A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily.

The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described. A cDNA clone of its gene, which was designated psp54, was also isolated. The psp54 cDNA contains an open reading frame coding for a 54.4-kDa polypeptide (p54). p16 corresponds to the C-terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known. The sequence of p54 is 60% identical with that of the precursor of a sucrose-binding soybean protein, and, to a lesser extent (31-34%), it shares homology with some storage proteins. p16 is also 30% homologous with Nhp2p, a yeast nuclear protein. The psp54 gene, present in a single copy in pea genome, starts being expressed during seed desiccation. Soon after rehydration in seed germination, p54 mRNA disappears and is no longer detectable in vegetative tissues, except in response to hydric stress (exposure to abscisic acid, osmolites or desiccation). p16 can be recovered from nuclei cross-linked to histone H3, when the disulfide bridges that occur in vivo are preserved. On the other hand, p16 shares some properties with dehydrins, which are thought to protect cellular structures against desiccation. We propose that the possible precursor polypeptide p54 belongs to the vicilin superfamily, members of which play a variety of roles. The function of p16 may be related to the protection of chromatin structure against desiccation during seed development.     

A multifunctional bicupin serves as precursor for a chromosomal protein of Pisum sativum seeds

The fact that the psp54 gene codes for p16, a seed chromatin protein of Pisum sativum, has been described previously. In the present paper it is shown that p54, the p16 precursor, also exists as a free polypeptide in pea and that it also yields p38, a second polypeptide from the N-terminal region of p54, which is co-localized at a subcellular level with p16. By using antibodies against pea p16 and p38, it was found that these proteins are present in the members of the tribe Viciae examined. Sequence analysis and 3D modelling indicates that p54 proteins belong to the cupin superfamily, and that they are related to sucrose binding proteins and, to a lesser extent, to vicilin-type seed storage proteins. Nevertheless, several distinctive characteristics of psp54 expression have been found: (i) the gene is differentially induced by ABA and several stress situations, in accordance with the presence of putative separate ABA and stress responsive elements in its promoter; (ii) the proteins are present in pods and seed coats, tissues of maternal origin; and (iii) p54 mRNA accumulates in the dry seeds. In view of both the functional properties of p54-derived proteins and the features of the psp54 gene expression, it is concluded that p54 represents a novel class within the cupin superfamily.     

Pathways mediating the nuclear import of histones H3 and H4 in yeast.

The correct assembly of chromatin is necessary for the maintenance of genomic stability in eukaryotic cells. A critical step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. Here we demonstrate that the nuclear import pathway of histones H3 and H4 is mediated by at least two karyopherins/importins, Kap123p and Kap121p. Cytosolic H4 is found associated with Kap123p and H3. Kap121p is also present in the H4-PrA-associated fractions, albeit in lesser amounts than Kap123p, suggesting that this Kap serves as an additional import receptor. We further demonstrate that cytosolic Kap123p is associated with acetylated H3 and H4. H3 and H4 each contain a nuclear localization signal (NLS) in their amino-terminal domains. These amino-terminal domains were found to be essential for the nuclear accumulation of H3 and H4-green fluorescent protein reporters. Each NLS mediated direct binding to Kap123p and Kap121p, and decreased nuclear accumulation of H3 and H4 NLS-green fluorescent protein reporters was observed in specific kap mutant strains. H3 and H4 are the first histones to be assembled onto DNA, and these results show that their import is mediated by at least two import pathways.     

Rapid dephosphorylation of H1 histones after apoptosis induction

H1 histones are involved in the formation of higher order chromatin structures and in the modulation of gene expression. Changes in chromatin structure are a characteristic initial feature of apoptosis. We therefore have investigated the histone H1 pattern of the human leukemic cell line HL60 undergoing programmed cell death, as induced by topoisomerase I inhibition. Histone H1 proteins were isolated and analyzed by high performance liquid chromatography and capillary zone electrophoresis. DNA fragmentation after apoptosis induction was monitored by agarose gel electrophoresis. The patterns of the three H1 histone subtypes extractable from apoptotic HL60 cells significantly differed from those of control cells in showing a decrease of phosphorylated H1 subtypes and an increase of the respective dephosphorylated forms. This dephosphorylation of H1 histones could be observed already 45 min after apoptosis induction and preceded internucleosomal DNA cleavage by approximately 2 h. We conclude that during apoptotic DNA fragmentation, the H1 histones become rapidly dephosphorylated by a yet unknown protein phosphatase.     

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.     

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.     

Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab,Eriocheir sinensis

During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.     

