Differing accessibility in chromatin of the antigenic sites of regions 1-58 and 63-125 of histone H2B

Experiments with antibodies induced by separated fragments 1-58 and 63- 125 of H2B histone indicated that the 1-58 portion of the molecule is much more accessible in chromatin than is the 63-125 region. In immunoabsorption and immunoelectron microscopic assays with bovine and chicken chromatins, anti-1-58 antibodies reacted with sheared or unsheared chromatin both at low ionic strength (1 mM Tris-HCl) and in 0.14 M NaCl. Anti-63-125 antibodies were bound only weakly by chromatin at low ionic strength and not at all in 0.14 M NaCl. Antibodies to whole H2B showed intermediate reactivity with chromatin in both assays. In tests of immunofluorescence with unfixed calf liver nuclei in suspension, anti-1-58 caused nucleolar as well as nucleoplasmic fluorescence, whereas anti-63-125 did not lead to detectable fluorescence; anti-H2B showed intermediate staining intensity. In control experiments, anti-H1 antibody was bound by chromatin at low ionic strength but not in 0.14 M NaCl; anti-H3 antibody was bound poorly under either condition.     

The H1 and H2 polypeptides associate to form the asialoglycoprotein receptor in human hepatoma cells

Antibody-induced degradation and chemical cross-linking experiments have been carried out to assess the nature of the interaction between the two asialoglycoprotein-receptor polypeptides, H1 and H2, synthesized in HepG2 cells. Incubation of HepG2 cell monolayers with anti-H1 antibody caused a specific and equal loss of both H1 and H2 polypeptides. The same result was obtained with anti-H2 antibody. Control serum did not affect the level of H1 or H2 not did anti-H1 or anti-H2 antibodies affect the level of the transferrin receptor. The chemical cross-linking reagent, difluorodinitrobenzene, has been used to demonstrate that H1 can be cross-linked to H2 in HepG2 cell microsomal membranes. Dimer and trimer species with apparent molecular masses of 93 and 148 kD, respectively, were readily observed upon chemical cross-linking and some dimers and trimers were immunoreactive with both anti-H1 and anti-H2 antibodies. The putative trimer, possibly two H1 and one H2 molecules, is a minimum estimate of the true size of the asialoglycoprotein receptor in intact HepG2 cell, and it is possible that larger hetero-oligomeric forms of the receptor exist. The results of both types of experiments indicate that H1 and H2 form an oligomeric complex in HepG2 cells and thus, both polypeptides constitute the human asialoglycoprotein receptor.     

Monoclonal antibody with specificity to a conserved epitope in the C-terminal domain of histone H1 variants.

A monoclonal type M-immunoglobulin (IgM) was generated in mice against a nuclease-urea extract of HeLa metaphase chromosomes. This antibody stains metaphase chromosomes from a variety of mammalian cultured cell types by indirect immunofluorescence. Antibody 12C7 reacts by western transfer technique with histone H1 in all the cell lines tested. The antibody cross-reacts with H1, and H1(0) in human cells. Proteolytic digestions of H1 suggest that the epitope is localized in the carboxy-terminal domain of the histone H1 molecule. Digestion with trypsin demonstrates that the antibody 12C7 does not react with the globular domain of histone H1. The C-terminal domain of H1 subtypes therefore seems to have a conserved determinant which does exist in H1, H1(0), and probably in H5. This antibody has applications in studying the role of that domain of H1 in processes like chromosome condensation and variations in chromatin structure which influence gene expression.     

Interphase chromosome structures of human cells

Major intermediates of chromosome condensation in erythroleukemia K562 cells are presented. Interphase chromatin structures became visible after reversal of permeabilization. Large-scale chromatin structures and the development of individual interphase chromosomes were observed by fluorescence microscopy. In the linear arrangement the following major intermediates of K562 chromatin condensation could be distinguished: (1) the most decondensed chromatin veil, (2) chromatin ribbon, (3) chromatin funnel, a new intermediate regarded as the earliest visible form of interphase chromosomes, (4) chromatin body, (5) 300 nm chromatin fiber, (6) u, v, or s forms of chromosomes, and (7) linear chromosomes. The observations made in nuclei of K562 cells conform to the model of helical coil chromosome condensation.     

Reformation of the nuclear envelope in melanophores during recovery from a hormone plus puromycin treatment

Previous experiments have shown that treatment of the melanophores of Pachymedusa (Agalychnis) dacnicolor with Melanophore-Stimulating Hormone (MSH) + puromycin causes the nuclear envelope to breakdown leading to the formation of discontinuous vesicles and the hyperdispersion of chromatin. We show here that these cells recover, reform their nuclear envelopes, and recondense their chromatin, both in the presence and in the absence of actinomycin D (actD). After recovery, these cells respond to MSH by melanosome dispersion. From these results, the following conclusions or observations are drawn:

1. 1, Reformation of nuclear envelope does not require that the chromatin be condensed into chromosomes as in mitosis.

2. 2, The new nuclear envelope is derived primarily from reutilization of the membrane vesicles produced during nuclear envelope breakdown, somewhat similar to mitosis. There may also be contributions from other membranous organelles.

3. 3, The hyperdispersed chromatin appears not to be subject to extensive attack by endogenous nucleases as the recovered cells are of good ultrastructure and can respond tropically to MSH.

4. 4, The presence of actD appears not to prevent the conversion of the hyperdispersed chromatin into the normal pattern.

