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.     

Plant histone acetylation: in the beginning ..

The study of histone acetylation in plants started with protein purification and sequencing, with gel analysis and the use of radioactive tracers. In alfalfa, acid urea Triton gel electrophoresis and in vivo labeling with tritated acetate and lysine quantified dynamic acetylation of core histones and identified the replication-coupled and -independent expression patterns of the histone H3.1 and H3.2 variants. Pulse-chase analyses demonstrated protein turnover of newly synthesized histone H3.2 and thereby identified the replacement H3 histones of plants which maintain the nucleosome density of transcribed chromatin. Sequence analysis of histone H4 revealed acetylation of lysine 20, a site typically methylated in animals and yeasts. Histone deacetylase inhibitors butyrate and trichostatin A are metabolized in alfalfa, but loss of TSA is slow, allowing its use to induce transient hyperacetylation of histones H2B, H4 and H3. This article is part of a Special Issue entitled: Epigenetic Control of cellular and developmental processes in plants.     

Regulation of histone acetylation during memory formation in the hippocampus

Formation of long term memory begins with the activation of many disparate signaling pathways that ultimately impinge on the cellular mechanisms regulating gene expression. We investigated whether mechanisms regulating chromatin structure were activated during the early stages of long term memory formation in the hippocampus. Specifically, we investigated hippocampal histone acetylation during the initial stages of consolidation of long term association memories in a contextual fear conditioning paradigm. Acetylation of histone H3 in area CA1 of the hippocampus was regulated in contextual fear conditioning, an effect dependent on activation of N-methyl-D-aspartic acid (NMDA) receptors and ERK, and blocked using a behavioral latent inhibition paradigm. Activation of NMDA receptors in area CA1 in vitro increased acetylation of histone H3, and this effect was blocked by inhibition of ERK signaling. Moreover, activation of ERK in area CA1 in vitro through either the protein kinase C or protein kinase A pathways, biochemical events known to be involved in long term memory formation, also increased histone H3 acetylation. Furthermore, we observed that elevating levels of histone acetylation through the use of the histone deacetylase inhibitors trichostatin A or sodium butyrate enhanced induction of long term potentiation at Schaffer-collateral synapses in area CA1 of the hippocampus, a candidate mechanism contributing to long term memory formation in vivo. In concert with our findings in vitro, injection of animals with sodium butyrate prior to contextual fear conditioning enhanced formation of long term memory. These results indicate that histone-associated heterochromatin undergoes changes in structure during the formation of long term memory. Mimicking memory-associated changes in heterochromatin enhances a cellular process thought to underlie long term memory formation, hippocampal long term potentiation, and memory formation itself.     

Acetylation of the Entamoeba histone H4 N-terminal domain is influenced by short-chain fatty acids that enter trophozoites in a pH-dependent manner

Treatment of higher eukaryotic cells with short-chain fatty acids (SCFA) such as butyrate causes decreased levels of histone deacetylase (HDAC) activity and hyperacetylation of histones, and thereby affects gene expression, cell growth and differentiation. Entamoeba parasites encounter high levels of SCFA in the host colon, and in vitro these compounds allow trophozoite stage parasites to multiply but prevent their differentiation into infectious cysts. The Entamoeba invadens IP-1 histone H4 protein has an unusual number of lysines in its N-terminus, and these become hyperacetylated in trophozoites exposed to the HDAC inhibitors trichostatin A (TSA) or HC-toxin, but not in trophozoites exposed to butyrate. We have now found that several other commonly studied isolates of Entamoeba parasites also have an extended set of histone H4 acetylation sites that become hyperacetylated in response to TSA, but hypoacetylated in response to butyrate, suggesting an unusual sensitivity of this parasite's histone modifying enzymes to SCFA. Butyrate was found to enter trophozoites in a pH-dependent manner consistent with diffusive entry of the un-ionised form of the fatty acid into the amoebae. Transit of the Entamoeba organism through areas of the host intestine with distinct pH and SCFA concentrations would therefore result in very different levels of SCFA within the parasite. Entamoeba appears to have acquired unique alterations of its histone acetylation mechanism that may allow for its growth in the presence of varying amounts of the bacterial fermentation products.     

Core histone acetylation is regulated by linker histone stoichiometry in vivo

We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo.     

K562 human erythroleukemia cell variants resistant to growth inhibition by butyrate have deficient histone acetylation

K562 is an established human erythroleukemia cell line, inducible for hemoglobin synthesis by a variety of compounds including n-butyrate. To elucidate the role of butyrate-induced histone acetylation in the regulation of gene expression in K562 cells, we isolated 20 variants resistant to the growth inhibitory effect of butyrate. Four variants having different degrees of resistance were selected for detailed study. All four were found to be resistant to the hemoglobin-inducing effect of butyrate, suggesting that the two aspects of butyrate response, restriction of growth and induction of hemoglobin synthesis, are coupled. Further, after (5 days) culture with butyrate, two of the four variants exhibit less acetylation of H3 and H4 histones than does the butyrate-treated parent. Analysis of histone deacetylases from the variants indicated that each variant was distinct and that butyrate resistance may be accounted for by decreased affinity of the variant enzymes for butyrate, increased affinity of the enzymes for acetylated histone, or both. The fact that variants selected for resistance to growth inhibition by butyrate are also deficient in butyrate-induced hemoglobin synthesis and have abnormal histone deacetylase activity argues for butyrate inducing K562 cells to synthesize hemoglobin and restrict growth via histone acetylation.     

Differentiation of eosinophilic leukemia EoL-1 cells into eosinophils induced by histone deacetylase inhibitors

EoL-1 cells differentiate into eosinophils in the presence of n-butyrate, but the mechanism has remained to be elucidated. Because n-butyrate can inhibit histone deacetylases, we hypothesized that the inhibition of histone deacetylases induces the differentiation of EoL-1 cells into eosinophils. In this study, using n-butyrate and two other histone deacetylase inhibitors, apicidin and trichostatin A, we have analyzed the relationship between the inhibition of histone deacetylases and the differentiation into eosinophils in EoL-1 cells. It was demonstrated that apicidin and n-butyrate induced a continuous acetylation of histones H4 and H3, inhibited the proliferation of EoL-1 cells without attenuating the level of FIP1L1-PDGFRA mRNA, and induced the expression of markers for mature eosinophils such as integrin beta7, CCR1, and CCR3 on EoL-1 cells, while trichostatin A evoked a transient acetylation of histones and induced no differentiation into eosinophils. These findings suggest that the continuous inhibition of histone deacetylases in EoL-1 cells induces the differentiation into mature eosinophils.     

Role of histone deacetylation in cell-specific expression of endothelial nitric-oxide synthase

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5'-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2'-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.