A role for nucleosome assembly protein 1 in the nuclear transport of histones H2A and H2B

Import of core histones into the nucleus is a prerequisite for their deposition onto DNA and the assembly of chromatin. Here we demonstrate that nucleosome assembly protein 1 (Nap1p), a protein previously implicated in the deposition of histones H2A and H2B, is also involved in the transport of these two histones. We demonstrate that Nap1p can bind directly to Kap114p, the primary karyopherin/importin responsible for the nuclear import of H2A and H2B. Nap1p also serves as a bridge between Kap114p and the histone nuclear localization sequence (NLS). Nap1p acts cooperatively to increase the affinity of Kap114p for these NLSs. Nuclear accumulation of histone NLS-green fluorescent protein (GFP) reporters was decreased in deltanap1 cells. Furthermore, we demonstrate that Nap1p promotes the association of the H2A and H2B NLSs specifically with the karyopherin Kap114p. Localization studies demonstrate that Nap1p is a nucleocytoplasmic shuttling protein, and genetic experiments suggest that its shuttling is important for maintaining chromatin structure in vivo. We propose a model in which Nap1p links the nuclear transport of H2A and H2B to chromatin assembly.     

Replacement of histone H1 by H5 in vivo does not change the nucleosome repeat length of chromatin but increases its stability.

In vivo competition between histones H1 and H5 for chromatin has been studied in rat sarcoma XC10 cells transfected with a glucocorticoid responsive MMTV-H5 gene. Activation of H5 expression results in accumulation of H5 in the nuclei where it partially replaces H1. H5 displaces H1 from its primary binding sites presumably during chromatin replication and also binds with high affinity to secondary chromatin sites normally not occupied by H1. Replacement of H1 by H5 to levels similar to those of mature chicken erythrocytes does not alter the nucleosome repeat length of chromatin. This indicates that H5 is not solely responsible for the increase in nucleosome spacing of maturing erythroid cells. Exchange of H1 by H5 in vivo or in vitro results in a higher compaction/stability of chromatin.     

The complete amino-acid sequence of a trout-testis non-histone protein, H6, localized in a subset of nucleosomes and its similarity to calf-thymus non-histone proteins HMG-14 and HMG-17.

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].     

Interactions of Hsp90 with histones and related peptides

The 90 kDa heat shock protein (Hsp90) induces the condensation of the chromatin structure [Csermely, P., Kajtár, J., Hollósi, M., Oikarinen, J., and Somogyi, J. (1994) Biochem. Biophys. Res. Commun. 202, 1657-1663]. In our present studies we used surface plasmon resonance measurements to demonstrate that Hsp90 binds histones H1, H2A, H2B, H3 and H4 with high affinity having dissociation constants in the submicromolar range. Strong binding of the C-terminal peptide of histone H1 containing the SPKK-motif and a pentaeicosa-peptide including the Hsp90 bipartite nuclear localization signal sequence was also observed. However, a lysine/arginine-rich peptide of casein, and the lysine-rich platelet factor 4 did not display a significant interaction with Hsp90. Histones and positively charged peptides modulated the Hsp90-associated kinase activity. Interactions between Hsp90, histones, and high mobility group (HMG) protein-derived peptides raise the possibility of the involvement of Hsp90 in chromatin reorganization during steroid action, mitosis, or after cellular stress.     

Histones H2a, H2b, H3, and H4 form a tetrameric complex in solutions of high salt.

In 2 M NaCl, histones H2b, H2a, H3, and H4 form a heterotypic tetrameric complex made up of one chain of each histone. This complex has been analyzed by hydrodynamic techniques. It is indistinguishable from histones in chromatin by its resistance to trypsin, pattern of reactivity with 125I. and ability to form specific crosslinked products after treatment with formaldehyde. It is proposed that this complex is responsible for protecting the small DNA fragments produced by exhausting nuclease digestion of nuclei and that on the average two of these complexes protect the larger 180-200 base pair unit produced by partial treatment of nuclei with nuclease.     

Dynamic behavior of histone H1 microinjected into HeLa cells

Histone H1 was purified from bovine thymus and radiolabeled with tritium by reductive methylation or with 125I using chloramine-T. Red blood cell-mediated microinjection was then used to introduce the labeled H1 molecules into HeLa cells synchronized in S phase. The injected H1 molecules rapidly entered HeLa nuclei, and a number of tests indicate that their association with chromatin was equivalent to that endogenous histone H1. The injected molecules copurified with HeLa cell nucleosomes, exhibited a half-life of approximately 100 h, and were hyperphosphorylated at mitosis. When injected HeLa cells were fused with mouse 3T3 fibroblasts less than 10% of the labeled H1 molecules migrated to mouse nuclei during the next 48 h. Thus, the intracellular behavior of histone H1 differs markedly from that of high mobility group proteins 1 and 2 (HMG1 and HMG2), which rapidly equilibrate between human and mouse nuclei after heterokaryon formation (Rechsteiner, M., and L. Kuehl, 1979, Cell, 16:901-908; Wu, L., M. Rechsteiner, and L. Kuehl, 1981, J. Cell Biol, 91: 488-496). Despite their slow rate of migration between nuclei, the injected H1 molecules were evenly distributed on mouse and human genomes soon after mitosis of HeLa-3T3 heterokaryons. These results suggest that although most histone H1 molecules are stably associated with interphase chromatin, they undergo extensive redistribution after mitosis.