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PubMed2004

Antimicrobial activity of histones from hemocytes of the Pacific white shrimp.

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp, Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. The L. vannamei histone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition against Micrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 microm while a mixture of histones H2B and H4 was active at 3 microm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought.

Amino Acid Sequence↗
PubMed2002

The elusive structural role of ubiquitinated histones.

It is increasingly apparent that histone posttranslational modifications are important in chromatin structure and dynamics. However, histone ubiquitination has received little attention. Histones H1, H3, H2A, and H2B can be ubiquitinated in vivo, but the most prevalent are uH2A and uH2B. The size of this modification suggests some sort of structural impact. Physiological observations suggest that ubiquitinated histones may have multiple functions and structural effects. Ubiquitinated histones have been correlated with transcriptionally active DNA, implying that it may prevent chromatin folding or help maintain an open conformation. Also, in some organisms during spermiogenesis, a process involving extensive chromatin remodeling, uH2A levels increase just prior to histone replacement by protamines. Determination of chromatin's structural changes resulting from histone ubiquitination is therefore important. Recent work using reconstituted nucleosomes and chromatin fibers containing uH2A indicate that in the absence of linker histones, ubiquitination has little structural impact. DNase I digests and analytical ultracentrifugation of reconstituted ubiquitinated nucleosomes show no structural differences. Solubility assays using reconstituted chromatin fibers in the presence of divalent ions demonstrate that uH2A fibers are slightly more prone to aggregation than controls, and analytical ultracentrifugation results with different MgCl2 and NaCl concentrations determined that chromatin folding is not affected by this modification. Additional work to assess possible synergistic affects with histone acetylation also precludes any structural implications. Protamine displacement experiments concluded that the presence of uH2A does not significantly affect the ability of the protamines to displace histones. In addition, uH2A does not interfere with histone H1 binding to the nucleosome. While work with uH2B remains insufficient to come to any definitive conclusions about its structural impact, current work with uH-2A indicates that, contrary to predictions, this histone modification does not affect either nucleosome or chromatin structure. Consequently, the search for a structural role for ubiquitinated histones continues and their effect on and importance in chromatin dynamics remains elusive.

Animals↗
PubMed2025

Trypanosomatid histones: the building blocks of the epigenetic code of highly divergent eukaryotes.

Histones play a fundamental role in eukaryotic organisms not only as scaffolding proteins in DNA packaging but also in regulating gene expression. They constitute the protein reel around which DNA wraps forming nucleosomes. This initial packing gives rise to the chromatin fiber which is next folded into three-dimensional arrangements. Additionally, histones have expanded their functions through the emergence of histone variants which have specialized purposes and can deeply affect chromatin organization and dynamics. Moreover, both canonical histones and histone variants comprise the building blocks of the histone code by being targets of different post-translational modifications (PTMs) that occur in a highly regulated manner both in place and time. Most of the above-mentioned about chromatin organization is conserved among eukaryotes. However, trypanosomatid histones have many peculiarities that entail a special description. In this review, we compile the current knowledge of canonical core histones, histone variants, and their PTMs in trypanosomatids. We highlight the similarities and differences between histone variants and their canonical counterparts in trypanosomatids, and we compare them with those from model organisms. Finally, we discuss the crosstalk between different histone marks and their genomic distribution underlying the uniqueness of trypanosomatids.

Histones↗
PubMed2004

Expression of the metastasis-associated MTA1 protein and its relationship to deacetylation of the histone H4 in esophageal squamous cell carcinomas.

