A novel yeast histone deacetylase: partial characterization and development of an activity assay

We have characterized a histone deacetylase activity associated with yeast nuclei. An unusual feature of the deacetylase is that it is not inhibited by the short-chain fatty acids n-butyrate and propionate. These short-chain fatty acids are typically potent inhibitors of histone deacetylases in eukaryotic systems. The deacetylase(s) were detected by monitoring the levels of acetylation of yeast histones during incubation of isolated yeast nuclei. The activity was optimal at 37 degrees C and at 0.1 M NaCl. The enzyme did not require divalent cations and was inhibited by Zn2+ and Cu2+. A simple activity assay was developed using as substrate, [3H]acetate-labeled histone in chicken erythrocyte nuclei. This assay was used to demonstrate that the deacetylase(s) can be extracted from yeast nuclei with 0.5 M NaCl. A gel electrophoretic analysis of the deacetylated chicken histones verified that the solubilization of incorporated radiolabel was a result of histone deacetylation, not an artifact of histone degradation by yeast proteinases.     

An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.     

Characterization of pea histone deacetylases

The present paper is the first report on histone deacetylases from plants. Three enzyme fractions with histone deacetylase activity (HD0, HD1 and HD2) have been partially purified from pea (Pisum sativum) embryonic axes. They deacetylate biologically acetylated chicken histones and, to a lesser extent, chemically acetylated histones, this being a criterion of their true histone deacetylase nature. The three enzymes are able to accept nucleosomes as substrates. HD1 is not inhibited by n-butyrate up to 50 mM, whereas HD0 and HD2 are only slightly inhibited, thereby establishing a clear difference to animal histone deacetylases. The three activities are inhibited by acetate, Cu²⁺ and Zn²⁺ ions and mercurials, but are only scarcely affected by polyamines, in strong contrast with yeast histone deacetylase. Several criteria have been used to obtain cumulative evidence that HD0, HD1 and HD2 actually are three distinct enzymes. In vitro experiments with free histones show that HD0 deacetylates all four core histones, whereas HD1 and HD2 show a clear preference for H2A and H2B, the arginine-rich histones being deacetylated more slowly.     

Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free 3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.     

Effect of valproic acid on radiation-induced DNA damage in euchromatic and heterochromatic compartments

The distinction between heterochromatin and euchromatin in the double-strand break (DSB) damage pathway is of interest, recent reports indicate that chromatin is not created equally nor is it acquiescent to DSBs. Using the classical histone deacetylase inhibitor, Trichostatin A, we have previously demonstrated that chromatin represents a heterogeneous substrate with respect to histone tail modification by histone deacetylase inhibitors and consequent responses to DNA damage and repair. Here, we extended the initial findings by investigating the radiation sensitizing properties of the widely used antiepileptic, valproic acid. Clonogenic survival assays confirm that valproic acid is an efficient sensitizer of radiation-induced cell death. The radiosensitizing effect is correlated with valproic acid-mediated histone hyperacetylation, chromatin decondensation and enhanced formation of radiation-induced γH2AX preferentially on euchromatic alleles. Heterochromatin was much more resistant to histone tail modification, changes in chromatin architecture and DNA damage. These findings are consolidated by studies with the structurally related analogue, valpromide, which does not inhibit histone deacetylase enzymes. At a relatively low concentration (1 mM) valpromide did not cause chromatin modifications and radiation sensitivity, providing further evidence that the radiation sensitizing properties of valproic acid are at least in part, due to histone modification-dependent effects on euchromatin. When higher concentrations (5 mM) were used, both compounds resulted in significant radiation sensitivity, albeit, with differing efficacy (dose modifying factors of 1.5 and 1.2 for valproic acid and valpromide, respectively). The findings imply that histone-modification independent mechanisms also contribute to the radiation sensitizing properties of valproic acid. Overall, our findings are consistent with the emerging interest in the use histone deacetylase inhibitors in combination with radiotherapy for the treatment of cancer.     

Interaction between dexamethasone and butyrate in apoptosis induction: non-additive in thymocytes and synergistic in a T cell-derived leukemia cell line.

In thymocytes butyrate and trichostatin A are unable to augment dexamethasone-induced apoptosis. In cultured rat thymocytes the extent of apoptosis induced by dexamethasone alone did not increase by addition of 0.1 - 10 mM butyrate. Even more pronounced was the non-additive interrelationship between dexamethasone and trichostatin A, as trichostatin A-induced apoptosis was not only blocked by the presence of dexamethasone but dexamethasone-induced apoptosis was also partially inhibited in the presence of 0.1 - 0.5 microM trichostatin A. The fact that the non-additive relationship with dexamethasone for apoptosis induction was observed with both histone deacetylase inhibitors suggests that in thymocytes this phenomenon is related to histone acetylation. In contrast to this, in the human T cell-derived leukemia cell line CEM-C7H2, dexamethasone did not block butyrate- or trichostatin A-induced apoptosis; moreover, butyrate, in the concentration range of 0.1 - 1 mM, had a marked synergistic effect on dexamethasone-induced apoptosis. This synergism, however, was not mimicked by trichostatin A, indicating that the effect is not related to histone acetylation but rather due to a pleiotropic effect of butyrate. Furthermore, in CEM-C7H2 cells, at higher concentrations of butyrate (5 - 10 mM) or trichostatin A (0.4 - 0.8 microM), there was a minor but reproducible antagonistic effect of dexamethasone on apoptosis induced by each of the two histone deacetylase inhibitors, suggesting that this antagonistic effect too, is related to histone hyperacetylation.     

