Specificity landscapes of 40 R2R3‐MYBs reveal how paralogs target different cis‐elements by homodimeric binding

Abstract Paralogous transcription factors (TFs) frequently recognize highly similar DNA motifs. Homodimerization can help distinguish them according to their different dimeric configurations. Here, by studying R2R3‐MYB TFs, we show that homodimerization can also directly change the recognized DNA motifs to distinguish between similar TFs. By high‐throughput SELEX, we profiled the specificity landscape for 40 R2R3‐MYBs of subfamily VIII and curated 833 motif models. The dimeric models show that homodimeric binding has evoked specificity changes for AtMYBs. Focusing on AtMYB2 as an example, we show that homodimerization has modified its specificity and allowed it to recognize additional cis‐ regulatory sequences that are different from the closely related CCWAA‐box AtMYBs and are unique among all AtMYBs. Genomic sites described by the modified dimeric specificities of AtMYB2 are conserved in evolution and involved in AtMYB2‐specific transcriptional activation. Collectively, this study provides rich data on sequence preferences of VIII R2R3‐MYBs and suggests an alternative mechanism that guides closely related TFs to respective cis‐ regulatory sites.