G1 phase-dependent nucleolar accumulation of human histone H1x

Background information: H1 histones are a protein family comprising several subtypes. Although specific functions of the individual subtypes could not be determined so far, differential roles are indicated by varied nuclear distributions as well as differential expression patterns of the H1 subtypes. Although the group of replication‐dependent H1 subtypes is synthesized during S phase, the replacement H1 subtype, H1°, is also expressed in a replication‐independent manner in non‐proliferating cells. Recently we showed, by protein biochemical analysis, that the ubiquitously expressed subtype H1x is enriched in the micrococcal nuclease‐resistant part of chromatin and that, although it shares common features with H1°, its expression is differentially regulated, since, in contrast to H1°, growth arrest or induction of differentiation did not induce an accumulation of H1x. Results: In the present study, we show that H1x exhibits a cell‐cycle‐dependent change of its nuclear distribution. This H1 subtype showed a nucleolar accumulation during the G₁ phase, and it was evenly distributed in the nucleus during S phase and G₂. Immunocytochemical analysis of the intranucleolar distribution of H1x indicated that it is located mainly in the condensed nucleolar chromatin. In addition, we demonstrate that the amount of H1x protein remained nearly unchanged during S phase progression, which is in contrast to the replication‐dependent subtypes. Conclusion: These results suggest that the differential localization of H1x provides a mechanism for a control of H1x activity by means of shuttling between nuclear subcompartments instead of a controlled turnover of the protein.     

Autonomous activation of histone H1 kinase, cortical activity and microtubule organization in one- and two-cell mouse embryos

The activation of M-phase promoting factor (MPF) in one-cell mouse embryo is independent from the nucleus. Other autonomous phenomena include the cortical activity observed at the end of the first cell cycle and the reorganization of the microtubule network. Here, we observed that the autonomous control of MPF activation is present also in two-cell mouse embryos (H1 kinase activity being higher in the first than in the second cell cycle). Moreover, the disappearance of the cortical activity in anucleated halves is observed when MPF activation takes place. The rounding up of the cytoplast and the mitotic reorganization of the microtubule network correlates with the maximum activity of H1 kinase in anucleated halves from one-cell embryos. In anucleated halves of two-cell stage blastomeres neither the cortical activity nor the microtubule reorganization were observed. The degree of activation of histone H1 kinase, and, as a consequence, the cortical activity and the microtubule reorganization, does not depend on the distribution of cyclin B. Finally, the level of cyclin B synthesis is similar in anucleated and nucleated halves derived from both one- and two-cell embryos.     

Possible role of H1 histone in replication timing

AT‐rich repetitive DNA sequences become late replicating during cell differentiation. Replication timing is not correlated with LINE density in human cells (Ryba et al. 2010). However, short and properly spaced runs of oligo dA or dT present in nuclear matrix attachment regions (MARs) of the genome are good candidates for elements of AT‐rich repetitive late replicating DNA. MAR attachment to the nuclear matrix is negatively regulated by chromatin binding of H1 histone, but this is counteracted by H1 phosphorylation, high mobility group proteins or, indirectly, core histone acetylation. Fewer MAR attachments correlates positively with longer average DNA loop size, longer replicons and an increase of late replicating DNA.     

Phosphorylation of histone H1 during the cell cycle of artichoke

Abstract. Histones have been extracted from tuber and cultured tuber explant material and separated by gel electrophoresis. Histone H1 is heterogenous with 3–4 components in addition to the widely recognized histone H1a and H1b. Using labelling procedures and alkaline phosphatase treatment, histone H1 has been shown to be phosphorylated on both serine and threonine residues and possibly other acid-labile linkages. Variations in histone H1 phosphorylation have been measured through the cell cycle and the evidence indicates enhanced phosphorylation occurring during the G2/M phase as in animal systems.     

