The condensation of chromatin and histone H1-depleted chromatin by spermine

At low ionic strength, spermine induces aggregation of native and H1-depleted chromatin at spermine/phosphate (Sp/P) ratios of 0.15 and 0.3, respectively. Physico-chemical methods (electric dichroism, circular dichroism and thermal denaturation) show that spermine, at Sp/P less than 0.15, does not appreciably alter the conformation of native chromatin and interacts unspecifically with all parts of chromatin DNA (linker as well as regions slightly or tightly bound to histones). In chromatin, the role of spermine could be more important in the stabilization of higher-order structure than in the condensation of the 30 nm solenoid. The addition of spermine to H1-depleted chromatin revealed two important features: (i) spermine can partially mimic the role of histone H1 in the condensation of chromatin; (ii) the core histone octamer does not appear to play any role in the aggregation process by spermine as DNA and H1-depleted chromatin aggregate at the same Sp/P ratio.     

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.     

The dynamics of histone H1 function in chromatin

Over 80% of the nucleosomes in chromatin contain histone H1, a protein family known to affect the structure and activity of chromatin. Genetic studies and in vivo imaging experiments are changing the traditional view of H1 function and mechanism of action. H1 variants are partially redundant, mobile molecules that interact with nucleosomes as members of a dynamic protein network and serve as fine tuners of chromatin function.     

SUMOylation of linker histone H1 drives chromatin condensation and restriction of embryonic cell fate identity

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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 distribution of the histone H1° in different brain cell types

H1^o levels were examined in two populations of nuclei from calf cerebral cortex which have chromatin of different repeat lengths. More H1^o was found in the nuclei with the shorter repeat length chromatin. These nuclei are also believed to be the more active in RNA synthesis of the two types. Thus H1^o contrasts with the avian erythrocyte-specific histone H5 in that the latter is associated with both increased repeat length and suppression of RNA synthesis. Since the central globular domains of H1^o and H5 are highly homologous, it is suggested that the non-homologous, extended ‘tails’ of H1^o and H5 are crucial to the function of these molecules.     

Characteristics of limited trypsinolysis of histone H5 in a solution and as a component of chromatin with different degrees of compactness

Trypsinolysis of histone H5 in solution and as a component of chromatin with different level of compactization was studied. It was demonstrated that the existence of supernucleosomal organization leads to a significant decrease of the degradation rate of histones H1 and H5 in comparison with histones H2A, H2B, H3 and H4. Analysis of trypsinolysis electrophoretic spectra of histone H5 revealed the existence of protease-resistant fragments in chromatin, but not in solution. These fragments contain not only the globular domain of histone H5 but also small-sized unstructured N- and/or C-terminal regions. The peptides were identified with the help of an immune serum specific for the globular region of histone H5. The possible role of resistant fragments in the nucleosomal organization of chromatin is discussed.     

Immunofractionation of chromatin regions associated with histone H1o

Two monoclonal antibodies, which were elicited against histone H5, bind to purified rat liver chromatin and to rat liver H1o but not to rat liver H1. The monoclonal antibodies were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to fractionate rat liver oligonucleosomes. Enzyme-linked immunoabsorbant assay (ELISA) and immunoblotting experiments indicate that the nucleosomes bound to the column were tenfold enriched in their content of H1o. Oligonucleosomes, prepared from the livers of either untreated or 3-methylcholanthrene-treated adult rats, were fractionated on the anti-H1o affinity column. The DNA purified from the unfractionated nucleosomes, from the unbound nucleosomes and from the nucleosomes which were bound to the column was examined with various 32P-labeled probes. A slight enrichment in H1o was detected in the coding region of the rat albumin gene. In contrast DNA which was bound to the column was significantly depleted in sequences hybridizing with total cellular RNA (which contains mostly ribosomal RNA) and with sequences hybridizing to the 3'-terminal region of a cytochrome P-450 gene, which is inducible by the chemical carcinogen 3-methylcholanthrene, regardless of whether isolated from control or from carcinogen-treated rat livers. Our experiments clearly demonstrate that chromatin can be efficiently immunofractionated. The results suggest that the H1o content of chromatin regions containing genes which are constitutively transcribed is not necessarily different from that of regions containing non-transcribed genes and that highly inducible genes may be segregated into chromatin regions which are depleted of H1o.     

