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.     

Mitosis-specific histone H3 phosphorylation in vitro in nucleosome structures

A mechanism of mitosis-specific enhancement of histone H3 phosphorylation was analyzed in vitro in terms of nucleosome structure. The incorporation of [32P]phosphate into DNA-bound H3 was approximately 5-7 times higher than in DNA-free H3 using the catalytic subunit of cAMP-dependent protein kinase. The two major N-terminal serine sites, including the mitosis-specific site (Ser10) and Ser28, were extensively phosphorylated in the DNA-bound forms. These phosphorylation patterns were identical to those of nucleosomal H3. In contrast, the H3 in DNA-free octamers was very slightly phosphorylated. The major site of H3 phosphorylation in DNA-free H3 was Thr118 in the C-terminus. Results indicate that DNA-binding is essential for the high level of mitosis-specific H3 phosphorylation, and that the nucleosome structure promotes H3 N-terminal phosphorylation in vitro. It also suggests the possibility that H1 prevents H3 phosphorylation during interphase of the cell cycle.     

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.     

The histone H5 gene is flanked by S1 hypersensitive structures.

The potential of the cloned histone H5 gene to form altered DNA structures has been examined by S1 nuclease digestion of supercoiled recombinant plasmids containing up to 8.8 kbp of chicken DNa. The three main nicking sites map at the upstream and downstream sequences flanking the structural gene. The cleavage sites share sequence homology, strand specificity, and do not seem to be single-stranded. The sequence of the S1-sensitive sites does not suggest that the fragments can adopt any of the known DNA secondary structures.     

In vitro induction of H1-H1 histone cross-linking by adenosine diphosphate-ribose polymers

It is well-known that H1-H1 interactions are very important for the induction of 30 nm chromatin fiber and that, among all posttranslational modifications, poly(ADP-ribosyl)ation is one of those capable of modifying chromatin structure, mainly through H1 histone. As this protein can undergo both covalent and noncovalent modifications by poly(ADP-ribosyl)ation, our aim was to investigate whether and how ADP-ribose polymers, by themselves, are able to affect the formation of H1-H1 oligomers, which are normally present in a condensed chromatin structure. The results obtained in our in vitro experimental system indicate that ADP-ribose polymers are involved in chromatin decondensation. This conclusion was reached as the result of two different observations: (a) H1 histone molecules can be hosted in clusters on ADP-ribose polymers, as shown by their ability to be chemically cross-linked, and (b) H1 histone has a higher affinity for ADP-ribose polymers than for DNA; ADP-ribose polymers compete, in fact, with DNA for H1 histone binding.     

[FeFe]-Hydrogenase models assembled into vesicular structures

Compartmentalization is a major prerequisite for the origin of life on earth according to Wächtershäuser “Iron-Sulfur-World”. The hypothesis is mainly based on an autocatalytic inorganic energy reproducing redox system consisting of iron and sulfur as requirement for the subsequent synthesis of complex organic structures. Here, we modified [FeFe]-hydrogenase models by means of covalent coupling to either oleic acid or the amphiphilic block copolymer polybutadiene-polyethyleneoxide (PB-PEO) and incorporated those into the membranes of vesicles composed of phospholipids (liposomes) or the unmodified amphiphilic polymer (polymersomes). We employed a [2Fe-2S] cluster as a hydrogenase model, since these structures are known to be suitable catalysts for the generation of H₂ in the presence of weak acids. Successful incorporation was confirmed by spectrophotometric iron quantification and the vesicles formed were characterized by size determination (photon correlation spectroscopy (PCS)), and zeta potential as well as by cryo-transmission electron microscopy (Cryo-TEM). The modified models could be incorporated into liposomes or polymersomes up to molar proportions of 3.15% and 28%, respectively. Due to the immobilization in vesicular bilayers the [FeFe]-hydrogenase models can even exhibit catalytic action under the particular conditions of the intravesicular microenvironment. Our results suggest that the vesicular systems described may be applied as a nanoreactor for the reduction of encapsulated substances by generating hydrogen and thus as a minimal cell model.     

Centromere identity maintained by nucleosomes assembled with histone H3 containing the CENP-A targeting domain

Active centromeres are marked by nucleosomes assembled with CENP-A, a centromere-specific histone H3 variant. The CENP-A centromere targeting domain (CATD), comprised of loop 1 and the alpha2 helix within the histone fold, is sufficient to target histone H3 to centromeres and to generate the same conformational rigidity to the initial subnucleosomal heterotetramer with histone H4 as does CENP-A. We now show in human cells and in yeast that depletion of CENP-A is lethal, but recruitment of normal levels of kinetochore proteins, centromere-generated mitotic checkpoint signaling, chromosome segregation, and viability can be rescued by histone H3 carrying the CATD. These data offer direct support for centromere identity maintained by a unique nucleosome that serves to distinguish the centromere from the rest of the chromosome.     

H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro

Nucleosome formation has been studied in a system containing relaxed Col E1 DNA, core histones and an extract of Drosophila embryos. The formation of nucleosomes was established by the introduction of supercoils into DNA. The degree of DNA supercoiling was shown to be higher if nucleosomes were assembled in the presence of the H1 histone, polylysine (Mr 20 000) or spermine. These agents do not stimulate relaxation and are the more effective the earlier they are added to the reaction. Thus, the H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro.     

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.     

