The distribution of histone H1 subfractions in chromatin subunits.

Rat liver chromatin was digested with micrococcal nuclease to various extents and fractionated into nucleosomes, di and trimers of nucleosomes on an isokinetic sucrose gradient. In conditions under which degradation of linker DNA within the particles was limited, the electrophoretic analysis of the histone content showed that the overall content of H1 histone increased from nucleosomes to higher order oligomers. Moreover, the histone H1 subfractions were found unevenly distributed among the chromatin subunits, one of them, H1--3 showing most variation. A more regular distribution of these subfractions was found in subunits obtained from a more extended digestion level of chromatin. It is suggested that the H1 subfractions differ in the protection they confer upon DNA.     

Heterogeneity of chromatin subunits in vitro and location of histone H1.

Chromatin subunits ("nucleosomes") which were purified by sucrose gradient centrifugation of a staphylococcal nuclease digest of chromatin have been studied. We found that such a preparation contains nucleosomes of two discrete types which can be separated from each other by polyacrylamide gel electrophoresis. Nucleosome of the first type contains all five histones and a DNA segment of approximately 200 base pairs long, whereas nucleosome of the second type lacks histone H1 and its DNA segment is approximately 170 base pairs long, i.e., about 30 base pairs shorter than the DNA segment of the nucleosome of the first type. Purified dimer of the nucleosome also can be fractionated by gel electrophoresis into three discrete bands which correspond to dinucleosomes containing two molecules of histone H1, one and no H1. These and related findings strongly suggest that the H1 molecule is bound to a short (approximately 30 base pairs) terminal stretch of the nucleosomal DNA segment which can be removed by nuclease (possibly in the form of H1-DNA complex) without any significant disturbance of main structural features of the nucleosome.     

Effects of cell cycle dependent histone H1 phosphorylation on chromatin structure and chromatin replication.

We have reconstituted salt-treated SV40 minichromosomes with differentially phosphorylated forms of histone H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies revealed a clear difference between minichromosomes reconstituted with S-phase histone H1 compared with histone H1 from G0- or M-phase cells, indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using reconstituted minichromosomes as substrate in the SV40 in vitro replication system, we measured a higher replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 compared with G0- or M-phase histone H1. These data indicate that the chromatin structure induced by the phosphorylation of histone H1 influences the replication efficiency of SV40 minichromosomes in vitro.     

Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly

We find that the remodeling of the condensed Xenopus laevis sperm nucleus into the paternal pronucleus in egg extracts is associated with phosphorylation of the core histones H2A, H2A.X and H4, and uptake of a linker histone B4 and a HMG 2 protein. Histone B4 is required for the assembly of chromatosome structures in the pronucleus. However neither B4 nor core histone phosphorylation are required for the assembly of spaced nucleosomal arrays. We suggest that the spacing of nucleosomal arrays is determined by interaction between adjacent histone octamers under physiological assembly conditions.     

Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl₂ and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation.     

Content of histone H1 and histone phosphorylation in relation to the higher order structures of chromatin in Drosophila

The amount of histone H1 relative to core histones has been determined in three Drosophila species (D. melanogaster, D. texana and D. virilis) in chromatin from several tissues differing in chromatin structure and genetic activity. Low levels of H1 were found in relatively undifferentiated, early embryos as well as in a line of cultured cells. In late embryos the content of H1 was highest in D. virilis which possesses larger amounts of and a partially more compacted constitutive heterochromatin than the two other species. Polytene chromatin from larval salivary glands showed increased levels of H1 compared with diploid chromatin and the degree of phosphorylation of this histone was relatively low. The degree of phosphorylation of H2A was found to be drastically reduced in polytene as compared with diploid embryonic chromatin, which parallels the extensive underreplication of constitutive heterochromatin. Also, in diploid chromatin a qualitative correlation was observed between the relative amounts of heterochromatin and the levels of H2A phosphorylation. These findings suggest a connection between H2A phosphorylation and heavy compaction of interphase chromatin.     

Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1.

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.     

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants

Chromatin immunoprecipitation (ChIP) is a powerful tool for the characterization of covalent histone modifications and DNA-histone interactions in vivo. The procedure includes DNA-histone cross-linking in chromatin, shearing DNA into smaller fragments, immunoprecipitation with antibodies against the histone modifications of interest, followed by PCR identification of associated DNA sequences. In this protocol, we describe a simplified and optimized version of ChIP assay by reducing the number of experimental steps and isolation solutions and shortening preparation times. We include a nuclear isolation step before chromatin shearing, which provides a good yield of high-quality DNA resulting in at least 15 mug of DNA from each immunoprecipitated sample (from 0.2 to 0.4 g of starting tissue material) sufficient to test > or =25 genes of interest. This simpler and cost-efficient protocol has been applied for histone-modification studies of various Arabidopsis thaliana tissues and is easy to adapt for other systems as well.     

Immunochemical detection of changes in chromatin subunits induced by histone H4 acetylation.

Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non-acetylated H3, H2A, and H2B with either mono-, di-, or tri-acetylated H4 isolated from cuttle -fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C-terminal) peptide of H3 was considerably decreased when di- or tri-acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.     

Use of bifunctional reagents in the study of histones topography in chromatin]

The ability of bifunctional reagents--diimidoesters of sebacinic acid and tetranitromethane--to cross-link the histones in rat thymus chromatin was studied. The histone polymers formed were extracted with 0,8 N H2SO4 and analyzed by polyacrylamide gel electrophoresis. The results obtained suggest that in chromatin some histone fractions are localized in immediate proximity to each other. This phenomenon may probably account for the formation of chromatin structure and its functioning in the cell.     

Immunochemical localization of the C-terminal hexapeptide of histone H3 at the surface of chromatin subunits.

The C-terminal hexapeptide of histone H3 of chicken erythrocytes (residues 130-135) corresponding to the sequence Ile-Arg-Gly-Glu-Arg-Ala ( IRGERA ) was prepared by solid-phase peptide synthesis and, after coupling to bovine serum albumin, was used to elicit antibodies in rabbits. The antigenic activity of the synthetic peptide IRGERA was found to be very similar to that of the natural CN3 fragment (residues 121-135), and it inhibited the H3-anti H3 reaction in complement fixation, solid-phase radioimmunoassay, and enzyme-linked immunosorbent assay. Antibodies induced by IRGERA were found to bind equally well to IRGERA coupled to hemocyanin, to the intact H3 molecule, and to chromatin subunits (nucleosomes and core particles). The results demonstrate that the C-terminal hexapeptide of histone H3 is located at the surface of chromatin subunits and agree with current models proposed for the spatial organization of the chromatin core particle.     

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.     

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.     

Monoclonal antibodies against histone H5. Epitope mapping and binding to chromatin

Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed.     

Asymmetry of chromatin subunits probed with histone H1 in an H1-DNA complex

Treatment of nucleosomes with a low concentration of sodium dodecyl sulfate removed all proteins except histone H1 from DNA, thus confirming our previous observation on sheared chromatin. No redistribution of H1 occurred during this procedure for isolation of the H1-DNA complex. The H1-DNA complex was isolated from a nucleosome monomer, doubly labeled in its protein and DNA and fractionated according to the length of DNA, and then the distribution of H1 was analyzed quantitatively. The results indicated that the monomer consisted of two subspecies, one containing 160 base pairs of DNA and one molecule of H1, and the other containing 140 base pairs of DNA and no H1. Since no monomer with two molecules of H1 was found, it is concluded that the nucleosome core has a binding site for H1 on only one side, and thus that the nucleosome is not a dyad.     

Heterochromatin and histone phosphorylation

Histone phosphorylation and nuclear structure have been compared in cultured cell lines of two related species of deer mice, Peromyscus crinitus and Peromyscus eremicus, which differ greatly in their heterochromatin contents but which contain essentially the same euchromatin content. Flow microfluorometry measurements indicated that P. eremicus contained 36% more DNA than did P. crinitus, and C-band chromosome staining indicated that the extra DNA of P. eremicus existed as constitutive heterochromatin. Two striking differences in interphase nuclear structure were observed by electron microscopy. Peromyscus crinitus nuclei contained small clumps of heterochromatin and a loose, amorphous nucleolus, while P. eremicus nuclei contained large, dense clumps of heterochromatin and a densely structured, well defined, nucleolonema form of nucleolus. Incorporation of ³²PO₄ into histones indicated that the steady-state phosphorylation of H1 was identical in P. crinitus and P. eremicus cells. In contrast, the phosphorylation rate of H2a was 58% greater in the highly heterochromatic chromatin of P. eremicus cells than in the lesser heterochromatic chromatin of P. crinitus cells, suggesting an involvement of H2a phosphorylation in heterochromatin structure. It is suggested that the three histone phosphorylations related to cell growth (H1, H2a, and H3) may be associated with different levels of chromatin organization: H1 interphase phosphorylation with some submicroscopic (molecular) level of organization, H2a phosphorylation with a higher level of chromatin organization found in heterochromatin, and H3 and H1 superphosphorylation with the highest level of chromatin organization observed in condensed chromosomes.     

Use of antibodies in the analysis of connexin 43 turnover and phosphorylation

A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the 41-kDa protein was detectable in heart homogenates. Cultured rat leptomeningeal cells expressed high levels of the gap junction protein Cx43 and were used to analyze its turnover and phosphorylation. Pulse-chase experiments in leptomeningeal cells with [(35)S]methionine indicated that the 41-kDa form of connexin 43 was the first immunoprecipitable translation product. Radiolabel subsequently appeared in the lower band of the doublet at 43 kDa, followed by a shift into the higher band and turnover of the protein with a t(1/2) of 2.7 h. Pulse-chase labeling with [(32)P]P(i) indicated that phosphorylation of connexin 43 was limited to the 43-kDa protein, with a t(1/2) of 1.7 h. Treatment with alkaline phosphatase shifted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on Western blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for most, if not all, of Cx43 phosphorylation in this system.