Understanding the Regulation of Chromatin by Phase Separation

Abstract

Chromatin is a highly dynamic DNA-protein complex that contributes to various essential processes in eukaryotes, including packaging of genomes. The fundamental unit of chromatin, the nucleosome core particle, comprises of the complex formed by DNA wrapped around the histone octamer (two copies each of H2A, H2B, H3, H4). Modifications of either the histones or DNA can regulate structure and function of chromatin, along with other downstream processes. Among them, the monoubiquitination of H2B modulate crucial pathways in various organisms. In yeast, Lge1 is a critical protein for monoubiquitination of the 123rd lysine of H2B and undergoes liquid-liquid phase separation. This physical property of Lge1 can concentrate the substrate chromatin and the ubiquitination machinery comprising of Rad6 (E2 conjugating enzyme) and Bre1 (E3 ligase) to give rise to condensed reaction chambers in vitro and is critical for maintaining optimal levels of ubiquitinated H2B in vivo. To elucidate the molecular mechanism of this process, single-molecule studies of reconstituted nucleosomal arrays, which have been used extensively to study chromatin and its interaction with different factors, can be done. We have optimized the reconstitution of nucleosome arrays using biotinylated λ phage DNA to generate a template to analyze how Lge1-Bre1 condensates encounter DNA that is in the form of chromatin.