Abstract
The Rpd3S histone deacetylase complex utilizes two subunits, Eaf3 and Rco1, to recognize nucleosomes methylated at H3K36 (H3K36me) with high affinity and strong specificity. However, the chromobarrel domain of Eaf3 (CHD) that is responsible for H3K36me recognition only binds weakly and with little specificity to histone peptides. Here, using deuterium exchange mass spectrometry (DXMS), we detected conformational changes of Rpd3S upon its contact with chromatin. Interestingly, we found that the Sin3-interacting domain of Rco1 (SID) allosterically stimulates preferential binding of Eaf3 to H3K36-methylated peptides. This activation is tightly regulated by an autoinhibitory mechanism to ensure optimal multivalent engagement of Rpd3S with nucleosomes. Lastly, we identified mutations at the interface between SID and Eaf3 that do not disrupt complex integrity but severely compromise Rpd3S functions in vitro and in vivo, suggesting that the nucleosome-induced conformational changes are essential for chromatin recognition.