Long‐term effects of a single exposure to immobilization: A C‐fos mRNA study of the response to the homotypic stressor in the rat brain

A single exposure to a severe emotional stressor such as immobilization in wooden boards (IMO) causes long‐term (days to weeks) peripheral and central desensitization of the hypothalamic‐pituitary‐adrenal (HPA) response to the same (homotypic) stressor. However, the brain areas putatively involved in long‐term desensitization are unknown. In the present experiment, adult male rats were subjected to 2 h of IMO and, 1 or 4 weeks later, exposed again to 1 h IMO together with stress‐naive rats. C‐fos mRNA activation just after IMO and 1 h after the termination of IMO (post‐IMO) were evaluated by in situ hybridization. Whereas in most brain areas c‐fos mRNA induction caused by the last IMO session was similar in stress‐naive (controls) and previously immobilized rats, a few brain areas showed a reduced c‐fos mRNA response: ventral lateral septum (LSv), medial amygdala (MeA), parvocellular region of the paraventricular hypothalamic nucleus (pPVN), and locus coeruleus (LC). In contrast, an enhanced expression was observed in the medial division of the bed nucleus stria terminalis (BSTMv). The present work demonstrates that a previous experience with a stressor can induce changes in c‐fos mRNA expression in different brain areas in response to the homotypic stressor and suggests that LSv, MeA, and BSTMv may be important for providing signals to lower diencephalic (pPVN) and brainstem (LC) nuclei, which results in a lower physiological response to the homotypic stressor. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Helicobacter pylori infection‐induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications

Background

Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori‐induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication.

Materials and Methods

Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low‐grade intraepithelial neoplasia, high‐grade intraepithelial neoplasia, and intestinal‐type gastric cancer groups. Helicobacter pylori infection was determined by either ¹³C‐urea breath test or immunohistochemistry staining.

Results

In Helicobacter pylori‐negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low‐grade intraepithelial neoplasia, and declined in high‐grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori‐infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori‐negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori‐negative control. Labeling index of Ki67 in Helicobacter pylori‐negative groups was higher in gastric cancer than chronic atrophic gastritis and low‐grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori‐positive groups, Ki67 labeling index was increased stepwise from nonatrophic gastritis to gastric cancer except slightly decrease in chronic atrophic gastritis group. In addition, we noted that histone H3 serine 10 phosphorylation staining is accompanied with its location changes from gastric gland bottom expanded to whole gland as disease stage progress.

Conclusions

These results indicate that stepwise gastric carcinogenesis is associated with altered histone H3 serine 10 phosphorylation, Helicobacter pylori infection enhances histone H3 serine 10 phosphorylation expression in these processes; it is also accompanied with histone H3 serine 10 phosphorylation location change from gland bottom staining expand to whole gland expression. The results suggest that epigenetic dysregulation may play important roles in Helicobacter pylori‐induced gastric cancer.     

Loss of nuclear flavanols during drought periods in Taxus baccata

Normally, needles of Taxus baccata during the growth period prominently stain blue for nuclear flavanols with the histochemical DMACA procedure. However, under excess heat and drought conditions, nuclear flavanols of current‐year needles decline to zero. Nevertheless, greenish‐yellow‐coloured flavonols (quercetin derivatives) were still observed in nuclei. All of these yellow nuclei were in a silenced state and without mitosis. This link between drought and loss of nuclear flavanols was found in 3 years, 2003, 2007 and 2010. In 2007, exceptional drought occurred in early spring, interrupted by short rains. This, in turn, led to flushing of new sprouts, a characteristic feature in which nuclei were overloaded with flavanols. By the end of three drought periods, all nuclei developed blue‐coloured nuclear flavanols. The flavanols seem to be associated with the histone proteins of chromatin. The oxidative degradation of catechin in Tris buffer (pH 8.0) containing MgCl₂ was studied in the presence of the H4‐core fragment TYTEHAKRKTVTAMD, modified according to the epigenetic histone code. The results show that catechin degradation can be significantly inhibited by the non‐modified peptides and the methylated peptides (methylation at both lysine residues). The acetylated and formylated peptides do not show this behaviour. These observations indicate that flavanol association at chromosomes appears to be regulated by the epigenetic histone code.     

Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser‐10 lowers the rate of chemical acetylation of Lys‐9, Lys‐14, and Lys‐18 by methyl acetyl phosphate in that order consistent with a higher pK_a of these Lys residues induced by phosphorylation; basicities increase up to 3 pK_a units as a function of distance from Ser‐10 phosphate. Enzymic acetylation of Lys residues with high pK_a values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N‐acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser‐10, the amines of Lys‐9, Lys‐14, and Lys‐18 have lowered pK_a values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9–10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate–Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.     

The CaMV 35S enhancer has a function to change the histone modification state at insertion loci in Arabidopsis thaliana

Chromatin regions with different states usually harbor distinct epigenetic information, through which gene expression is regulated. Recent studies using mammalian cells showed that a chromatin state signature is associated with active developmental enhancers, defined by high levels of histone H3 lysine 27 acetylation (H3K27ac) and strong depletion of H3K27 trimethylation (H3K27me3). These findings also imply that active enhancers may play a role in creating a chromatin state by changing histone modification markers, which in turn affects gene expression. To explore whether an active enhancer in plants affect histone modifications, we investigated the cauliflower mosaic virus 35S enhancer (35S_enh) for understanding its action model in Arabidopsis. We report that the 35S_enh has a function to change the histone modification pattern at its presenting loci, by characterization of the 35S_enh activated BREVIPEDICELLUS (BP) silencing lines and the randomly selected 35S_enh activation tagging lines. By analyzing histone modification markers reflecting the plant chromatin state, we show that the 35S_enh is generally correlated with the reduced level of H3K27me3 and the increased level of H3K4me3 at the insertion loci. Our data are consistent with those in mammals and suggest that the enhancer sequence correlating with the active chromatin state signature may be generally present in the eukaryotic kingdom.     

Domain Requirements of the JIL-1 Tandem Kinase for Histone H3 Serine 10 Phosphorylation and Chromatin Remodeling in Vivo

The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH₂-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH₂-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH₂-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein.     

Factors affecting the formation of phytol and its incorporation into chlorophyll by homogenates of the leaves of the french bean Phaseolus vulgaris

The biosynthesis and phosphorylation of histone fractions were measured in synchronized CHO Chinese hamster cells arrested in late G₁ by hydroxyurea treatment. Hydroxyurea was found to inhibit the initiation of both DNA and histone synthesis, thus confirming the conclusion that it arrests cells in G₁ slightly before the $\text{G}{}_1\text{S}$ boundary. However, hydroxyurea did not inhibit the phosphorylation of histone f1 or histone f2a2. The phosphorylation of histone f1, which normally is absent in early G₁, begins 2 hr prior to DNA synthesis. In the presence of hydroxyurea, f1 phosphorylation occurs on schedule at this same time in G₁, resulting in significant G₁-phase f1 phosphorylation. This offers strong evidence that (a) f1 phosphorylation is not restricted to S phase; (b) “old” f1 which was synthesized in previous cell cycles is phosphorylated in G₁ before “new” f1 which is synthesized in S phase; and (c) G₁-phase f1 phosphorylation does not require new histone or new DNA synthesis.Histone f1 phosphorylation was observed to occur at accelerated rates in S phase over phosphorylation rates observed in late G₁-arrest. Data support the proposal that three different levels of f1 phosphorylation occur during the cell cycle: (1) a G₁-related phosphorylation of “old” f1; (2) an S-related phosphorylation of both “old” and “new” f1; and (3) a superphosphorylation of f1 associated with chromosome condensation during the G₂ to M transition. It is also possible that a limited proportion of f1 may be phosphorylated in G₁, perhaps at the initial DNA synthesis sites, and that an increased proportion of f1 is phosphorylated in S as DNA is synthesized. Similarities between the kinetics of histone f1 phosphorylation and the association of DNA with lipoprotein in synchronized control and hydroxyurea-treated cells suggest an involvement of f1 phosphorylation in cell-cycle-dependent chromatin structural changes.     

