A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

Abstract

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimicks class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

GSE1 links the HDAC1/CoREST co-repressor complex to DNA damage

“…When comparing global acetylation patterns of HDAC1 CI to GSE1 KO cells, we found 102 sites of different abundance (fold change ≥ 2, adjusted p-value ≤ 0.01), suggesting that the mutants have to a certain extent individual effects on the acetylome (Supplementary Figure 3C). In line with this, recently described substrate sites of HDAC1 (36) show higher acetylation levels in the HDAC1 CI mutant (Supplementary Figure 3C), which also supports our observation that GSE1 does not affect HDAC activity (Figure 2D). We next wanted to get an insight into DNA damage-induced processes that are commonly dysregulated in GSE1 KO and HDAC1 CI.…”

“…Cell extract and mass spectrometry sample preparation. MS sample preparation was performed as previously described (36). Briefly, 2 x 10 7 cells per replicate were resuspended in lysis buffer (8 M urea (VWR, 0568-500G), 50 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM PMSF, 5 mM sodium butyrate (Sigma-Aldrich,303410), 10 ng/ml NAM (Sigma Aldrich, 72340), 10 ng/ml trichostatin A (TSA) (Sigma Aldrich, T8552-1MG), 1x cOmplete protease inhibitor cocktail (+EDTA) (Roche, 11697498001), 250 U/replicate benzonase (Merck, 1.01695.0001)), lysed using a Bioruptor sonication device (Diagenode) (settings: 30 sec sonication (power level H, 30 sec cooling, 5 cycles), centrifuged at 15,000 x g for 10 min at 4°C and precipitated with 4x volumes of cold (-20°C) 100% acetone (overnight, 4°C).…”

“…Acetylated peptide enrichment. Acetylated peptides were enriched, washed, eluted and desalted as described in (36), using the PTMScan Acetyl-Lysine Motif Kit (Cell Signaling Technology, 11663V1). The flow-through of the Acetyl-Lysine immunoprecipitation was kept for phosphorylated peptide enrichment.…”

DOT1L inhibition does not modify the sensitivity of cutaneous T cell lymphoma to pan-HDAC inhibitors in vitro

“…To investigate this possibility, more specific inhibitors could be used in future studies. Class-I specific HDAC inhibitors are available for such studies, but inhibiting HDAC1 or other specific class-I HDACs remains a challenge ( Schölz et al, 2015 ; Eckschlager et al, 2017 ; Shortt et al, 2017 ; Pride and Summers, 2018 ; Hess et al, 2022 ; Siklos and Kubicek, 2022 ). Interestingly, recent efforts have led to the development of more selective HDAC inhibitors, some of which inhibit certain class-I HDACs more than others ( Yang et al, 2019 ; Hess et al, 2022 ; Siklos and Kubicek, 2022 ).…”

Targeting the catalytic activity of HDAC1 in T cells protects against experimental autoimmune encephalomyelitis

“…Expression of HDAC1 On and HDAC1 Off proteins was confirmed upon transient transfection in HeLa cells and stable expression in HAP1 cells [31]. The ORFs encoding FLAG-tagged HDAC1 WT and HDAC1 CI were cloned into the exchange vector pBTV2-11.…”