Tom1p ubiquitin ligase structure, interaction with Spt6p, and function in maintaining normal transcript levels and the stability of chromatin in promoters

Abstract

Phosphorylation-dependent binding of the S. cerevisiae Spt6p tSH2 domain (Spt6ptSH2) to the Rbp1p subunit of RNA polymerase II supports efficient transcription. Here, we report that Spt6ptSH2 also binds the HECT-family E3 ubiquitin ligase Tom1p, a homolog of human HUWE1. Tom1p/HUWE1 have been implicated in targeting many small basic proteins for degradation, including excess ribosomal subunits and histones, although the mechanism of substrate recognition is not known. Our cryo-EM data revealed that Tom1p can adopt a compact -solenoidal "basket" similar to the previously described structure of HUWE1, with the central cavity partially occupied by a disordered acidic domain. Sub-regions of this acidic domain supported binding to Spt6p or histones/nucleosomes in vitro, and the histone-binding region was important for Tom1p function in vivo. We also visualized Tom1p in more extended forms, and speculate that transitions among these forms could be important for substrate selection and ubiquitylation. Genomic analyses provided additional support for the previously observed role for Tom1p in maintaining ribosomal protein pools, and also demonstrated a role in maintaining chromatin structure near genes. This suggests that the interaction with Spt6ptSH2 affects substrate specificity by anchoring Tom1p to localized environments where histone ubiquitylation alters chromatin architecture.