Apoptotic Phosphorylation of Histone H3 on Ser-10 by Protein Kinase C��

Phosphorylation of histone H3 on Ser-10 is regarded as an epigenetic mitotic marker and is tightly correlated with chromosome condensation during both mitosis and meiosis. However, it was also reported that histone H3 Ser-10 phosphorylation occurs when cells are exposed to various death stimuli, suggesting a potential role in the regulation of apoptosis. Here we report that histone H3 Ser-10 phosphorylation is mediated by the pro-apoptotic kinase protein kinase C (PKC) δ during apoptosis. We observed that PKCδ robustly phosphorylates histone H3 on Ser-10 both in vitro and in vivo. Ectopic expression of catalytically active PKCδ efficiently induces condensed chromatin structure in the nucleus. We also discovered that activation of PKCδ is required for histone H3 Ser-10 phosphorylation after treatment with DNA damaging agents during apoptosis. Collectively, these findings suggest that PKCδ is the kinase responsible for histone H3 Ser-10 phosphoryation during apoptosis and thus contributes to chromatin condensation together with other apoptosis-related histone modifications. As a result, histone H3 Ser-10 phosphorylation can be designated a new ‘apoptotic histone code’ mediated by PKCδ.     

Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila

Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a ;kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.     

Comprehensive Histone Phosphorylation Analysis and Identification of Pf14-3-3 Protein as a Histone H3 Phosphorylation Reader in Malaria Parasites

The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.     

Difference in larval type explains patterns of nonsynonymous substitutions in two ancient paralogs of the histone H3 gene in sea stars

SUMMARY Paralogous genes frequently show differences in patterns and rates of substitution that are typically attributed to different selection regimes, mutation rates, or local recombination rates. Here, two anciently diverged paralogous copies of the histone H3 gene in sea stars, the tandem‐repetitive early‐stage gene and a newly isolated gene with lower copy number that was termed the “putative late‐stage histone H3 gene” were analyzed in 69 species with varying mode of larval development. The two genes showed differences in relative copy number, overall substitution rates, nucleotide composition, and codon usage, but similar patterns of relative nonsynonymous substitution rates, when analyzed by the d_N/d_S ratio. Sea stars with a nonpelagic and nonfeeding larval type (i.e., brooding lineages) were observed to have d_N/d_S ratios that were larger than for nonbrooders but equal between the two paralogs. This finding suggested that demographic differences between brooding and nonbrooding lineages were responsible for the elevated d_N/d_S ratios observed for brooders and refuted a suggestion from a previous analysis of the early‐stage gene that the excess nonsynonymous substitutions were due to either (1) gene expression differences at the larval stage between brooders and nonbrooders or (2) the highly repetitive structure of the early‐stage histone H3 gene.     

Secondary structure and dynamics study of the intrinsically disordered silica‐mineralizing peptide P5S3 during silicic acid condensation and silica decondensation

