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.

The Arabidopsis transcription factor WUSCHEL-related homeobox 14 (AtWOX14) plays versatile roles in plant growth and development. However, its biochemical specificity of DNA binding, its genome-wide regulatory targets, and how these are affected by DNA methylation remain uncharacterized. To clarify the biochemistry underlying the regulatory function of AtWOX14, using the recently developed 5mC-incorporation strategy, this study performed SELEX and DAP-seq for AtWOX14 both in the presence and absence of cytosine methylation, systematically curated 65 motif models and identified 51,039 genomic binding sites for AtWOX14, and examined how 5mC affects DNA binding of AtWOX14 through bioinformatic analyses. Overall, 5mC represses the DNA binding of AtWOX14 monomers but facilitates the binding of its dimers, and the methylation effect on a cytosine’s affinity to AtWOX14 is position-dependent. Notably, we found that the most preferred homodimeric configuration of AtWOX14 has changed from ER1 to ER0 upon methylation. This change has the potential to rewire the regulatory network downstream of AtWOX14, as suggested by the GO analyses and the strength changes in the DAP-seq peaks upon methylation. Therefore, this work comprehensively illustrates the specificity and targets of AtWOX14 and reports a previously unrecognized effect of DNA methylation on transcription factor binding.

A distinct epigenetic feature of plants is the DNA methylation in non-CG contexts. Although the physiological roles of non-CG methylation have been elucidated, its direct impact on transcription factor (TF)–DNA interactions remains largely unexplored. Focusing on WRKY-family TFs, here we investigated how non-CG methylation influences their DNA binding specificity and genome-wide cis-regulatory elements (CREs). By generating 461 SELEX and DAP-seq libraries for 54 AtWRKYs, we show that DNA methylation alters both monomeric and dimeric binding specificities of WRKYs, leading to an overall increase in specificity divergence among family members. We curated 201 WRKY motifs and clustered them into 11 classes, 5 of which represent previously unreported specificities. Notably, the known WRKY cis-element PRE4 was found to be recognized only when methylated. The comprehensive dataset of accurate WRKY motifs also enabled the identification of the amino acid discriminants of W-box and WT-box. Expanding on prior knowledge, we demonstrate that methylation not only decreases but can also increase the affinity of WRKYs. This bidirectional effect has globally reshaped the genomic binding landscape of WRKYs upon methylation. Finally, we constructed the WRKY Regulatory Code Database (https://transysbio.cn/WRKYRCDB.php) to facilitate data access.

The Arabidopsis transcription factor WUSCHEL-Related Homeobox 14 (AtWOX14) has versatile roles in plant growth and development. However, its biochemical specificity of DNA-binding and genome-wide regulatory targets remain uncharacterized. With the recently developed 5mC-incorporation strategy[1], this study performed SELEX and DAP-seq for AtWOX14 both in the presence and absence of cytosine methylation, and systematically curated 65 motif models and identified 51,039 genomic binding sites for AtWOX14. Overall, 5mC represses DNA binding of AtWOX14 monomers but facilitates binding of its dimers, and the methylation effect on a cytosine’s affinity to AtWOX14 is positional dependent. Notably, we found that the most preferred homodimeric configuration of AtWOX14 has changed from ER1 to ER0 upon methylation. Such change has the potential to rewire the regulatory network downstream of AtWOX14, as suggested by the GO analyses and the strength changes of the DAP-seq peaks upon methylation. Therefore, this work comprehensively illustrates the specificity and targets of AtWOX14, and reports a yet unrecognized effect of DNA methylation on transcription factor binding.