Metastasis-associated protein MTA1 and histone deacetylase form a protein complex with histone deacetylase activity that plays an important role in histone deacetylation, alteration of chromatin structure and transcriptional control. The precise role of the MTA1 protein in the malignant progression of human cancers remains unknown, however, especially its overexpression and relationship with histone acetylation/deacetylation in experimental and clinical tumors. The expression levels of MTA1 protein and the acetylation levels of histone H4 were examined in 70 cases of surgically resected esophageal squamous cell carcinomas, using immunohistochemistry. The intensities of immunostaining of MTA1 protein and acetylated histone H4 in carcinoma tissues (Ca) were compared to normal epithelium (N) contained in the same section. Thirty of 70 cases (42.9%) displayed overexpression of MTA1 protein (N < Ca). Cancers overexpressing MTA1 protein invaded deeper into the esophageal wall (p < 0.005) and showed significantly higher degrees of lymph node metastasis (p < 0.01), higher pathological stage, more lymphatic involvement and poorer prognosis (p < 0.05) than the remaining cases. The acetylation levels of histone H4 inversely correlated to the depth of cancer invasion and pathological stage (p < 0.05), and the patients with higher level of histone H4 acetylation had a better prognosis (p < 0.05). Furthermore, immunostaining patterns of MTA1 and acetylated histone H4 were inversely correlated (p < 0.001), demonstrating the relationship of deacetylation of histone H4 in MTA1-overexpressing carcinomas. In conclusion, the data suggest that the overexpression of MTA1 protein and acetylation level of histone H4 protein, both of which are closely related, might be useful predictors of malignant potential of esophageal squamous cell carcinomas. Thus, strategies involving inhibition of MTA1 function as well as inhibition of histone deacetylation could be novel approaches for the treatment of esophageal squamous cell carcinomas.

Acetylation↗
PubMed2007

Dynamic histone acetylation/deacetylation with progesterone receptor-mediated transcription.

Histone acetylation is a highly dynamic posttranslational modification that plays an important role in gene expression. Previous work showed that promoter histone deacetylation is accompanied by progesterone receptor (PR)-mediated activation of the mouse mammary tumor virus (MMTV) promoter. We investigated the role of this deacetylation and found that this histone deacetylation is not a singular event. In fact, histone acetylation at the MMTV promoter is highly dynamic, with an initial increase in acetylation followed by an eventual net deacetylation of histone H4. The timing of increase in acetylation of H4 coincides with the time at which PR, RNA polymerase II, and histone acetyltransferases cAMP response element-binding protein (CREB)-binding protein and p300 are recruited to the MMTV promoter. The timing in which histone H4 deacetylation occurs (after PR and RNA polymerase II recruitment) and the limited effect that trichostatin A and small interfering RNA knockdown of histone deacetylase (HDAC)3 have on MMTV transcription suggests that this deacetylation activity is not required for the initiation of PR-mediated transcription. Interestingly, two HDACs, HDAC1 and HDAC3, are already present at the MMTV before transcription activation. HDAC association at the MMTV promoter fluctuates during the hormone treatment. In particular, HDAC3 is temporarily undetected at the MMTV promoter within minutes after hormone treatment when the histone H4 acetylation increases but returns to the promoter near the time when histone acetylation levels start to decline. These results demonstrate the dynamic nature of coactivator/corepressor-promoter association and histone modifications such as acetylation during a transcription activation event.

Acetylation↗
PubMed1979

Purification and properies of cAMP dependent and independent histone kinases from human leukocytes.

Histone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 x g supernatant, followed by DEAE-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had Mr 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had Mr 33 000 upon gelfiltration, but sedimented at 2.8--3.0S and 3.0--3.2S, respectively. R2 and R4 subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)--5.1S, respectively. The holoenzyme from peak I had Mr 165 000, 6.7S, which suggest a R2C2 structure, while that of peak III sedimented at 6.7S, but eluted at Mr 330 000 (2R2C2) by gelfiltration. The Kmapp for peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 microM and 23 microM, and cAMP 5 X 10(-8) M and 6.3 x 10(-8) M. Both enzymes had pH optimum 6.7--6.9 and were equally sensitive to Ca2+, temperature and protein kinase inhibitor. The substrate specificity was histone VS greater than histone IIA = histone VIS greater than casein greater than phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20--30% of cAMP dependent protein kinase activity and is absent from the 180 000 x g supernatant of gently disrupted cells. Purified catalytic subunit had Kmapp (ATP) 20 microM with rabbit muscle glycogen synthease I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07). cAMP independent histone kinase activity eluted in one peak (Peak II) at3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with Mr 78 000. Kmapp for ATP was 78 microM and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca2+, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA greater than histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.

Chromatography, DEAE-Cellulose↗
PubMed2004

Expression and purification of recombinant human histones.

Nucleosomes reconstituted from bacterially expressed histones are useful for functional and structural analyses of histone variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human histone H2A, H2B, and H3 genes were expressed well in Escherichia coli cells, but the human histone H4 gene was poorly expressed. Therefore, we designed a new histone H4 gene with codons optimized for the E. coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histones were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed, and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B, and H4.

