Histone H2B monoubiquitination is a key modification that regulates chromatin accessibility and mediates histone crosstalk. The deposition and removal of this mark is highly dynamic and modulates different cellular processes ranging from transcription to DNA replication, repair, and heterochromatin silencing. In Saccharomyces cerevisiae , two deubiquitinating enzymes (DUBs), Ubp8 and Ubp10, play distinct roles in chromatin-mediated processes. Ubp10 promotes subtelomeric gene silencing and is required to modulate transcription in mid-coding regions. Bulk assays revealed that Ubp10 deubiquitination rate is slow when ubiquitin is incorporated into nucleosomes; however, its activity is stimulated by the histone chaperone FACT (facilitating chromatin transcription). FACT is responsible for maintaining the integrity of genic nucleosomes during transcriptional elongation, as well as ensuring heterochromatin spread by histone turnover regulation. The mechanism by which FACT promotes the activity of Ubp10 on nucleosomes is still unclear. Here, we used a three-color ubiquitinated nucleosome (Ub-Nuc) construct to directly monitor histone H2B deubiquitination and nucleosome conformational dynamics at a single molecule level. Unexpectedly, this assay revealed that Ubp10 deubiquitinates nucleosomes efficiently in a FACT-independent manner. Tethering Ubp10 to the surface through antibody interaction allowed real-time visualization of Ub-Nuc pulldown. Deubiquitination occurred almost immediately after Ubp10 interaction—within two seconds. Nucleosomes remained stably bound to Ubp10 even after ubiquitin removal (up to an hour). Bulk assays using streptavidin coated magnetic beads further confirm Ubp10 tethering to biotinylated nucleosomes and a robust DUB activity at equimolar concentrations. Notably, FACT is able to efficiently evict nucleosomes from surface immobilized Ubp10. Altogether, this study sheds light on the mechanism underlying FACT stimulation of Ubp10 and proposes that FACT promotes turnover by releasing Ubp10 from product inhibition following deubiquitination.
Urteaga et al. (Sun,) studied this question.