Insights into the Recruitment of Class IIa Histone Deacetylases (HDACs) to the SMRT/NCoR Transcriptional Repression Complex

Abstract

Class IIa histone deacetylases repress transcription of target genes. However, their mechanism of action is poorly understood because they exhibit very low levels of deacetylase activity. The class IIa HDACs are associated with the SMRT/NCoR repression complexes and this may, at least in part, account for their repressive activity. However, the molecular mechanism of recruitment to co-repressor proteins has yet to be established. Here we show that a repeated peptide motif present in both SMRT and NCoR is sufficient to mediate specific interaction, with micromolar affinity, with all the class IIa HDACs (HDACs 4, 5, 7, and 9). Mutations in the consensus motif abrogate binding. Mutational analysis of HDAC4 suggests that the peptide interacts in the vicinity of the active site of the enzyme and requires the "closed" conformation of the zinc-binding loop on the surface of the enzyme. Together these findings represent the first insights into the molecular mechanism of recruitment of class IIa HDACs to the SMRT/NCoR repression complexes.

Keywords: epigenetics; histone acetylation; histone deacetylase 4 (HDAC4); protein-protein interaction; transcription corepressor.

FIGURE 1.

A repeated sequence motif within the class IIa HDAC binding region of SMRT/NCOR co-repressors. A, domain arrangement of SMRT indicating regions known to interact with partner proteins and schematic of RD3 of SMRT/NCoR showing the location of identified GSI repeats and the BCL6 binding domain with a similar arrangement and spacing. B, Weblogo plot of RD3 GSI motifs from SMRT and NCoR of 6 species demonstrates a well conserved 8 amino acid residue motif. NR, nuclear receptor binding region.

FIGURE 2.

Investigating the interaction between class IIa HDACs and a GSI motif peptide (CPRPLKEGSITQGTPLKYDTG). A, a peptide representative of a wild type GSI motif and 12 mutant peptides were fluorescently labeled for use in fluorescence anisotropy to define and characterize the interaction between the peptide and HDAC4. B, fluorescence anisotropy was performed using the wild type peptide with the catalytic domains of class IIa HDACs HDAC4, HDAC5, HDAC7, and HDAC9 and the class I HDAC8.

FIGURE 3.

Comparison of the closed (A) and open (B) loop conformations of HDAC4. Views of a wild type HDAC4 structure (Protein Data Bank code 2vqj) with an open loop conformation (cyan) and an L728A mutant structure (Protein Data Bank code 4cby), which demonstrates a closed loop conformation (green). Side chains of the residues discussed in the text are highlighted in magenta. Zinc atoms are colored yellow. C, fluorescence anisotropy binding experiments of GSI peptide to wild type HDAC4cd, L728A mutant, which is reported to stabilize the class IIa loop, and three mutants of the zinc chelating residues, C667A, H675A, and C751A.

FIGURE 4.

Investigating the effect of inhibitors on the HDAC4-GSI interaction. A, fluorescence anisotropy of wild type (WT) and the H976Y gain-of-function (GOF) HDAC4 catalytic domain with 2-fold molar excess of different HDAC inhibitors. B, schematic representation of how the different HDAC inhibitors occupy the active site.

FIGURE 5.

Investigating the effect of co-repressor interaction on class IIa HDAC activity. To determine whether interaction with co-repressor affects the deacetylation potential of the class IIa HDACs, an assay was performed using 5 μm HDAC4 catalytic domain in the presence and absence of GSI peptide (20:1) and inhibitor (2:1) compared with 5 μg of HeLa nuclear extract. NE, nuclear extract; WT, wild type HDAC4; GSI, GSI motif peptide; TSA, HDAC inhibitor trichostatin A.