Amino Acid Sequence↗
PubMed2003

Alteration of GCN5 levels in maize reveals dynamic responses to manipulating histone acetylation.

The role played by histone acetyltransferase (HAT), GCN5, in transcriptional co-activation has been analysed in detail in yeast and mammals. Here, we present the cloning and expression pattern of Zmgcn5, the maize homologue. The enzymatic activity of the recombinant ZmGCN5 was analysed with histone and nucleosome substrates. In situ hybridisation of developing maize kernels using Zmgcn5 as probe shows that the transcript is concentrated in rapidly dividing cells. To investigate the role of ZmGCN5 in the transcription of specific plant genes, direct protein-protein interactions were tested. A cDNA clone encoding a putative interacting partner in GCN5-adapter complexes, ZmADA2, was isolated and the interaction between ZmGCN5 and ZmADA2 was confirmed by a GST-spin down experiment. Co-immunoprecipitation of the plant transcriptional activator Opaque-2 and ZmADA2 in nuclear extracts suggests ADA2/GCN5-containing complexes to mediate transcriptional activation by binding of this bZIP factor. For a more general analysis of the effects of histone acetylation on plant gene expression, 2500 ESTs spotted on filters were hybridised with cDNA probes derived either from maize cell lines treated with Trichostatin A (TSA), or from a transgenic line expressing the ZmGCN5 antisense transcript. Several sequences showing marked changes in abundance were confirmed by RNA blot analysis. Inhibition of histone deacetylation with TSA is accompanied by a decrease in the abundance of ZmGCN5 acetylase protein, but by increases in mRNAs for histones H2A, H2B, H3 and H4. The elevated histone mRNA levels were not reflected in increasing histone protein concentrations, suggesting hyperacetylated histones arising from TSA treatment may be preferentially degraded and substituted by de novo synthesised histones. The ZmGCN5 antisense material showed suppression of the endogenous ZmGCN5 transcript and the profiling analysis revealed increased mRNA levels for H2A, H2B and H4. Furthermore, in the antisense line, a reduction in the amount of the RPD3-type HD1B-I histone deacetylase protein was observed. A model for linked regulation of histone acetylation and histone mRNA transcription is discussed.

Acetyltransferases↗
PubMed1985

Deposition-related histone acetylation in micronuclei of conjugating Tetrahymena.

Macro- and micronuclei of the ciliated protozoan, Tetrahymena thermophila, afford a unique opportunity to study histone acetylation under conditions where acetylation associated with the regulation of transcription and acetylation associated with the deposition of histones on the DNA are separable. In this study we demonstrate that histone H3 and histone H4 synthesized in young (5 hr) conjugating Tetrahymena are deposited into micronuclei in acetylated forms. Most of the newly synthesized histone H3 migrates as a monoacetylated form while essentially all of the new histone H4 is deposited as a diacetylated species. Since micronuclei replicate rapidly during this stage of the life cycle, but are transcriptionally inactive, these data suggest that histone acetylation is related functionally to histone deposition and chromatin assembly. Pulse-chase experiments show that micronuclei also contain a butyrate-sensitive deacetylase activity(ies) which operates to remove the deposition-related acetate groups from newly synthesized and deposited H3 and H4. This enzymatic activity probably contributes to the steady state level of micronuclear histone acetylation that is low or nonexistent. Thus, evidence is emerging for at least two independent systems of histone acetylation in Tetrahymena. The first system is specific to macronuclei and may be related to gene expression. The second system is common to macro- or micronuclear histones (H3 and H4) and may be related to histone deposition during DNA replication.

Acetylation↗
PubMed1958

Bactericidal action of histone.

