Structural basis of nucleosomal H4K20 methylation by methyltransferase SET8

Histone H4 lysine 20 monomethylation (H4K20me1) plays a crucial role in multiple processes including DNA damage repair, DNA replication, and cell cycle control. Histone methyltransferase SET8 (previously named PR‐Set7/KMT5A) mediates the chromatin deposition of H4K20me1, but how SET8 recognizes and modifies H4 in the context of the nucleosome is not fully understood. Here, we developed a simple chemical modification approach for H4K20 substitution by using the lysine analog S‐ethyl‐L‐cysteine (Ecx). Substitution of H4K20 with H4Ecx20 improves the stability of the SET8‐nucleosome complex, allowing us to determine the cryo‐EM structure at 3.2 Å resolution. Structural analyses show that SET8 directly interacts with the H4 tail and the H2A‐H2B acidic patch to ensure nucleosome binding. SET8 residues R339, K341, K351 make contact with nucleosomal DNA at the super helical location 2 (SHL2). Substitution of SET8 DNA‐binding residues with alanines decreases the SET8‐nucleosome interaction and impairs the methyltransferase activity. Disrupting the binding between SET8 R192 and H2A‐H2B acidic patch decreases the cellular level of H4K20me1. Together, these results reveal a near‐atomic resolution structure of SET8‐bound nucleosome and provide insights into the SET8‐mediated H4K20 recognition and modification. The lysine‐to‐Ecx substitution approach can be applied to the study of other methyltransferases.