Quantitative mass spectrometry analysis of Rpd3L histone deacetylase activity on mono- and di-nucleosome substrates

The Rpd3L (Rpd3 Large) complex is a Sin3 family histone deacetylase (HDAC) complex that dynamically adapts to chromatin states to reinforce transcriptional silencing, yet the mechanisms governing its catalytic activation in a chromatin context-dependent manner remain unclear. In the preliminary stage of this study, we resolved the near-atomic resolution cryo-EM structures of Rpd3L bound to mono-nucleosome and di-nucleosome substrates, revealing a substrate-guided allosteric activation mechanism.

To validate the functional hypotheses proposed by the structural models, we performed quantitative proteomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, mass spectrometry was used to examine the histone deacetylase activity and substrate preference of the Rpd3L complex under different nucleosome substrate conditions. The experimental results showed that, compared with mono-nucleosome binding, simultaneous binding of di-nucleosomes significantly broadened the substrate specificity of Rpd3L towards different histone sites.