Cytosine Methylation Changes the Preferred Cis-Regulatory Configuration of Arabidopsis WUSCHEL-Related Homeobox 14

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.