Somatic histone H1 microinjected into fertilized mouse eggs is transported into the pronuclei but does not disrupt subsequent preimplantation development

We injected somatic subtypes of histone H1 into newly fertilized mouse eggs, which do not naturally contain this chromosomal protein, and examined the fate of the injected protein and its effect on preimplantation development of recipient eggs. Rhodamine-labelled H1 injected into the cytoplasm of 53 eggs was transported into the pronuclei in 51 cases, and this nuclear accumulation could be detected within 15 min of injection. Unlabelled histone H1, which was detected using immunofluorescence, was also transported following microinjection to the pronuclei, where it colocalized with the chromatin and remained associated with the nuclei following cleavage to the two-cell stage. Nuclear accumulation of injected H1 was inhibited when injected eggs were incubated in the presence of drugs that prevent mitochondrial electron transport or glycolysis, which indicates that nuclear transport occurs through an energy-dependent process, as previously observed in tissue culture cells. To determine whether the presence of somatic H1 in early embryonic nuclei would influence subsequent development, fertilized eggs were injected with an approximately physiological quantity (1–5 pg) of somatic H1 or, as controls, with another small basic protein, cytochrome c. Fifty-three eggs were injected with cytochrome c, of which 51 divided to the two-cell stage, and 32 (60%) reached the blastocyst stage, after 5 days in culture. One hundred and eleven eggs were injected with somatic H1, of which 95 divided to the two-cell stage, and 53 (48%) reached the blastocyst stage, after 5 days in culture. The two groups did not differ statistically (X², P > 0.1) with respect to the fraction of injected embryos that developed to the blastocyst stage. These results show that, although mouse embryos lack the somatic subtypes of histone H1 until the four-cell stage of development, they are able to progress through preimplantation development when these subtypes are present beginning at the one-cell stage. This may imply that the distinctive chromatin composition that characterizes early embryos of a variety of species is not essential for early development in mammals. © 1996 Wiley-Liss, Inc.     

Structure of transfection-active histone H1/DNA complexes

Relationships between the structure of transfecting complexes of histone H1 and DNA and their transfection efficiency were studied. Transfection activity proved to be connected to complex aggregates. Low speed centrifugation of the complexes resulted in loss of the transfection activity. The complexes/aggregates were active with high efficiency in a broad range of weight input ratios r _i (0.1<r _i<30). Using atomic force microscopy (AFM), the complexes were imaged at negative, nearly electroneutral and positive charge conditions. Electroneutral complexes at r _i=1 showed a multitude of different complex forms. Fibrillar, network-like and branched structures were frequently present in one complex. Strongly positive charged complexes had a toroidal appearance. All these different forms contributed to the high transfection efficiency. Cellular uptake is supposed to be by phagocytosis.     

Unambiguous identification of histone H1 in Trypanosoma cruzi

The existence of histone H₁ has been questioned in Trypanosomatids. We report here the presence of a histone H₁ in the chromatin of Trypanosoma cruzi. This protein was purified by narrow-bore reversed phase HPLC and its amino acid composition analyzed and compared with histones H₁ from other species. Furthermore, the purified chromosomal protein was digested with proteases and the amino acid sequences of the resulting peptides were analyzed by the automated Edman degradation. The sequences obtained were found to present a high degree of homology when compared to the carboxy terminal domain of other known histones H₁.     

Molecular characterization and expression of a tobacco histone H1 cDNA

We have isolated a 1104 bp tobacco cDNA clone (H1c12) which includes an 846 bp open reading frame. This encodes a polypeptide of 282 amino acid residues and represents the largest plant H1 histone identified so far. The structure of the deduced protein shows the classical tripartite organization of the H1-type linker histones. The expression of the tobacco H1 histone gene(s) corresponding to the H1c12 cDNA clone was examined during different developmental stages. We found that, at the level of steadystate mRNA, expression of gene(s) encoding this H1 histone was rapidly induced in germinating seeds. The H1 gene was expressed in all tissues examined. However, its expression was higher in tissues known to contain meristematic cells. Furthermore, in the leaves of mature plants accumulation of the H1 mRNA exhibits a very characteristic oscillation. This latter finding indicates that, at least in fully developed plants, the expression of this type of H1 histone gene(s) is modulated by a diurnal cycle.     