The C-terminal domain is the primary determinant of histone H1 binding to chromatin in vivo

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.     

Histone H1 regulates chromatin condensation in Leishmania parasites

We investigated the functional role of the Leishmania histone H1 and demonstrate for the first time that addition of histone H1 has a strong effect on microccocal digestion, chromatin condensation of parasite nuclei and that its overexpression can modulate parasite infectivity in vivo.     

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.     

Assembly and properties of chromatin containing histone H1

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.     

Influence of histone H1 on chromatin structure

Removal of histone H1 produces a transition in the structure of chromatin fibers as observed by electron microscopy. Chromatin containing all histone proteins appears as fibers with a diameter of about 250 A. The nucleosomes within these fibers are closely packed. If histone H1 is selectively removed with 50-100 mM NaCl in 50 mM sodium phosphate buffer (pH 7.0) in the presence of the ion-exchange resin AG 50 W - X2, chromatin appears as "beads-on-a-string" with the nucleosomes separated from each other by distances of about 150-200 A. If chromatin is treated in the presence of the resin with NaCl at concentrations of 650 mM or more, the structural organization of the chromatin is decreased, yielding fibers of irregular appearance.     

On the binding of histone H1 in chromatin

Nucleosomal subunits isolated from rabbit thymus nuclei in 0.04 M K₂SO₄-0.02 M Tris, pH 7.4 were devoid of histone H1, while whole chromatin prepared in the same buffer contained the full complement of histone H1. The question is asked why histone H1 dissociates from the subunits but not from the high molecular weight material. We propose that, at physiological salt concentrations, histone H1 is not bound to linker DNA as depicted in the current models; rather, alternate attachment sites, present only in the polymer, are involved.     

Synergistic effect of histone H1 and nucleolin on chromatin condensation in mitosis: role of a phosphorylated heteromer

Repeated motifs, rich in basic residues, are characteristic of both the N-terminal domain of the nucleolus-specific protein, nucleolin, and the second half of the C-terminal domain of histone H1. These repeats are also the target for phosphorylation by the mitosis-specific p34cdc2 kinase. We have previously shown that synthetic peptides [(KTPKKAKKP)2 for histone H1 and (ATPAKKAA)2 for nucleolin] corresponding to these two repeated motifs are able to act in synergy to induce DNA hypercondensation (Erard et al., 1990). In order to determine the molecular basis of this synergistic interaction, we have studied the condensation of the homopolymer poly(dA).poly(dT) in the presence of the two synthetic peptides. Circular dichroism has been used to monitor the psi (+)-type condensation and has revealed that phosphorylation enhances the synergistic effect of the two peptides. Analysis of different combinations of the two peptides suggests that there is a direct interaction between them which is stabilized by phosphorylation. Furthermore, there is a striking correlation between the degree of homopolymer condensation and the stability of the heteromeric complex. Phosphorylation takes place on the threonine residues on the repeat motifs within a region which is likely to adopt a beta-turn structure. Circular dichroism and infrared spectroscopy provide evidence that phosphorylation stabilizes the beta-turn structure of both peptides, and computer modeling shows that this may be due to steric hindrance imposed by the phosphate group. We suggest that phosphorylated nucleolin and histone H1 interact through their homologous domain structured in beta-spirals in order to condense certain forms of DNA during mitosis.     

The interaction of the histone H1-related protein phi 0 with chromatin

Protein phi 0 is a unique protein which is present in the sperm of the sea cucumber, Holothuria tubulosa. It associates with histones, but its physiological role is unknown. From its amino acid composition and sequence, protein phi 0 can be considered as an H1-related protein. In this paper, we have studied its interaction with chicken erythrocyte chromatin particles of different complexity, from core particles to polynucleosomes. Addition of protein phi 0 results in marked chromatin insolubilization. The higher the molecular weight of the chromatin fragment, the lower is the phi 0/nucleosome molar ratio at which precipitation occurs, so that complete insolubilization of polynucleosomes is achieved at a phi 0/nucleosome molar ratio which is identical to that found in mature H. tubulosa spermatozoa. We have also found that the interaction of protein phi 0 with chromatin is cooperative. These findings contribute to clarification of the peculiar physico-chemical properties shown by H. tubulosa sperm chromatin and the role played by the phi 0 protein.