Abstract

Abstract The mono-ubiquitination of the histone protein H2B (H2BUb1) has important functions in transcription, DNA repair, and other chromatin-related processes. The reaction is catalyzed by Bre1 and the homologous RNF20/RNF40 complex in the budding yeast and human cells, respectively, and is promoted by their respective interaction partners, Lge1 and WAC. The mechanism of the Bre1–Lge1 and RNF20/RNF40–WAC interactions is poorly understood. Here, we present the crystal structure of the Bre1–Lge1 complex and an AlphaFold predicted structure model of the RNF20/RNF40 complex bound with WAC, as well as in vitro and in vivo experiments to assess the interaction mechanism and function. Our study revealed extensive Bre1–Lge1 and RNF20/RNF40–WAC interfaces and a structural homology shared by these interfaces, but completely different sets of key electrostatic interactions at these interfaces that are crucial for the binding and encode the binding specificity. We further found that these interactions play critical roles in the Bre1-catalyzed H2BUb1 reaction and processes it regulates. Our data provide insights into the mechanism of the Bre1–Lge1 and RNF20/RNF40–WAC interactions.