     

Chromatin organization in the male germ line of Drosophila hydei

The chromatin organization in developing germ cells of Drosophila hydei males was studied with the highly sensitive DNA stain DAPI (4, 6-diamidino-2-phenylindole dichloride). The prophase of meiosis I is characterized by decondensed chromosomes and only late during this stage do they condense rapidly. The sex chromosomes show allocycly. During postmeiotic development the final condensation of chromatin is preceded by a cycle of condensation and subsequent decondensation. Meiotic chromosomes were studied in more detail after orcein staining. Pairing sites of the sex chromosomes could be localized in the distal end of the heterochromatic arm of the X chromosome and distally in both arms of the Y chromosome. The various heterochromatic parts of the genome condense differentially in meiosis. Chromatin reorganization was studied cytochemically with antibodies raised against histones H1 and H2A of D. melanogaster. The core histone H2A is present in spermatid nuclei until the late elongation stage. However, histone H1 is not found in the chromatin later than the early primary spermatocyte stage. Thus, chromatin reorganization during spermatogenesis in D. hydei is complex. The process is discussed with regard to possible functions.     

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI/SNF or NURF

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.     

Cytochemical properties of interphase chromatin condensed as a result of treatment with caffeine

Treatment of living cultured cells with caffeine (10 mg/ml, 2 h, 37 °C) brings about marked chromatin condensation which results in the appearance of small, distinct chromatin clumps in the majority of interphase nuclei. The changes taking place in the chromatin properties under the action of caffeine are rather similar to those observed in mitotic condensation (an increase in acridine orange and berberine binding and a sharp decrease in [³H]actinomycin D binding in situ; inhibition of [³H]uridine incorporation in vivo) and are reversible from the point of view of the criteria studied (nuclear morphology, ligand binding, [³H]uridine incorporation, culture viability). It is concluded that caffeine treatment can be regarded as a promising approach to the study of events occurring in chromatin condensation.     

M33 condenses chromatin through nuclear body formation and methylation of both histone H3 lysine 9 and lysine 27

Heterochromatin, a type of condensed DNA in eukaryotic cells, has two main categories: Constitutive heterochromatin, which contains H3K9 methylation, and facultative heterochromatin, which contains H3K27 methylation. Methylated H3K9 and H3K27 serve as docking sites for chromodomain-containing proteins that compact chromatin. M33 (also known as CBX2) is a chromodomain-containing protein that binds H3K27me3 and compacts chromatin in vitro. However, whether M33 mediates chromatin compaction in cellulo remains unknown. Here we show that M33 compacts chromatin into DAPI-intense heterochromatin domains in cells. The formation of these heterochromatin domains requires H3K27me3, which recruits M33 to form nuclear bodies. G9a and SUV39H1 are sequentially recruited into M33 nuclear bodies to create H3K9 methylated chromatin in a process that is independent of HP1α. Finally, M33 decreases progerin-induced nuclear envelope disruption caused by loss of heterochromatin. Our findings demonstrate that M33 mediates the formation of condensed chromatin by forming nuclear bodies containing both H3K27me3 and H3K9me3. Our model of M33-dependent chromatin condensation suggests H3K27 methylation corroborates with H3K9 methylation during the formation of facultative heterochromatin and provides the theoretical basis for developing novel therapies to treat heterochromatin-related diseases.     

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

Influenza neuraminidase (NA) proteins expressed in TK⁻ cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity.Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.     

Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation

We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that “drive” chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase. Received: 4 September 1997; in revised form: 14 September 1997 /Accepted: 14 September 1997     

PLANT MITOSIS PROMOTING FACTOR DISASSEMBLES THE MICROTUBULE PREPROPHASE BAND AND ACCELERATES PROPHASE PROGRESSION IN TRADESCANTIA

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase-arresting (met1) mutant ofChlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34^cdc2/cyclin B-like kinase. The enzyme was injected into living stamen hair cells ofTradescantia virginianain which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34^cdc2/cyclin B-like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase-cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically-active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.     

Changes of nuclear structure induced by increasing temperatures

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.     

Induction of Cyclin B and H1 Kinase Activity in Apoptotic PC12 Cells

The present study examines whether cyclin B may be involved in apoptosis of neuronally differentiated PC12 cells following withdrawal of NGF. Cyclin B mRNA increased approximately 10-fold 4 days after NGF withdrawal, as indicated by competitive RT/PCR. Sequencing of the PCR product confirmed that it was derived from cyclin B mRNA. Cyclin B protein increased in parallel with cyclin B mRNA, as shown by immunoblotting. Immunoprecipitation with anti-cyclin B antibody demonstrated that cyclin B was associated with H1K activity, which reached a maximum 5 days after NGF withdrawal. When proteins immunoprecipitated with anti-cyclin B antibody were immunoblotted with anti-PSTAIR antibody, a protein with apparent molecular weight of 34 kDa was detected. This protein was identified as p34^cdc2 on the basis of immunoreactivity with antibody against the C-terminal portion of mouse p34^cdc2. Since cyclin B/p34^cdc2 complexes are known to catalyze chromosomal condensation and nuclear envelope breakdown during mitosis, these results suggest that cyclin B/p34^cdc2 may play some role in the nuclear changes accompanying apoptosis of PC12 cells.     

The cell cycle dependent phosphorylation of histone H3 is correlated with the condensation of plant mitotic chromosomes

Mitotically dividing cells of Secale cereale, Hordeum vulgare and Vicia faba were studied by indirect immunofluorescence using an antibody recognizing phosphorylated histone H3. The study revealed the following features: (i) the H3 phosphorylation starts at prophase and ends at telophase in the pericentromeric chromatin, is associated with the condensation of mitotic chromosomes and is independent of the distribution of late replicating heterochromatin. (ii) Compared with other chromosome regions, the pericentromeric chromatin is histone H3 hyperphos- phorylated. (iii) The study of a semi-dicentric chromo- some revealed that only at intact centromeres is the chromatin hyperphosphorylated at H3.     

Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice

The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by high-titer antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT50] = 44 microg/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.