The arginine-rich fraction of calf thymus histone (histone B) exerts bactericidal activity on various coliform bacilli and micrococci under certain conditions in vitro. Final concentrations of less than 1 microg. histone per ml. kill susceptible microbes without detectable morphological alteration or lysis. Among the microorganisms highly susceptible to histone are Escherichia, Salmonella, Shigella, Pseudomonas, Klebsiella, and Micrococcus pyogenes var. albus. Less susceptible or completely resistant are Proteus, Serratia, Micrococcus pyogenes var. aureus, and various types of hemolytic streptococci. Coliforms grown on solid media are much more resistant to the lethal effect of histone than are those cultured in liquid media. This difference is apparently related to the physiological state of the bacteria; agar grown microorganisms washed with water remain resistant to histone, whereas incubation in broth rapidly renders them more susceptible. Histone is adsorbed onto heat-killed E. coli K-12 under conditions suitable for lethal action on this organism. The bactericidal activity of histone is but little affected by pH of the test system, but ionic strength of the medium exerts a marked influence, the lethal action being reduced or blocked as the salt concentration reaches levels higher than that of 0.15-0.2 M NaCl. Relatively high concentrations of rabbit serum or of bovine plasma albumin reduce the bactericidal activity of histone in a medium at pH 7; these serum preparations are, however, essentially without effect in the test system at pH 5.6. The bactericidal effect of histone is antagonized by addition to the medium of small amounts of certain basic substances (protamine, spermine), or of various acid polysaccharides (heparin, nucleic acid, bacterial lipopolysaccharides). The rate of killing of E. coli K-12 by histone increases as the temperature and the concentration of histone are raised. Within the limits studied, this rate also appears to be directly proportional to the concentration of bacteria in the system.

Animals↗
PubMed2003

The transcriptional enhancer of the pea plastocyanin gene associates with the nuclear matrix and regulates gene expression through histone acetylation.

The influence of the transcriptional enhancer of the pea plastocyanin gene (PetE) on the acetylation of histones was examined with chromatin immunoprecipitation (ChIP) experiments using antibodies that recognize acetylated or nonacetylated histones H3 and H4. In transgenic tobacco plants containing the pea PetE promoter fused to uidA, both acetylated and nonacetylated histones H3 and H4 were present on the integrated transgene. Linking the PetE enhancer to the transgene resulted in increased beta-glucuronidase activity and increased amounts of acetylated histones H3 and H4 present on the promoter, suggesting that the enhancer may increase transcription by mediating the acetylation of histones. Trichostatin A and sodium butyrate, which are potent inhibitors of histone deacetylases (HDAs), activated expression from the PetE promoter by fourfold, with a concomitant increase in the acetylation states of histones H3 and H4, as determined by ChIP, indicating that the acetylation of histones has a direct positive effect on transcription. The HDA inhibitors did not increase expression from the PetE promoter when it was linked to the enhancer, consistent with preexisting hyperacetylated histones on the transgene. Mapping of histone acetylation states along the reporter gene indicated that the histones H3 and H4 associated with the promoter and the 5' region of uidA were hyperacetylated in the presence of the PetE enhancer. The PetE enhancer bound to isolated tobacco nuclear matrices in vitro and was associated with the nuclear matrix in nuclei isolated from transgenic tobacco plants. These results suggest that the pea PetE enhancer activates transcription by associating with the nuclear matrix, mediating the acetylation of histones on the promoter and the nearby coding region and resulting in an altered chromatin structure.

Acetylation↗
PubMed2004

Microarray analysis of chromatin-immunoprecipitated DNA identifies specific regions of tobacco genes associated with acetylated histones.

The acetylation states of histones present on the upstream, promoter, coding or intronic regions of 88 tobacco genes were examined with chromatin immunoprecipitation (ChIP) experiments using antibodies that recognised acetylated histone H4. The DNA sequences enriched in the immunoprecipitates were amplified by ligation-mediated PCR, labelled with Cy-dUTP and hybridised to DNA microarrays. In green tobacco shoots, histone H4 acetylation was localised to 300-600-bp sequences in the promoters or coding regions of 31 genes, or occurred extensively over several kilobase-pair regions containing the upstream, promoter and/or coding regions of 25 genes. Genes associated with high histone H4 acetylation levels at promoters were actively expressed, whereas genes depleted in acetylated histone H4 were non-transcribed or expressed at very low levels, suggesting a correlation between histone H4 acetylation and gene activity. Trichostatin A (TA), an inhibitor of histone deacetylases (HDAs), did not alter histone H4 acetylation states globally but increased acetylation levels at specific tobacco sequences, suggesting that HDAs are targeted to particular nucleosomes. Genes that were upregulated by TA were associated with increased histone H4 acetylation at promoter or coding regions, indicating that acetylation of histones on coding regions may activate transcription. Increased histone H4 acetylation leading to elevated expression was observed on genes with diverse functions, suggesting that histone H4 acetylation is involved in regulation of many plant processes.

Acetylation↗
PubMed2006

Histone modifications in kainate-induced status epilepticus.

