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

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.