Alwin Köhler

The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle¹. Although processive ubiquitination might—in principle—arise from Bre1 and Rad6 travelling with RNA polymerase II², the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid–liquid phase separation³ as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated ‘reaction chambers’, with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.

Significance: Histone H2B monoubiquitination is important for gene expression. It is mediated by the yeast E3 ligase Bre1 (human RNF20/40) and the E2 conjugating enzyme Rad6. These enzymes associate with RNA polymerase II and their activity is counteracted by the H2B deubiquitinase of the Spt–Ada–Gcn5 acetyltransferase (SAGA) complex. In contrast to many other E3 ligases, the Rad6–Bre1 machinery exclusively modifies a single lysine on the nucleosome. Given the number of other nearby lysines, it was unknown how such specificity could be achieved. Our study shows how Rad6–Bre1 recognition of the nucleosome is mechanistically coupled to ubiquitin transfer, how a single target lysine is chosen, and how Rad6–Bre1 compete with SAGA for regulatory access to a specific region on the nucleosome.