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.     

Histone H1 in the centromeric heterochromatin of Glyptotendipes barbipes larval polytene chromosomes

Immunofluorescent analysis with antibodies against histone H1 failed to detect H1 in the centromeric heterochromatin blocks of the polytene chromosomes of Glyptotendipes barbipes larvae. Centromeric regions were dissected microsurgically and acid-extracted. Electrophoresis in SDS and acid-urea gels revealed a band comigrating with H1 of calf thymus and of Gl. barbipes salivary gland nuclei. ELISA dot assay of the extracted material gave a positive reaction with anti-H1 monoclonal antibodies and with anti-H1 affinity-purified polyclonal antibodies. This shows that the centromeric heterochromatin contains histone H1 but packed in a way which prevents the H1 antigenic determinants from reacting in situ with the specific antibodies.     

Accessibility of histone H1° and its structural domains to antibody binding in extended and folded chromatin

The aim of this work was to study the accessibility of histone H1° and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1° which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1° which recognized epitopes located in the globular region of H1° and (3) anti-C-tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1° did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1° in chromatin was much less than that of histone H1.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - G-domain Globular domain - IgG Immunoglobulin G - SDS Sodium Dodecylsulphate     

Histone autoantigens in murine lupus. Definition of a major epitope within an accessible region of chromatin.

In SLE and in the (NZB x NZW)F1 murine model of this disease, IgG autoantibodies are frequently produced to DNA and histones. In the present study, we define a linear epitope on histone H2B that is recognized by (NZB x NZW)F1 mice. IgG antibodies from anti-H2B positive (but not anti-H2B negative) mice bound strongly to a peptide containing the first 15 N-terminal amino acids, a region that is exposed in chromatin. Competitive inhibition studies showed that the binding of autoantibodies to H2B in ELISA as well as the binding to soluble H2B was substantially blocked by this peptide. Studies with smaller peptides mapped the epitope to residues 3-12. Individual mice recognized different residues within this region, and a sequence search did not reveal proteins other than H2B that could elicit this spectrum of antibodies. Interestingly, these autoantibody specificities were not a component of those induced in preautoimmune mice by immunization with H2B/RNA complexes or with H2B peptide 1-30 containing the autoantigenic sequence. These findings argue that recognition of a specific N-terminal region of self histone contributes to the anti-H2B autoantibody response in lupus. Autoreactive B cells with specificity for this sequence seem to develop only after the autoimmune process has been initiated.     

Binding form of pollen mother cell protein in the nucleosomes of lily

We have previously reported the existence of pollen mother cell nuclear protein (PMCP) which appears during microsporogenesis in lily (Lilium spesiosum). It is very similar to mammalian testis specific H1 histone, H1t. In this paper, we describe the PMCP distribution in lily nucleosomes. Isolated nuclei were treated with micrococcal nuclease, and template active and inactive chromatin fractions were prepared. The nucleosome repeat length of pollen mother cells was determined to be 210 base pairs. The majority of the PMCP was found in the template inactive chromatin fraction, similar to other histones. PMCP was contained in the nucleosome monomer, but not in the core particle. However, PMCP was mainly found in the nucleosome dimer when slightly digested. Salt extraction from isolated nuclei indicated that PMCP and H1 histone share similar binding affinities to DNA. Judging from our results, it seems probable that PMCP links two core particles more strongly than H1 histone does. Since it is known that meiotic chromatin includes nick transferase and nuclease activity, one possible role of PMCP is the protection of its own chromatin. Other possible functions of PMCP are also discussed.     

Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation

Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes. The histone H3 kinase was not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity could be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity was regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibited kinase activity. The presence of PP1 on chromatin suggested that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generated an H3 kinase activity that was also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, we found that stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1.     

Nucleosomes containing histones H1 or H5 are closely interspersed in chromatin

The distribution of histones H1 and H5 along chromatin fibers has been examined in the nucleated hen erythrocyte. Nucleosome oligomers, produced by micrococcal nuclease digestion of nuclei, were sequentially reacted with affinity-chromatography purified rabbit anti-H5 and sheep anti-rabbit antibodies. Quantitation of the relative amounts of H1 and H5 in the precipitated and supernatant fractions as a function of the oligomer number was consistent with a close interspersion of both types of histones, probably a random one. This conclusion was supported by the immunoprecipitation of longer chromatin fibers. This pattern of distribution appears to apply both to bulk chromatin and to chromatin inactivated during the maturation of the erythrocyte.     

Histone crosslinking patterns indicate dynamic binding of histone H1 in chromatin

Crosslinking of histone H1 molecules to each other and to the core histones with bifunctional reagents in mouse liver nuclei and chromatin was compared with that under the conditions of random 'contacts' between these molecules. The patterns of crosslinking of the H1 subfractions (H1A, H1B, and H10) to each other in nuclei, chromatin and in solution at different ionic strengths due to random collisions were essentially the same. Moreover, the contacts between the H1 molecules were qualitatively the same in nuclei, chromatin and in solution also at the level of the chymotryptic halves of the H1 molecules. The contacts between the H1 molecules and the core histones in nuclei were similar to those obtained in chromatin at 70 mM NaCl, when H1 molecules readily migrate, and at 0.6 M NaCl, when H1 molecules are dissociated from chromatin. We conclude that spatial arrangement of H1 subfractions and mutual orientation of H1 molecules in isolated nuclei are random-like at least in terms of cross-linking. The static and dynamic models of histone H1 binding to chromatin compatible with the known data are considered. Although unequivocal verification of the models is not possible at present, the dynamic models do correspond better to recent data on the location of the histone H1 in nuclei and chromatin.     

High Mobility Group 1 Protein Is Not Stably Associated with the Chromosomes of Somatic Cells

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.     

