Chemical Synthesis of Asymmetric Nucleosomes Reveals Intramolecular Activation of UHRF1 E3 Ligase by H3K9me3.

The activation of UHRF1 E3 ligase by the H3K9me3 mark is critical for epigenetic inheritance; however, its precise mode of action within asymmetric nucleosomes remains unclear. Here, we leveraged the SpyTag/SpyCatcher bioorthogonal system to synthesize nucleosomes with predefined chemical asymmetry─an approach that enables clear spatial discrimination between the H3K9me3 recognition site and ubiquitin-acceptor lysines. Employing these synthetic, structurally defined nucleosomes, we found that H3K9me3 preferentially activates UHRF1 via an intramolecular (in cis) mechanism. Subsequent biochemical analyses demonstrated that this process depends on the physical coupling of two key UHRF1 functions: its synergistic recognition of the H3K9me3 modification and UHRF1's catalytic activity on the same H3 tail. In contrast, an H3K9me3 mark present on the opposing H3 monomer fails to significantly stimulate ubiquitination. To elucidate the structural basis of this cis activation, we further chemically synthesized a stable mimetic of the cis-activated UHRF1/UbcH5b-Ub/H3K9me3 nucleosome complex using CAET-mediated conjugation and split-intein splicing chemistry. Cross-linking mass spectrometry (XL-MS) analyses of this complex mapped the interaction networks among domains within UHRF1, between UHRF1 and histones, and between the E2-Ub module and the nucleosome core. These data yield spatial insights into UHRF1 activity on the nucleosome. Our study provides the first direct evidence for cis-activation of UHRF1 by H3K9me3 at the nucleosomal level, while highlighting the utility of chemical biology for deciphering complex epigenetic processes.