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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
Structural basis of nucleosome deubiquitination by the bidentate Calypso/Asx complex
Chi Wang1,2, Fahui Sun2, Heyu Zhao2,3
1Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China.
None:
The Polycomb repressive complex 1 (PRC1) and PR-DUB constitute a canonical pair of histone-modifying enzymes that deposit and remove monoubiquitinated H2A at lysine 119 (H2AK119ub1), serving as a model of dynamic epigenetic regulation. In humans, PR-DUB, composed of BAP1 and ASXL1, functions as a monomeric complex, while the Drosophila homolog Calypso/Asx forms a bidentate dimer (Calypso2: Asx2) with an unclear chromatin engagement mechanism. Here, we present its cryo-EM structure bound to a nucleosome, revealing the molecular basis of interaction. Surprisingly, only one Calypso/Asx unit engages the nucleosome in a conformation similar to human BAP1/ASXL1, while the second remains disengaged. Structural and biochemical analysis of the positively charged Calypso C terminus suggests a "spreading" potential of the bidentate complex along chromatin, which was validated in vitro using nucleosome arrays. These findings support a model in which the bidentate Calypso/Asx complex enables processive deubiquitination along chromatin via alternating or cooperative engagement.
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