On March 29, 2023, the journal Nature published an online article, entitled "Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase", by the group of researchers lead by Drs. XU Rui-Ming and ZHU Bing from the Institute of Biophysics, Chinese Academy of Sciences. In this study, the researchers reported the 3.0 Å-resolution cryo-EM structure of human Polycomb repressive deubiquitinase (PR-DUB) complex bound to the chromatosome particle with mono-ubiquitinated H2AK119 (H2AK119ub1), revealing the mechanism by which PR-DUB specifically removes H2AK119ub1 in the context of nucleosome/chromatosome.

Polycomb group (PcG) proteins are crucial for embryonic development, cell fate determination and gene repression in somatic cells. Major PcG protein complexes include PRC1 (Polycomb Repressive Complex 1), PRC2 (Polycomb Repressive Complex 2) and PR-DUB (Polycomb Repressive Deubiquitinase) complexes. PR-DUB specifically removes H2AK119ub1 on the nucleosome, counteracting the ubiquitin E3 ligase activity of PRC1. The human PR-DUB complex is composed of ubiquitin C-terminal hydrolase BAP1 (BRCA1 associated protein 1) and one of the three Additional Sex comb Like (ASXL) proteins ASXL1/2/3. BAP1 is also a tumor suppressor that is frequently mutated in many cancers, and ASXL protein mutations are associated with varieties of leukemia. Although the crystal structure of Drosophila PR-DUB complex has been previously reported, the solved structure lacked the ubiquitinated nucleosome substrate. This deficiency has severely limited the understanding of the molecular mechanism by which PR-DUB specifically removes H2AK119ub1 in the context of the nucleosome.

By determining the cryo-EM structure of PR-DUB in complex with the chromatosome, researchers found that amino acid residues 699-706 located on the positively charged BAP1 C-terminal extension (CTE) form a finger-like structure, and it interacts with histone H3-H4 and DNA next to the nucleosome dyad. In the catalytic domain of BAP1, a highly conserved sequence motif, RRSRR, spanning residues 56-60 is placed in the vicinity of the acidic patch of the nucleosome. This bidentate binding mode stabilizes the interaction between BAP1 and the nucleosome. The ASXL1 subunit does not substantially interact with the nucleosome, but it forms a complete ubiquitin binding pocket together with BAP1. Additionally, the interaction between ASXL1 and BAP1 directs the BAP1 CTE for correct nucleosome binding. In this binding mode, the active site of BAP1 is located far away from the conventional H2AK119 position on the nucleosome, and the H2A C-terminal tail undergoes a large conformational change, placing the H2AK119-ubiquitin isopeptide bond in the catalytic active site for hydrolysis. The observed structural feature clearly accounted for the nucleosomal H2AK119 specificity of PR-DUB. The researchers also validated the structural observations by mutagenesis and in vitro and in vivo deubiquitinase assays of key residues at the interface between PR-DUB and nucleosome, and clarified the role of ASXL1 truncation mutants in the regulation of BAP1 enzymatic activity.

Taken together, this study unveiled the particular nucleosome binding mode and the dramatic conformational change of ubiquitinated C-terminal tail of H2A in the nucleosome, revealing the structural basis for PR-DUB's specificity for nucleosomal H2AK119ub1. This revelation significantly expanded the understanding of the molecular mechanism governing deubiquitination of nucleosomal H2A, and the knowledge should also benefit the understanding of tumorigenesis effects of BAP1 and ASXL mutations and related drug development.

Drs. XU Rui-Ming and ZHU Bing are co-corresponding authors, and Ph.D. students GE Weiran, YU Cong and LI Jingjing are co-first authors of this study. Dr. LI Guohong helped the in vitro deubiquitinase activity experiments, Dr. ZHANG Xinzheng supervised the cryo-EM data analysis, and Dr. TIAN Changlin advised chemical synthesis of ubiquitinated H2A. The research was supported in part by the Natural Science Foundation of China, the Ministry of Science and Technology and the Chinese Academy of Sciences. The Protein Science Core Facility and the Center for Biological Imaging at the Institute of Biophysics, CAS, provided support for multiangle light scattering analyses and cryo-EM data collection.

Figure: Overall structure

Article link: https://www.nature.com/articles/s41586-023-05841-y

Contact: XU Ruiming

Institute of Biophysics, Chinese Academy of Sciences

Beijing 100101, China

Email: rmxu@ibp.ac.cn

（Reported by Dr. XU Ruiming's group）