TRiC-assisted folding of class I HDAC family proteins regulated by distinct co-chaperone and cofactor networks.

Class I histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC8) are key chromatin regulators, but how they are activated by chaperonin TRiC remains elusive. Using cryo-electron microscopy, cross-linking mass spectrometry, and biochemistry analyses of tagged HDACs overexpressed in HEK293F cells, we identify class I HDACs as TRiC substrates and elucidate the TRiC-assisted folding pathways of HDAC1 and HDAC3 across ATPase cycle, orchestrated by distinct co-chaperone/cofactor networks. In closed TRiC chamber, both clients adopt near-native conformations and engage similar binding interfaces. In the open state, however, their pathways diverge: HDAC3 involves Hsp70 atop TRiC and PDCD5 within the chamber, whereas HDAC1 involves prefoldin atop TRiC, revealing distinct mechanisms of substrate delivery and folding modulation. We also identify an unexpected bent conformation of CCT4 in TRiC-HDAC1 complex that may relate to co-chaperone release. By contrast, HDAC8 folds independently of TRiC. Together, these findings reveal client-specific co-chaperone/cofactor networks governing TRiC-assisted folding of class I HDACs, shedding light on the sophisticated regulatory landscape of TRiC.