Differential HDAC1/2 network analysis reveals a role for prefoldin/CCT in HDAC1/2 complex assembly

Charles A S Banks,Sayem Miah,Michael P Washburn,Laurence Florens,Cassandra G Eubanks,Mark K Adams,Janet L Thornton

doi:10.1038/s41598-018-32009-w

Charles A S Banks, Sayem Miah + Show 5 more

Open Access

https://doi.org/10.1038/s41598-018-32009-w

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Abstract

HDAC1 and HDAC2 are components of several corepressor complexes (NuRD, Sin3, CoREST and MiDAC) that regulate transcription by deacetylating histones resulting in a more compact chromatin environment. This limits access of transcriptional machinery to genes and silences transcription. While using an AP-MS approach to map HDAC1/2 protein interaction networks, we noticed that N-terminally tagged versions of HDAC1 and HDAC2 did not assemble into HDAC corepressor complexes as expected, but instead appeared to be stalled with components of the prefoldin-CCT chaperonin pathway. These N-terminally tagged HDACs were also catalytically inactive. In contrast to the N-terminally tagged HDACs, C-terminally tagged HDAC1 and HDAC2 captured complete histone deacetylase complexes and the purified proteins had deacetylation activity that could be inhibited by SAHA (Vorinostat), a Class I/II HDAC inhibitor. This tag-mediated reprogramming of the HDAC1/2 protein interaction network suggests a mechanism whereby HDAC1 is first loaded into the CCT complex by prefoldin to complete folding, and then assembled into active, functional HDAC complexes. Imaging revealed that the prefoldin subunit VBP1 colocalises with nuclear HDAC1, suggesting that delivery of HDAC1 to the CCT complex happens in the nucleus.

Highlights

Levels of gene expression can be modulated by controlling the acetylation state of lysine residues within unstructured histone N-terminal tails
To investigate protein complexes associating with human HDAC1 using Halo affinity chromatography, we first examined the structure of HDAC1 to assess whether either terminus of HDAC1 appeared to be accessible for tagging
We have been able to isolate different populations of HDAC1 bound preferentially to either the chaperonin containing T (CCT) complex or assembled into active histone deacetylase complexes depending on the placement of the Halo affinity tag used for isolation

Summary

Results

To investigate protein complexes associating with human HDAC1 using Halo affinity chromatography, we first examined the structure of HDAC1 to assess whether either terminus of HDAC1 appeared to be accessible for tagging. To identify HDAC1 interaction partners, we affinity purified protein complexes using Halo-HDAC1 and HDAC1-Halo expressed in HeLa cells and first analysed the copurifying proteins by SDS PAGE and silver staining (Fig. 1C) Both Halo-HDAC1 and HDAC1-Halo preparations contained factors not present in the control, there appeared to be distinct differences between the populations of proteins detected with each HDAC1 bait. We used Halo-HDAC2 and HDAC2-Halo to capture protein complexes from HeLa cells as we had done for HDAC1 and quantified the relative amounts of Sin[3], NuRD, CCT, and prefoldin, complex subunits in each purification by calculating dBNSAF values as before. As we had seen previously in experiments using tagged versions of either HDAC1 or HDAC2 in HeLa cells, relatively larger amounts of NuRD complex subunits copurified with C terminally tagged HDAC1-Halo in HEK293T cells compared with the amounts copurifying with N-terminally tagged Halo-HDAC1 (Fig. 3C, Supplementary Tables S3–S5). An expanded explanation of this differential interaction network analysis can be found in

Discussion

Materials and Methods

Data Availability Statement