Involvement of histone hypoacetylation in Ni<Superscript>2+</Superscript>-induced<Emphasis Type="Italic"> bcl</Emphasis>-<Emphasis Type="Italic">2</Emphasis> down-regulation and human hepatoma cell apoptosis

Although induction of cell apoptosis is known to be involved in the cytotoxicity of Ni(2+), little research has been aimed at the mechanism of Ni(2+)-induced apoptosis. Recent studies showed that Ni(2+) induces histone hypoacetylation in different cell lines. Since histone hypoacetylation plays important roles in the control of cell cycle progress and apoptosis, we hypothesized that histone hypoacetylation may be an unrevealed pathway in Ni(2+)-induced apoptosis. To address this, effects of Ni(2+) on cell apoptosis, bcl- 2 gene expression and histone acetylation were examined in human hepatoma Hep3B cells. We found that Ni(2+) treatment resulted in cell proliferation arrest, the appearance of detached cells, condensed chromatin, apoptotic bodies and specific DNA fragmentation, indicating the occurrence of cell apoptosis. At the same time, Ni(2+) induced a significant decrease in bcl- 2 expression and histone acetylation; the decrease of histone H4 acetylation in nucleosomes associated with the bcl- 2 promoter region was also proven by a chromatin immunoprecipitation assay, indicating the involvement of histone hypoacetylation in Ni(2+)-induced bcl- 2 down-regulation. Further studies showed that increasing histone acetylation by either 100 nM of trichostatin A or over-expressing histone acetyltranferase p300 in Hep3B cells obviously attenuated the bcl- 2 down-regulation and cell apoptosis caused by Ni(2+). Considering the importance of bcl- 2 in determining cell survival and apoptosis, the data presented here suggest that histone hypoacetylation may represent one unrevealed pathway in Ni(2+)-induced cell apoptosis, where bcl- 2 is one of its targets.     

Chromatin decondensation and nuclear reprogramming by nucleoplasmin

Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1beta and TIF1beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.     

Epigenetic aspects of HP1 exchange kinetics in apoptotic chromatin

Apoptotic bodies are the most condensed form of chromatin. In general, chromatin structure and function are mostly dictated by histone post-translational modifications. Thus, we have analyzed the histone signature in apoptotic cells, characterized by pronounced chromatin condensation. Here, H2B mono-acetylation, and H3K9 and H4 acetylation was significantly decreased in apoptotic cells, which maintained a high level of H3K9 methylation. This phenotype was independent of p53 function and distinct levels of anti-apoptotic Bcl2 protein. Interestingly, after etoposide treatment of leukemia and multiple myeloma cells, H3K9 and H4 hypoacetylation was accompanied by increased H3K9me2, but not H3K9me1 or H3K9me3. In adherent mouse fibroblasts, a high level of H3K9me3 and histone deacetylation in apoptotic bodies was likely responsible for the pronounced (∼40%) recovery of GFP-HP1α and GFP-HP1β after photobleaching. HP1 mobility in apoptotic cells appeared to be unique because limited exchange after photobleaching was observed for other epigenetically important proteins, including GFP-JMJD2b histone demethylase (∼10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (∼20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic.     

Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.     

Global assessment of combinatorial post-translational modification of core histones in yeast using contemporary mass spectrometry. LYS4 trimethylation correlates with degree of acetylation on the same H3 tail

A global view of all core histones in yeast is provided by tandem mass spectrometry of intact histones H2A, H2B, H4, and H3. This allowed detailed characterization of >50 distinct histone forms and their semiquantitative assessment in the deletion mutants gcn5Delta, spt7Delta, ahc1Delta, and rtg2Delta, affecting the chromatin remodeling complexes SAGA, SLIK, and ADA. The "top down" mass spectrometry approach detected dramatic decreases in acetylation on H3 and H2B in gcn5Delta cells versus wild type. For H3 in wild type cells, tandem mass spectrometry revealed a direct correlation between increases of Lys(4) trimethylation and the 0, 1, 2, and 3 acetylation states of histone H3. The results show a wide swing from 10 to 80% Lys(4) trimethylation levels on those H3 tails harboring 0 or 3 acetylations, respectively. Reciprocity between these chromatin marks was apparent, since gcn5Delta cells showed a 30% decrease in trimethylation levels on Lys(4) in addition to a decrease of acetylation levels on H3 in bulk chromatin. Deletion of Set1, the Lys(4) methyltransferase, was associated with the linked disappearance of both Lys(4) methylation and Lys(14) and Lys(18) or Lys(23) acetylation on H3. In sum, we have defined the "basis set" of histone forms present in yeast chromatin using a current mass spectrometric approach that both quickly profiles global changes and directly probes the connectivity of modifications on the same histone.     

Histone acetylation and gene expression analysis of sex lethal mutants in Drosophila

The evolution of sex determination mechanisms is often accompanied by reduction in dosage of genes on a whole chromosome. Under these circumstances, negatively acting regulatory genes would tend to double the expression of the genome, which produces compensation of the single-sex chromosome and increases autosomal gene expression. Previous work has suggested that to reduce the autosomal expression to the female level, these dosage effects are modified by a chromatin complex specific to males, which sequesters a histone acetylase to the X. The reduced autosomal histone 4 lysine 16 (H4Lys16) acetylation results in lowered autosomal expression, while the higher acetylation on the X is mitigated by the male-specific lethal complex, preventing overexpression. In this report, we examine how mutations in the principal sex determination gene, Sex lethal (Sxl), impact the H4 acetylation and gene expression on both the X and autosomes. When Sxl expression is missing in females, we find that the sequestration occurs concordantly with reductions in autosomal H4Lys16 acetylation and gene expression on the whole. When Sxl is ectopically expressed in Sxl(M) mutant males, the sequestration is disrupted, leading to an increase in autosomal H4Lys16 acetylation and overall gene expression. In both cases we find relatively little effect upon X chromosomal gene expression.     

State of histone modification in the rat Ig-beta/growth hormone locus.

The state of acetylation in H3 and H4 histones and dimethylation in the H3 histone Lys4 residue were examined by chromatin immunoprecipitation (ChIP) at 11 targets in the rat Ig-beta/growth hormone locus. Marked enhancement of the acetylation of histones H3 and H4 and the dimethylation of H3 Lys4 was observed in the chromatin situated close to the promoter of an actively transcribed gene. Chromatin positioned near a cell-type-specific DNase I-hypersensitive site with enhancer activity had the same histone modifications as the active promoter. In one transcribed intron, chromatin with fewer histone modifications was found, and in another transcribed intron, chromatin with markedly enhanced modifications was found. In most cases, no appreciable difference in the acetylation of histones H3 and H4 was found at prominently enhanced targets. However, different acetylation levels of H3 and H4 were found at one target. The targets with enhanced dimethylation of the H3 Lys4 residue coincided with those with prominently enhanced acetylation of histones H3 and H4.