Laura D. Gallego,1,* Maren Schneider,1,* Chitvan Mittal,2,* Anete Romanauska,1 Ricardo M. Gudino Carrillo,1 Tobias Schubert,1 B. Franklin Pugh,2 and Alwin Köhler1,#
1 Max Perutz Labs, Medical University of Vienna, Vienna Biocenter Campus (VBC), Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria
2 Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, PA 16802, USA
- These authors contributed equally to this work
Correspondence: alwin.koehler@mfpl.ac.at
Abstract
The conserved yeast E3 ligase Bre1 and its partner E2 Rad6 monoubiquitinate histone H2B across gene bodies during the transcription cycle. While processive ubiquitination might in principle arise from Bre1/Rad6 traveling with RNA polymerase II, we provide a different explanation. Here we implicate liquid-liquid phase separation as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, whose intrinsically disordered region 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, accelerating H2B ubiquitination. 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, causing neurological disease when impaired.