Semisynthesis of Multiphosphorylated and Acetylated Histone Methyltransferase Dot1L Reveals Synergistic Inhibition of Nucleosomal H3K79 Methylation

Abstract Histone methyltransferase Dot1L is the sole non-SET-domain lysine methyltransferase that exclusively catalyzes nucleosomal histone H3 lysine 79 (H3K79) methylation, and its dysregulation is tightly associated with leukemogenesis and other human diseases. While the regulation of Dot1L activity by histone substrate modifications (e.g., H2BK120Ub, H2BK34Ub, and H4K16ac) has been well characterized biochemically and structurally, the regulatory mechanisms underlying intrinsic phosphorylation and acetylation within its catalytically essential lysine-rich region (LRR) remain poorly understood, largely limited by the lack of homogeneous Dot1L proteins with site-specific post-translational modifications (PTMs). Here, we develop a modular semisynthetic strategy combining peptide hydrazide ligation and Sortase A-mediated chemoenzymatic ligation to construct homogeneous Dot1L catalytic domain (1–416) variants bearing site-specific PTM patterns: unmodified, penta-phosphorylated, penta-acetylated, and dual penta-phosphorylated/penta-acetylated. By splitting Dot1L(1–416) into a recombinant N-terminal fragment and a chemically synthesized C-terminal LRR fragment, we further optimize a hybrid microwave/manual solid-phase peptide synthesis to overcome the β-elimination of phosphoserine and phosphothreonine residues, enabling efficient and precise preparation of multiphosphopeptides. Biochemical characterizations demonstrate that both phosphorylation and acetylation individually repress Dot1L-catalyzed nucleosomal H3K79 methylation, and dual modifications exert a synergistic inhibitory effect on its catalytic activity. Mechanistically, these intrinsic PTMs attenuate the binding affinity of Dot1L to nucleosomes, which directly accounts for the impaired catalytic activity. This work establishes a generalizable semi-synthetic platform for generating PTM-defined epigenetic enzymes and unveils the synergistic inhibitory regulation of Dot1L activity on nucleosomal H3K79 methylation by its intrinsic phosphorylation and acetylation.