Dynamic and multivalent engagement determines context-dependent nucleosomal deacetylation by the Rpd3S complex

Abstract Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation. The Rpd3S complex recognizes H3K36 trimethylation (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions. Here we solved the cryo-EM structures of yeast Rpd3S in its free and H3K36me3-nucleosome bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3-Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex. Multivalent recognition of two H3K36me3 marks, nucleosomal and linker DNAs by Eaf3, Sin3 and Rco1 positions the catalytic center of Rpd3 next to the histone H4 N-terminal tail for deacetylation. In an alternative catalytic mode, combinatorial readout of unmethylated H3K4 and H3K36me3 by Rco1 and Eaf3 directs histone H3-specific deacetylation except for the registered H3K9ac. Collectively, our work illustrates dynamic and diverse modes of multivalent nucleosomal engagement and methylation-guided deacetylation by Rpd3S, highlighting the exquisite complexity of epigenetic regulation with delicately designed multi-subunit enzymatic machineries in transcription and beyond.