Possible role of histone H1 in the regulation of furin-dependent proprotein processing

Histone H1 and its C-terminal lysine rich fragments were recently found to be potent inhibitorsof furin,a mammalian proprotein convertase.However,its role in the regulation of furin-dependent proproteinprocessing remains unclear.Here we report that histone H1 efficiently blocks furin-dependent pro-yonWillebrand factor(pro-vWF)processing in a dose-dependent manner.Coimmunoprecipitation and immunof-luorescence studies confirmed that histone H1 could interact with furin,and the interaction mainly took placeon the cell surface.We noted that histone H1 was released from cells undergoing necrosis and apoptosisinduced by H_2O_2.Our findings suggested that histone H1 might be involved in extracellular and/or intracellu-lar furin regulation.     

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.     

Purification and characterization of histone H1 variants from human placenta

Three histone H1 variants were extracted from human placental tissue in a single process using a high-salt buffer solution, and purified by ion exchange, hydroxyapatite, and reversed-phase chromatography. In the first chromatographic step, a cation exchanger resin, SP-Sepharose FF, was used to remove impurities having molecular weights higher than those of histones. In the second chromatographic step, hydroxyapatite resin was used to remove impurities with relatively low molecular weights. A second round of cation exchange chromatography using high-grade HS POROS resin resulted in two main fractions, each of which appeared as a single band following SDS-PAGE. The first fraction showed a single peak in RP-HPLC, while the second fraction showed two main peaks. These three peaks were further separated and polished by semi-preparative RP-HPLC, and their molecular masses and sequences were determined using MALDI-TOF-MS and N-terminal amino acid sequencing, respectively. The sequences and masses of these three variants corresponded with those of histones H1.2, H1.4, and H1.5. Moreover, all three purified histone subtypes demonstrated cytotoxicity in an MTT assay.     

Water-deficit induction of a tomato H1 histone requires abscisic acid

Many genes are induced by periods of water deficit, and a subset of these are dependent on elevated ABA content for expression. A number of drought-induced genes are not induced in leaves of the ABA-deficient mutant flacca from tomato (Lycopersicon esculentum) but are induced in detached, wilted wild-type leaves and ABA-treated leaves of both genotypes. The nucleotide sequence of the cDNA and corresponding genomic DNA fragment of one of these genes, his1-s (formerly called le20), encodes an amino acid sequence that is rich in Lys, Ala, and Ser. The predicted protein contains the tripartite structure of H1 histone and is similar to other H1 histones, especially in the globular domain. Since, his1-s is more closely related to a stress-induced gene from Lycopersicon pennellii than to another H1 histone in the tomato genome it is considered a stress-induced variant of H1 histone. his1-s mRNA accumulated in vegetative plants in response to other abiotic stress treatments, including application of polyethylene glycol, and salt. The mRNA preferentially accumulated in leaves as compared to roots. his1-s mRNA accumulation was controlled during development; the level was higher in developing seeds of mature green fruit than in detached wilted leaves. H1 histones have been implicated in the general repression of gene expression and in the regulation of specific genes. The rapid accumulation of his1-s mRNA during stress may indicate that this unique, stress-induced H1 histone is involved in controlling gene expression during plant stress.     

A proposal for a coherent mammalian histone H1 nomenclature correlated with amino acid sequences.

Bio-Rex 70 chromatography was combined with reverse-phase (RP) HPLC to fractionate histone H1 zero and 4 histone H1 subtypes from human placental nuclei as previously described (Parseghian MH et al., 1993, Chromosome Res 1:127-139). After proteolytic digestion of the subtypes with Staphylococcus aureus V8 protease, peptides were fractionated by RP-HPLC and partially sequenced by Edman degradation in order to correlate them with human spleen subtypes (Ohe Y, Hayashi H, Iwai K, 1986, J Biochem (Tokyo) 100:359-368; 1989, J Biochem (Tokyo) 106:844-857). Based on comparisons with the sequence data available from other mammalian species, subtypes were grouped. These groupings were used to construct a coherent nomenclature for mammalian somatic H1s. Homologous subtypes possess characteristic patterns of growth-related and cAMP-dependent phosphorylation sites. The groupings defined by amino acid sequence also were used to correlate the elution profiles and electrophoretic mobilities of subtypes derived from different species. Previous attempts at establishing an H1 nomenclature by chromatographic or electrophoretic fractionations has resulted in several misidentifications. We present here, for the first time, a nomenclature for somatic H1s based on amino acid sequences that are analogous to those for H1 zero and H1t. The groupings defined should be useful in correlating the many observations regarding H1 subtypes in the literature.