The silica forming repeat R5 of sil1 from Cylindrotheca fusiformis was the blueprint for the design of P₅S₃, a 50‐residue peptide which can be produced in large amounts by recombinant bacterial expression. It contains 5 protein kinase A target sites and is highly cationic due to 10 lysine and 10 arginine residues. In the presence of supersaturated orthosilicic acid P₅S₃ enhances silica‐formation whereas it retards the dissolution of amorphous silica (SiO₂) at globally undersaturated concentrations. The secondary structure of P₅S₃ during these 2 processes was studied by circular dichroism (CD) spectroscopy, complemented by nuclear magnetic resonance (NMR) spectroscopy of the peptide in the absence of silicate. The NMR studies of dual‐labeled (¹³C, ¹⁵N) P₅S₃ revealed a disordered structure at pH 2.8 and 4.5. Within the pH range of 4.5‐9.5 in the absence of silicic acid, the CD data showed a disordered structure with the suggestion of some polyproline II character. Upon silicic acid polymerization and during dissolution of preformed silica, the CD spectrum of P₅S₃ indicated partial transition into an α‐helical conformation which was transient during silica‐dissolution. The secondary structural changes observed for P₅S₃ correlate with the presence of oligomeric/polymeric silicic acid, presumably due to P₅S₃‐silica interactions. These P₅S₃‐silica interactions appear, at least in part, ionic in nature since negatively charged dodecylsulfate caused similar perturbations to the P₅S₃ CD spectrum as observed with silica, while uncharged ß‐d ‐dodecyl maltoside did not affect the CD spectrum of P₅S₃. Thus, with an associated increase in α‐helical character, P₅S₃ influences both the condensation of silicic acid into silica and its decondensation back to silicic acid.     

Differential effects of stress and amphetamine administration on Fos-like protein expression in corticotropin releasing factor-neurons of the rat brain

Corticotropin releasing factor (CRF) appears to be critical for the control of important aspects of the behavioral and physiological response to stressors and drugs of abuse. However, the extent to which the different brain CRF neuronal populations are similarly activated after stress and drug administration is not known. We then studied, using double immunohistochemistry for CRF and Fos protein, stress and amphetamine-induced activation of CRF neurons in cortex, central amygdala (CeA), medial parvocellular dorsal, and submagnocellular parvocellular regions of the paraventricular nucleus of the hypothalamus (PVNmpd and PVNsm, respectively) and Barrington nucleus (Bar). Neither exposure to a novel environment (hole-board, HB) nor immobilization (IMO) increased Fos-like immunoreactivity (FLI) in the CeA, but they did to the same extent in cortical regions. In other regions only IMO increased FLI. HB and IMO both failed to activate CRF+ neurons in cortical areas, but after IMO, some neurons expressing FLI in the PVNsm and most of them in the PVNmpd and Bar were CRF+. Amphetamine administration increased FLI in cortical areas and CeA (with some CRF+ neurons expressing FLI), whereas the number of CRF+ neurons increased only in the PVNsm, in contrast to the effects of IMO. The present results indicate that stress and amphetamine elicited a distinct pattern of brain Fos-like protein expression and differentially activated some of the brain CRF neuronal populations, despite similar levels of overall FLI in the case of IMO and amphetamine.     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

Posttranslational Modifications of the Histone 3 Tail and Their Impact on the Activity of Histone Lysine Demethylases In Vitro

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k _cat/K _m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K _m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.     

Effects of two types of restraint stress on spontaneous behavior of Sprague-Dawley and Lewis rats.

The purpose of this study was to evaluate the action of two types of stressors in Sprague-Dawley (S-D) and Lewis (LEW) rats differing in their hypothalamic-pituitary-adrenal axis activity on locomotion and rearing in an open space. Exposure to restraint immobilization alone (IMO) or this immobilization combined with cold water (22 degrees C) immersion (IMO+C) lasted for 1 h and started 2 or 5 h before the test. To evaluate the acute and persisting effects of both stressors, four trials were performed in one-week intervals; rats were exposed to the stressors in trial 1 and 3. While in LEW rats both acute stressors produced reduction of locomotion and rearing in all stressed groups, S-D rats responded with a decrease of both parameters only after IMO+C presented 2 h before testing. Neither strain displayed a changed performance one week after the first stress exposure. One week after the second stress exposure rats of both strains exhibited a tendency to an increase of both parameters reaching the significance in several experimental groups. The findings indicate: a) the IMO+C produced stronger behavioral alteration than IMO alone; b) the behavioral responses to stressors were more pronounced in LEW compared to S-D strain; c) change from the reduction of activity to its increase may be interpreted as bi-directional manifestation of long-term effects of immobilization stress.