H 1 histone and histone variants in Trypanosoma cruzi

Trypanosoma cruzi chromatin is not condensed in chromosomes during mitosis. In previous studies a characteristic H 1 was not found in SDS or in acid-urea-PAGE. Consequently, it was proposed that the particular behavior of T. cruzi chromatin in dividing cells was due to the absence of an H 1 histone. In the present work, histones from this parasite were systematically characterized by spectrofluorometric analysis, amino acid composition, PAGE in one and in two dimensions, differential extraction with PCA and TCA, immunological cross-reactivity with antisera, and immunoblotting. We conclude that T. cruzi contains all five histones, H 1 presenting solubility and immunological properties similar to those in other species, but with a particular electrophoretic mobility in Triton-PAGE. Thus an explanation other than the absence of H 1 should be offered in order to understand the behavior of T. cruzi chromatin during mitosis. Moreover, histone variants were described by two-dimensional PAGE. The presence of histone variants suggests that they may participate in the regulation of cell proliferation and differentiation of this parasite, as it has been postulated for higher eukaryotes.     

Organization and expression of H1 histone and H1 replacement histone genes

The H1 family is the most divergent subgroup of the highly conserved class of histone proteins [Cole: Int J Pept Protein Res 30:433–449, 1987]. In several vertebrate species, the H1 complement comprises five or more subtypes, and tissue specific patterns of H1 histones have been described. The diversity of the H1 histone family raises questions about the functions of different H1 subtypes and about the differential control of expression of their genes. The expression of main type H1 genes is coordinated with DNA replication, whereas the regulation of synthesis of replacement H1 subtypes, such as H1° and H5, and the testis specific H1t appears to be more complex. The differential control of H1 gene expression is reflected in the chromosomal organization of the genes and in different promoter structures. This review concentrates on a comparison of the chromosomal organization of main type and replacement H1 histone genes and on the differential regulation of their expression. General structural and functional data, which apply to both H1 and core histone genes and which are covered by recent reviews, will not be discussed in detail.     

Nucleosomes stacked with aligned dyad axes are found in native compact chromatin in vitro

In this study, electron tomograms of plunge-frozen isolated chromatin in both open and compacted form were recorded. We have resolved individual nucleosomes in these tomograms in order to provide a 3D view of the arrangement of nucleosomes within chromatin fibers at different compaction states. With an optimized template matching procedure we obtained accurate positions and orientations of nucleosomes in open chromatin in "low-salt" conditions (5 mM NaCl). The mean value of the planar angle between three consecutive nucleosomes is 70°, and the mean center-to-center distance between consecutive nucleosomes is 22.3 nm. Since the template matching approach was not effective in crowded conditions, for nucleosome detection in compact fibers (40 mM NaCl and 1 mM MgCl(2)) we developed the nucleosome detection procedure based on the watershed algorithm, followed by sub-tomogram alignment, averaging, and classification by Principal Components Analysis. We find that in compact chromatin the nucleosomes are arranged with a predominant face-to-face stacking organization, which has not been previously shown for native isolated chromatin. Although the path of the DNA cannot be directly seen in compact conditions, it is evident that the nucleosomes stack with their dyad axis aligned in forming a "double track" conformation which is a consequence of DNA joining adjacent nucleosome stacks. Our data suggests that nucleosome stacking is an important mechanism for generating chromatin compaction in vivo.     

Condensation of polynucleosome by histone H1 binding

The complete amino acid sequence of the oligomycin sensitivity-conferring protein (OSCP) of beef-heart mitochondria is reported. The protein contains 190 amino acids and has a molecular mass of 20 967. Its structure is characterized by a concentration of charged amino acids in the two terminal segments (N 1-77 and C 128-190) of the protein, whereas its central region is more hydrophobic. The earlier reported homology of the protein with the delta-subunit of E. coli F1, based on the terminal amino acid sequences of OSCP, is further substantiated.     

p34cdc2 phosphorylation sites in histone H1 are dephosphorylated by protein phosphatase 2A1.

The protein phosphatase activity in rat liver cytosol or nuclear extracts that dephosphorylates histone H1 which has been phosphorylated by p34cdc2 is inhibited completely by okadaic acid, but unaffected by inhibitor-2 or magnesium ions, demonstrating that the only enzyme in this tissue capable of dephosphorylating this substrate is a type 2A phosphatase. Fractionation of the cytosol by anion-exchange chromatography and gel filtration demonstrated that histone H1 phosphatase activity coeluted with the major species of protein phosphatase 2A, termed PP2A1 and PP2A2. PP2A1 was the most active histone H1 phosphatase, its histone phosphatase phosphorylase phosphatase activity ratio being 6-fold higher than PP2A2 and 30-fold higher than the free catalytic subunit PP2AC. It is concluded that PP2A1 is likely to be the enzyme which dephosphorylates p34cdc2-labelled histone H1 in vivo and that the A and B subunits which interact with PP2AC in this species each play a key role in facilitating dephosphorylation of this substrate. The results demonstrate that PP2A, in addition to being involved in suppressing the activation of p34cdc2 in vivo, can also function to reverse at least one of its actions.     

Hypophysiotropic activity of histone H3 in vitro

To assess the effect of histone H3 on pituitary hormone secretion, rat anterior pituitary (AP) cells were used and growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle stimulating hormone measured by radioimmunoassay. Incubation of cells with H3 (1, 6, and 30 microM) stimulated the release of all five hormones in a dose-dependent manner. This effect was blocked by preincubation of H3 with an anti-H3 antibody. Incubation of AP cells with 6 microM H3 in the presence of specific AP hormone secretagogues (GRP-6, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH)) showed additive effects on hormone secretion. Pharmacological experiments suggested that calcium- and diacylglycerol- (DAG) associated pathways, but not cAMP, participate in the hypophysiotropic activity of H3. Our results confirm previous evidence that histones may act as hypophysiotropic signals.     

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.