Bhat, Y. A. | Search bioRxiv

bioRxiv2026

Functional interplay between H2B ubiquitylation and H2A.Z deposition

The Bre1 ubiquitin conjugating enzyme catalyzes the monoubiquitylation of histone H2B-K120/K123 at promoter proximal nucleosomes, contributing to transcriptional regulation. These same nucleosomes are targeted by the yeast SWR1C chromatin remodeling enzyme that deposits the histone variant H2A.Z. Yeast strains that lack both Bre1 and Swr1 are inviable, indicating that together they contribute to an essential cell function. Interestingly, H2B-K123ub and H2A.Z levels are anticorrelated, and recent biochemical studies suggest a model in which H2B-K123ub inhibits SWR1C activity by blocking access to the nucleosome acidic patch. Here, we exploit recombinant nucleosomes to show that the H2A.Z deposition activity of SWR1C is strongly inhibited by H2B-K123ub. Surprisingly, the loss of H2B-K123ub does not lead to significant changes in the genomic organization of H2A.Z in cells grown in normal media, but we find that H2B-K123ub is required for a re-localization of H2A.Z from promoters to coding regions during replication stress. Together, these data indicate a complex functional interplay between H2B ubiquitylation and H2A.Z deposition.

biochemistry↗

bioRxiv2023

HDX-MS reveals concealed conformations of ISWI during different stages of nucleosome sliding

Nucleosome spacing across the genome is regulated by the adenosine 5-triphosphate (ATP)- dependent nucleosome sliding activity of Imitation Switch, ISWI. ISWI is believed to be auto-inhibited in the resting-state by the binding of its N-and C-terminal regulatory regions to its central ATPase-domain, attaining a "closed" conformation. To slide nucleosomes ISWI must i) transition to the state competent for nucleosome binding, ii) bind to nucleosome and iii) carry the ATP-dependent nucleosome sliding. The conformations attained by full-length ISWI (FL-ISWI) during the entire sliding process have remained inaccessible by the methods used so far. Using Hydrogen/Deuterium-exchange coupled to Mass-Spectrometry (HDX-MS), we monitored conformational dynamics of the Drosophila FL-ISWI at all the stages of sliding process. HDX-MS data show that in the resting state, ISWI samples an ensemble of conformations showing varying levels of deuterium uptake in many regions including N-and C-terminal regulatory regions, suggesting ISWI intrinsically samples relatively "open-states". In addition to substantiating previous nucleosome binding studies, HDX-MS reveals that during actual sliding-step, regions of ATPase-domain which bind to the nucleosomal DNA undergo major conformational change. The C-terminal HSS domain switches from the solvent protected stable state to a more dynamic state, implying several interactions established by ISWI with the nucleosome upon binding are relieved during sliding. In sum, this study provides mechanistic insights into how ISWI can switch from an auto-inhibited "closed-state" to an "open-state" competent for nucleosome binding, and reveals the conformation attained by ISWI during the actual nucleosome sliding step. We propose that, like ISWI, intrinsic dynamics may be involved in functioning of other Rec-like ATPase-domain containing protein families.

biochemistry↗