Molecular basis for the methylation specificity of ATXR5 for histone H3

In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELATED PROTEIN 5 and 6 (ATXR5/6) regulate heterochromatic DNA replication and genome stability. Our initial studies showed that ATXR5/6 discriminate between histone H3 variants and preferentially methylate K27 on H3.1. In this study, we report three regulatory mechanisms contributing to the specificity of ATXR5/6. First, we show that ATXR5 preferentially methylates the R/F-K*-S/C-G/A-P/C motif with striking preference for hydrophobic and aromatic residues in positions flanking this core of five amino acids. Second, we demonstrate that post-transcriptional modifications of residues neighboring K27 that are typically associated with actively transcribed chromatin are detrimental to ATXR5 activity. Third, we show that ATXR5 PHD domain employs a narrow binding pocket to selectively recognize unmethylated K4 of histone H3. Finally, we demonstrate that deletion or mutation of the PHD domain reduces the catalytic efficiency (kcat/Km of AdoMet) of ATXR5 up to 58-fold, highlighting the multifunctional nature of ATXR5 PHD domain. Overall, our results suggest that several molecular determinants regulate ATXR5/6 methyltransferase activity and epigenetic inheritance of H3.1 K27me1 mark in plants.