To understand the molecular actions of status epilepticus at the chromatin level, we studied the effects of kainate-induced status epilepticus on two different histone modifications at amino terminal tails: histone H3 phosphorylation at serine 10 and histone H4 acetylation. In addition to induction of c-fos and c-jun immediate early genes (IEGs) expression in mouse hippocampus, we also found the upregulation of acetylation and phosphorylation of histones, coupled with status epilepticus after kainate administration. c-fos and c-jun mRNA were sequentially induced in response to kainate, in different hippocampal subpopulations, starting from the dentate gyrus (DG) and spreading to the cornus ammonis regions. Immunohistochemical analysis showed that the spatio-temporal distribution of histone H4 hyperacetylation after kainate treatment was well correlated with the expression of c-fos and c-jun genes. Additionally, there was a transient appearance of phosphorylated histone H3 specifically in the DG region. CREB-binding protein or CBP, a well-known transcriptional co-activator with histone acetyltransferase (HAT) activity, was also induced by kainate and its expression pattern well correlated with histone H4 hyperacetylation in the hippocampus. Chromatin immunoprecipitation analysis showed that both histone modifications were associated with c-fos gene promoter after kainate stimulation, but only histone acetylation with c-jun gene. Pretreatment with curcumin, which has a HAT inhibitory activity specific for CBP/p300, attenuated histone modifications, IEGs expression and also the severity of status epilepticus after kainate treatment. Our findings suggest the involvement of histone modifications induced by kainate not only in IEGs expression but also in the development of epilepsy.

Animals↗
PubMed1988

Histone H3 thiol reactivity and acetyltransferases in chicken erythrocyte nuclei.

Chicken erythrocyte nuclei previously incubated separately with two novel mercury compounds (N-chloromercuribenzoyl)-biocytin and bis(p-(chloromercuribenzoyl))-[3H]lysine diamide) were digested with micrococcal nuclease and the digest products fractionated according to their solubility in 0.15 M NaCl and molecular size. The identity and quantitation of the chromatin fractions and proteins containing covalently bound mercury were determined by Western blotting, autoradiography, and scintillation counting. The most highly acetylated species of histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction also contained the highest proportion of bound mercury. This fraction contains hyperacetylated core histones, is depleted in linker histones, and enriched in nonhistone proteins. Histone H3 in the 0.15 M NaCl-soluble mononucleosomes, which are unacetylated and lack linker histones, was 45% less labeled than histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction. In the 0.15 M NaCl-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker histones, histone H3 was 63% less labeled. Allowing for the differential abundance of these subfractions in the nucleus, the relative H3 reactivities are 50, 7, and 1 for 0.15 M NaCl-soluble polynucleosomes, mononucleosomes, and 0.15 M NaCl-insoluble polynucleosomes, respectively. Thus a gradation of reactivities exists which correlates with increasing hyperacetylation and linker histone depletion. High mobility group proteins 1 and 2, found in subnucleosome particles in the 0.15 M NaCl-soluble fraction, are extensively mercury-labeled. Distribution of histone acetyltransferase activity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold greater in the 0.15 M NaCl-soluble supernatant than in the 0.15 M NaCl-insoluble pellet. Furthermore, the acetyltransferase activity, which is tightly bound to undigested chromatin, is rapidly released by both micrococcal nuclease and DNase I. For short digestion times the enzyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme appears to be free from intact nucleosomes. The enzyme may be localized in the globin domain in erythrocytes and maintains that region in a hyperacetylated state which results in an altered linker histone binding reflected in a change in the reactivity of the usually inaccessible H3 cysteine 110.

Acetyltransferases↗
PubMed1988

At least 60 ADP-ribosylated variant histones are present in nuclei from dimethylsulfate-treated and untreated cells.

The level of histone adenosine diphospho (ADP) ribosylation was studied in isolated nuclei from mouse myeloma cells in culture. The cells were treated with dimethylsulfate (DMS), a DNA-methylating agent, and histones were analyzed using two-dimensional gel electrophoresis. Seventeen or more bands probably representing mono-to heptadeca (ADP-ribosylated) histones could be visualized for each major variant histone. DMS treatment, by increasing the number of chromatin sites undergoing repair, greatly enhanced histone ADP-ribosylation. When histones were labeled in a cell lysate rather than in isolated nuclei, mono- and oligo(ADP-ribosylated) histone forms prevailed. The presence of approximately 87 ADP-ribosylated variant histone forms in cell lysates and of approximately 170 in isolated nuclei is shown for the first time in this work. Previous studies show multiple ADP-ribosylated forms for only histone H1. The theoretical number of variegated nucleosomes is thus much higher than previously thought, provided that histone-histone contacts are not disrupted at up to a certain level of histone ADP-ribosylation.