Analysis of Histones Associated with Neuronal and Glial Nuclei Exhibiting Divergent DNA Repeat Lengths

Abstract: Total cerebral hemisphere nuclei purified from adult rabbit brain were subfractionated into neuronal and glial populations. Previous studies have shown that chromatin in neuronal nuclei is organized in an unusual nucleosome conformation compared with glial or kidney nuclei, i.e., a short DNA repeat length is present. We now analyze whether this difference in chromatin organization is associated with an alteration in the histone component of nucleosomes. Total histone isolated by acid/urea-protamine extraction of purified neuronal, glial, and kidney nuclei was analyzed by electrophoresis on SDS-polyacrylamide slab gels. Histone H1 that was selectively extracted from nuclei was also examined. Differences were not observed on SDS gels in the electrophoretic mobilities of histones associated with either the nucleosome core particle (histones H2A, H2B, H3, H4) or the nucleosome linker region (histone H1). Total histone and selectively extracted histone H1 were also analyzed on acid/urea slab gels that resolve histones on the basis of both molecular weight and charge differences. When analyzed in this system, differences with respect to electrophoretic mobility were not detected when comparing either selectively extracted histone H1 or total histone from neuronal and glial nuclei. Quantitative analyses were also performed and neuronal nuclei were found to contain less histone H1 per milligram DNA compared with glial or kidney nuclei. Neuronal nuclei also demonstrated a lower ratio of histone H1/core histone. These results suggest that the pronounced difference in chromatin organization in neuronal compared with glial nuclei, which is reflected by a short DNA repeat length in neurons, appears to be associated with quantitative differences in neuronal histone H1.     

Monoclonal antibodies against distinct determinants of histone H5 bind to chromatin

A series of monoclonal antibodies specific for distinguishable epitopes in chromosomal protein histone H5 were obtained from mice immunized with either free H5 or H5 . RNA complexes. The antibodies elicited by H5 could be distinguished from those elicited by H5 . RNA by their binding to native or acid-denatured H5, by their interaction with the globular region of H5, and by their cross-reactivity with H1o. The specificity of the antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting experiments. The antibodies could distinguish between H5 and the closely related histones H1 and H1o. The binding of some of the antibodies to the antigens was dependent on the type of assay used, suggesting nonrandom binding of the antigen to the solid supports used in ELISA and immunoblotting. Competitive ELISA experiments indicate that 8 of the 11 antibodies characterized bind to distinct epitopes. Three monoclonal antibodies bind to epitopes which are in close spatial proximity, causing mutual steric hindrance. The monoclonal antibodies bind to nuclei of fixed cells and to isolated chromatin, indicating that the epitopes are present both in the purified protein and in chromatin-complexed H5. These monoclonal antibodies can be used to study the organization of distinct regions of histones H5 and H1o in chromatin and chromosomes.     

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly(ADP-ribosyl)ation on core nucleosome structure

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin was analyzed by gel electrophoresis, electron microscopy, and velocity sedimentation. In parallel, the interaction of automodified poly(ADP-ribose) polymerase with native and H1-depleted chromatin was analyzed. In H1-depleted chromatin histone H2B becomes the major poly(ADP-ribose) histone acceptor protein, whereas in native chromatin histone H1 was the major histone acceptor. Poly(ADP-ribosyl)ation of H1-depleted chromatin prevented the recondensation of polynucleosomes reconstituted with exogenous histone H1. This is probably due to the presence of modified poly(ADP-ribose) polymerase and hyper(ADP-ribosyl)ated histone H2B. Indeed, about 40% of the modified enzyme remained associated with H1-depleted chromatin, while less than 1% of the modified enzyme was bound to native chromatin. The influence of poly(ADP-ribosyl)ation on the chromatin conformation was also studied at the level of nucleosome in using monoclonal and polyclonal antibodies specific for individual histones and synthetic peptides of histones. In native chromatin incubated in the presence of Mg2+ there was a drop in the accessibility of histone epitopes to monoclonal and polyclonal antibodies whereas upon poly(ADP-ribosyl)ation their accessibility was found to remain even in the presence of Mg2+. In poly(ADP-ribosyl)ated H1-depleted chromatin an increased accessibility of some histone tails to antibodies was observed.     

Core histone acetylation is regulated by linker histone stoichiometry in vivo

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

The localization of histone H3.3 in germ line chromatin of Drosophila males as established with a histone H3.3-specific antiserum

A rabbit antiserum, specific for the histone H3.3 replacement variant, was raised with the aid of a histone H3.3-specific peptide. Immuno blot experiments demonstrated the specificity of this polyclonal antiserum. In addition, we showed on immuno blots that two monoclonal antibodies isolated from mice with systemic lupus erythematosus (SLE) display strong reactivity with the H3.3 histone, but not with its replication-dependent counterparts. Our observations indicate that histone H3.3 might play a role as autoantigen in SLE. We used the histone H3.3-specific antiserum to characterize the germ line chromatin in cytological preparations of Drosophila testes, because our previous studies had shown that a histone H3.3-encoding gene is strongly expressed in the germ line of Drosophila males. The antiserum reacted with some of the lampbrush loops in spermatocytes and with chromatin of the postmeiotic germ cells of males. Our data indicate that histone H3.3 is not evenly distributed throughout the chromatin of germ cells, but is concentrated in distinct regions. Histone H3.3 disappears from the spermatid nuclei, along with the other core histones, during the late stages of spermatogenesis. In Drosophila polytene chromosomes, however, a rather uniform distribution of the histone H3.3 was observed. The possible role of histone H3.3 is discussed. Received: 12 May 1997 / Accepted: 4 July 1997