Inhibitor-mediated stabilization of the conformational structure of a histone deacetylase-like amidohydrolase

Histone deacetylases are major regulators of eukaryotic gene expression. Not unexpectedly, histone deacetylases are among the most promising targets in cancer therapy. However, despite huge efforts in histone deacetylase inhibitor design, very little is known about the impact of histone deacetylase inhibitors on enzyme stability. In this study, the conformational stability of a well-established histone deacetylase homolog with high structural similarity (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188) was investigated using denaturation titrations and stopped-flow kinetics. Based on the results of these complementary approaches, we conclude that the interconversion of native histone deacetylase-like amidohydrolase into its denatured form involves several intermediates possessing different enzyme activities and conformational structures. The refolding kinetics has shown to be strongly dependent on Zn(2+) and to a lesser extent on K(+), which underlines their importance not only for catalytic function but also for maintaining the correct conformational structure of the enzyme. Two main unfolding processes of histone deacetylase-like amidohydrolase were differentiated. The unfolding occurring at submolar concentrations of the denaturant guanidine hydrochloride was not affected by inhibitor binding, whereas the unfolding at higher concentrations of guanidine hydrochloride was strongly affected. It was shown that the known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate are capable of stabilizing the conformational structure of histone deacetylase-like amidrohydrolase. Judging from the free energies of unfolding, the protein stability was increased by 9.4 and 5.4 kJ.mol(-1) upon binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate, respectively.     

Subcellular localization and nucleosome specificity of yeast histone acetyltransferases

We have previously reported [López-Rodas et al. (1989) J. Biol. Chem. 264, 19028-19033] that the yeast Saccharomyces cerevisiae contains four histone acetyltransferases, which can be resolved by ion-exchange chromatography, and their specificity toward yeast free histones was studied. In the present contribution we show that three of the enzymes are nuclear, type A histone acetyltransferases and they are able to acetylate nucleosome-bound histones. They differ in their histone specificity. Enzyme A1 acetylates H2A in chicken nucleosomes, although it is specific for yeast free H2B; histone acetyltransferase A2 is highly specific for H3, and histone acetyltransferase A3 preparations acetylate both H3 and H4 in nucleosomes. The fourth enzyme, which is located in the cytoplasm, does not accept nucleosomes as substrate, and it represents a canonical type B, H4-specific histone acetyltransferase. Finally, histone deacetylase activity is preferentially found in the nucleus.     

Alpha-keto amides as inhibitors of histone deacetylase

Alpha-keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The alpha-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.     

Epigenetic upregulation of Bak by ZBP-89 inhibits the growth of hepatocellular carcinoma

Zinc-binding protein-89 regulates Bak to facilitate apoptosis in cancer cells. This study examined if zinc-binding protein-89 regulates Bak through an epigenetic mechanism in hepatocellular carcinoma. We first demonstrated that the expression of Bak was reduced but the levels of deoxyribonucleic acid methyltransferase 1 and histone deacetylase 3 were increased in hepatocellular carcinoma cancer tissues compared to the corresponding non-cancer tissues. Moreover, there was a negative correlation between Bak expression and deoxyribonucleic acid methyltransferase 1 levels in hepatocellular carcinoma. Administration of zinc-binding protein-89 downregulated histone deacetylase 3 expression and suppressed the activities of histone deacetylase and deoxyribonucleic acid methyltransferase, which led to maintenance of histone acetylation status, inhibited the binding of methyl-CpG-binding protein 2 to genomic deoxyribonucleic acid and demethylated CpG islands in the Bak promoter in hepatocellular carcinoma cells. Using the xenograft mouse tumor model, we demonstrated that zinc-binding protein-89 or inhibitors of either epigenetic enzymes could stimulate Bak expression, induce apoptosis, and arrest tumor growth and that the maximal effort was achieved when zinc-binding protein-89 and the enzyme inhibitors were used in combination. Conclusively, zinc-binding protein-89 upregulates the expression of Bak by targeting multiple components of the epigenetic pathway in hepatocellular carcinoma.     

Functional interplay between histone demethylase and deacetylase enzymes

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.     

Casein kinase 2 inhibition differentially modulates apoptotic effect of trichostatin A against epithelial ovarian carcinoma cell lines

Histone deacetylase inhibitors and casein kinase 2 inhibitors have been shown to induce apoptosis. However, the combined effect of casein kinase 2 inhibition on the apoptotic effect of histone deacetylase inhibitor is unknown. We assessed the effect of casein kinase 2 inhibition on the apoptotic effect of trichostatin A in human epithelial carcinoma cell lines with respect to cell death signaling pathways. At concentrations that did not induce cell death, the casein kinase 2 inhibitor 4,5,6,7-tetrabromobenzotriazole inhibited activation of apoptotic proteins and changes in mitochondrial membrane permeability induced by the histone deacetylase inhibitor trichostatin A. These results suggest that casein kinase 2 inhibition may reduce trichostatin A-induced apoptosis in ovarian carcinoma cell lines by suppressing activation of apoptotic proteins and changes in mitochondrial membrane permeability, which both lead to caspase-3 activation. Casein kinase 2 inhibition, which does not induce a cytotoxic effect, may prevent histone deacetylase inhibitor-mediated apoptosis.     

Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.