Adenosine Diphosphate Ribose↗
PubMed1994

Histone acetylation influences both gene expression and development of Xenopus laevis.

We examine the potential role of histone hyperacetylation in gene activation during Xenopus development using Trichostatin A, (TSA), a specific inhibitor of histone deacetylase. We find that TSA is very effective in inducing both core histone hyperacetylation and histone H1(0) gene expression in a Xenopus somatic cell line. In contrast, TSA does not induce histone hyperacetylation or histone H1(0) transcription in Xenopus oocytes. Histone hyperacetylation is developmentally regulated during Xenopus embryogenesis; hyperacetylated histones first accumulate early in gastrulation. The capacity of TSA to induce histone H1(0) gene expression correlates with the induction of histone hyperacetylation. Concentrations of TSA sufficient to induce histone hyperacetylation in Xenopus embryos delay gastrulation and cause diminished midtrunk and posterior formation, suggesting defects in mesoderm formation. Although the constitutive hyperacetylation of the histones does not prevent either the cell division or differentiation sufficient for early morphogenesis it has a role in establishing stable states of differential gene activity during gastrulation.

Acetylation↗
PubMed1999

Subnuclear distribution of the entire complement of linker histone variants in Arabidopsis thaliana.

Linker histones (e.g. H1, H5, H1 degrees ) are thought to exert control on chromatin function by restricting nucleosomal dynamics. All higher eukaryotes possess a diverse family of linker histones, which may exhibit functional specialization. Arabidopsis thaliana apparently contains a minimal complement of linker histone structural variants and therefore is an ideal model for investigating functional differentiation among linker histones. Histones H1-1 and H1-2 are relatively similar proteins that are expressed in a wide variety of tissues and make up the majority of linker histone while H1-3 is a highly divergent minor variant protein that is induced by drought stress. We are interested in determining whether the in vivo distribution of each of these proteins also differs. To this end, we have produced subtype-specific antibodies and have localized each of the three proteins at the intranuclear and DNA sequence levels by indirect immunofluorescence and immunoprecipitation, respectively. Antibodies against linker histones H1-1 and H1-2 decorate nuclei in patterns very similar to 4',6-diamidino-2-phenylindole (DAPI) staining, but different than the staining pattern of total histones. In contrast, antibodies made against two regions of H1-3 bind to chromatin in a diffuse pattern distinct from the DAPI-staining pattern. We also describe a technique to determine the localization of plant linker histone variants along regions of chromatin, employing in vivo chemical DNA-protein cross-linking to preserve native associations followed by immunoprecipitation with subtype-specific antibodies. We use this technique to demonstrate that, in contrast to the major linker histones, H1-3 does not bind the repetitive sequences pAL1 and 5S rDNA. In addition, we show that linker histones are bound to the compacted nucleosomal arrays at the telomere but with reduced stoichiometry. Taken together, our results suggest that plants, as has been shown for animals, possess a variant linker histone that is differentially localized.

Antibody Specificity↗
PubMed1987

Histone variants and acetylated species from the alfalfa plant Medicago sativa.

The histones from the alfalfa plant Medicago sativa have been characterized in terms of type variants and levels of acetylation. Histones were isolated directly from total plant tissue (callus), eliminating the need to develop methods for nuclear isolation. An acid-urea-polyacrylamide gel with a transverse Triton X-100 gradient resolved and identified in a single gel at least one type of histone H4, two variant forms of histone H2B, two variant forms of histone H3, and four variant forms of histone H2A from a crude histone preparation. Histone H4 was present 25% in an unmodified state and 75% as monomodified, presumably as monoacetylated histone. Both histone H3 variants displayed five bands, consistent with up to four internal sites of acetylation. The two H3 variants differed in their steady-state level of acetylation, suggesting that they may reside in different chromatin environments. Several histone H1 species were identified by solubility and cross-reactivity with antiserum raised against the globular part of bovine H1(0), indicating conservation of epitopes between histone H1 of mammals and higher plants.

Acetylation↗