Identification of histone deacetylase (HDAC)-associated proteins with DNA-programmed affinity labeling

Abstract

Histone deacetylase (HDAC) is a large class of deacetylation enzymes that perform important roles in gene transcription control, as well as many other cellular processes. Many HDACs reside in large protein complexes in cells, and the biological roles of HDAC are highly contingent on the complex’s unique composition. Identification of HDAC-associated proteins is also of considerable significance for studying the molecular function of HDACs. Previously, small-molecule-based affinity tests were used to classify HDAC complexes. Nevertheless, these experiments were developed specifically to engage the primary target HDAC. Identification of other proteins in the complex remains relatively undeveloped. In fact, conventional affinity probes have a fixed design, so the proteins that they may reach are constrained by the configuration of the probe. Here we present a DNA-based affinity labeling method able to provide variable probe configurations without the need for multiple probes to be prepared. To illustrate the efficiency of this process, we developed 9 separate probe configurations for profiling HDAC-associated proteins using just one single binding probe. A significant number of possible novel HDAC interactors have been found by the DNA-based probes, which are markedly different from those found by small molecule-based probes. In particular, we demonstrate that the proposed method is ideal for the detection of indirect HDAC binders which could be challenging to reach with conventional affinity probes. Lastly, the approach also identified important biological implications in various cellular contexts for the HDAC-associated proteins. This research presented a clear and commonly applicable approach for characterizing protein complexes and interactions between protein and protein, complementing conventional small-molecule-based affinity studies.