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bioRxiv · 10.1101/2024.07.22.604500

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally-defined K63-Polyubiquitylated Linker Histone H1

Shi, Q.·Deng, Z.·Zhang, L.·Tong, Z.·Li, J.-B.·Chu, G.-C.·Ai, H.·Liu, L.
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Abstract

The chemical synthesis of histones with homogeneous modifications is a potent approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). Here, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine-Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on ubiquitylated chromatosomes with different ubiquitin lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally-defined polyubiquitylated H1.0 by CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

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Shi, Q., Deng, Z., Zhang, L., Tong, Z., Li, J.-B., Chu, G.-C., Ai, H., Liu, L.. 2024-07-22. Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally-defined K63-Polyubiquitylated Linker Histone H1. https://doi.org/10.1101/2024.07.22.604500

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