differential DNA-binding of MADS-domain protein complexes plays a role in the specificity of target gene regulation. Overall design: SELEX-seq for MADS-domain protein homo- and heterocomplexes using random pool of DNA and in vitro synthetized proteins.