Investigating the roles of HDAC1/2 in regulating the embryonic stem cell acetylome

Histone deacetylase 1 (HDAC1) and HDAC2 are critical catalytic subunits of six distinct coregulator complexes (SIN3, NuRD, CoREST, MiDAC, MIER, and RERE) that help regulate histone acetylation levels across the genome. One of the major unanswered questions in the HDAC field is why the cell needs such an array of HDAC-containing complexes? To try to understand how these different molecular machines are assembled and what they all do, we have been using the structure of HDAC1 bound to MTA1 (part of the NuRD complex) to design mutations (Y48E, L161E/Y166E, Y333D/Y336D, E63R, K126E, and K144E) on the surface of HDAC1 that discriminate binding to the different complexes. We have identified two HDAC1 mutants, Y48E, which binds only to SIN3 and E63R which interacts with SIN3 and CoREST complexes but not with NuRD or MiDAC. Surprisingly, retention of SIN3 binding alone is sufficient for cells to retain their viability, demonstrating the essential nature of this complex. Both Y48E and E63R cause differential gene expression and a gradual loss of pluripotency in embryonic stem cells. As a result, we developed new tools and the first-of-their-kind mutations that will help in distinguishing the functions of these various chromatin-modifying machinery. The MOZ/KAT6B complex provides an image of a chromatin-binding assembly that can be modified in terms of its activity and recruitment to particular genomic areas through post-translational modifications of histones. Here we validated the acetylation of two members of the KAT6B complex, KAT6B and BRPF1. We have demonstrated that BRPF1 alone is not acetylated, and its expression level is low. But when we co-transfected it with KAT6B, we were able to detect an acetylation signal. Moreover, the expression level increased with the presence of KAT6B, suggesting that KAT6B does not only acetylate BRPF1, it might also stabilise it. Additionally, KAT6B’s autoacetylation behaviour prevented us from switching off the acetylation signal in both targets.