ABSTRACT Histone H3 lysine 9 methylation (H3K9me) is a central epigenetic mark governing heterochromatin formation. Although the H3K9 methyltransferase Clr4 has been extensively characterized using short histone peptide substrates, how it recognizes and coordinates different structural domains to engage physiological substrate nucleosomes remains poorly understood. Here, we employed chemical protein synthesis to generate site‐specifically ubiquitinated H3K14Ub histones and nucleosomes, enabling quantitative biochemical and structural investigations. Using a CAET handle‐assisted strategy, we obtained homogeneous H3K14Ub nucleosomes and demonstrated that ubiquitination enhances Clr4 activity by ∼350‐fold on nucleosomes relative to unmodified substrates. Clr4 domain deletion analyses revealed that, the intrinsically disordered region (IDR) of Clr4 is critical for nucleosome binding and ubiquitin‐dependent stimulation. Through site‐directed photo‐crosslinking, we identified specific IDR residues mediating interactions with nucleosomal surfaces. Furthermore, using an isoUb‐based synthetic approach, we generated H3K9NleK14Ub nucleosomes and determined cryo‐EM structures of Clr4–nucleosome complexes, unveiling multivalent nucleosome recognition by the IDR via four distinct interfaces: the H2A–H2B acidic patch, the H2A/H2B basic groove, the H2B and H3 elbow regions. Methyltransferase activity assays confirmed that mutations disrupting these interfaces impair Clr4 activity. Our study provides mechanistic insights into the ubiquitin‐dependent activation mechanism of Clr4, highlighting the power of chemical biology in deciphering epigenetic regulation.
Sun et al. (2026) studied this question.