An intrinsically disordered region of Ubp10 regulates its binding and activity on ubiquitinated histone substrates.

Monoubiquitinated histone H2B at K123 in yeast (K120 in humans) is a transient modification that is both attached and removed during transcription. H2B is ubiquitinated in yeast by the E2/E3 pair, Rad6/Bre1, and deubiquitinated by two enzymes, Ubp8 and Ubp10. Previous studies had shown that Ubp10 has higher activity on ubiquitinated H2A/H2B dimers than on intact nucleosomes, but that activity on nucleosomes is higher in the presence of the histone chaperone, FACT. By contrast, the Ubp8 complex has equal activity on both histone substrates and is unaffected by FACT. We report here the results of single-molecule FRET experiments showing that FACT unwraps DNA and evicts ubiquitinated H2A/H2B dimers, the preferred substrate of Ubp10. To explore the basis for the differing activity of Ubp10 on ubiquitinated H2A/H2B dimers and nucleosomes, we employed crosslinking mass spectrometry combined with structural modeling. These studies revealed that Ubp10 forms a different set of interactions with H2A/H2B in free versus nucleosomal states. Acidic stretches within the N-terminal intrinsically disordered region (IDR) of Ubp10 interact extensively with H2A/H2B heterodimers, whereas this portion of Ubp10 interacts more with the tails of histones H3 and H4 in the nucleosome. The importance of these interactions for affinity is consistent with binding studies showing the IDR is necessary for substrate interactions. Structural modeling using the crosslinks as constraints suggests that the complex formed by Ubp10 with free H2A/H2B dimers could not be formed within a nucleosome due to steric clash with the DNA, H3, and H4, thereby explaining its low activity on ubiquitinated nucleosomes.