近日，上海交通大学艾华松团队报道了化学合成的H3K14Ub揭示了Clr4介导的H3K9甲基化多价核小体识别。该项研究成果发表在2026年3月24日出版的《德国应用化学》杂志上。论文共同第一作者为孙茂甡、杜运祥、李政卿；通讯作者为孙茂甡、艾华松。

组蛋白H3赖氨酸9甲基化（H3K9me）是调控异染色质形成的关键表观遗传标记。尽管利用短肽底物对H3K9甲基转移酶Clr4已开展了广泛研究，但该酶如何识别并协调不同结构域以结合生理底物核小体，其机制仍不甚明晰。

研究组采用化学蛋白质合成技术，制备了位点特异性泛素化的H3K14Ub组蛋白及核小体，从而实现了定量生化和结构研究。借助CAET手柄辅助策略，研究组获得了均质的H3K14Ub核小体，并证明与未修饰底物相比，泛素化可将核小体上Clr4的活性提高约350倍。Clr4结构域删除分析显示，其内在无序区（IDR）对核小体结合及泛素依赖性激活至关重要。通过定点光交联技术，研究组鉴定了IDR中介导与核小体表面相互作用的特定残基。

此外，利用基于isoUb的合成策略，研究组生成了H3K9NleK14Ub核小体，并解析了Clr4-核小体复合物的冷冻电镜结构，揭示了IDR通过四个不同界面实现对核小体的多价识别：H2A-H2B酸性斑块区、H2A/H2B碱性沟槽区、以及H2B与H3的肘部区域。甲基转移酶活性测定证实，破坏这些界面的突变会削弱Clr4活性。该研究为理解Clr4的泛素依赖性激活机制提供了结构基础，彰显了化学生物学在解析表观遗传调控中的强大作用。

附：英文原文

Title: Chemically Synthesized H3K14Ub Unveils Clr4's IDR-Mediated Multivalent Nucleosome Recognition in H3K9 Methylation

Author: Maoshen Sun, Yunxiang Du, Zhengqing Li, Akejiang Aderjiang, Meixuan Xin, Huasong Ai

Issue&Volume: 2026-03-24

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.

DOI: 10.1002/anie.202520817

Source: https://onlinelibrary.wiley.com/doi/10.1002/anie.202520817