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.     

Immunochemical analysis of histone H1 and H5 from pigeon erythroid cells]

An antiserum with the antibody titer of 1 : 4096 was obtained by immunization of rabbits with the tRNA-histone H5 complex from pigeon erythrocytes. The specificity of the antiserum was studied quantitatively from the reaction of the complement binding to a homologous antigen (histone H5) and its modifications (I, II, III), differing in the degree of phosphorylation. It was shown that phosphorylation of histone H5 increases the ability of the antigen to bind to antibodies, which is especially well-pronounced at the antiserum dilutions as high as 20480. The comparison of the antigenic properties of histones H5 from pigeon and chicken erythrocytes revealed beside structural differences of the proteins the presence of common antigenic determinants. A similar observation was made when histones H5 and H1 from pigeon erythrocytes were compared. Histone H1 from chicken erythrocytes and histone H1 from calf thymus did not produce criss-cross reactions with antiserum H5.     

Species variability of N-terminal sequence of avian erythrocyte-specific histone H5

A comparison of the N-terminal amino acid sequence of H5 isolated from chicken, quail, duck, goose and pigeon shows considerable sequence variation. With the possible exception of the very lysine-rich histone H1, H5 variation is much more extensive than that reported previously for other histones. The observed sequence diversity is in agreement with the known taxonomic grouping of these birds.     

Insights into erythroid differentiation obtained from studies on avian erythroblastosis virus

Analysis of the oncogenes v-erbB and v-erbA and their normal proto-oncogene counterparts has revealed several novel aspects of erythroid differentiation. A new erythroid progenitor capable of extended self-renewal has been described, tyrosine kinase receptors and steroid hormone receptors have been found to cooperate in controlling self-renewal, and dramatic alterations in the cell cycle have been found to accompany induction of terminal differentiation.     

The histone H1°/H5 variant and terminal differentiation of cells during development of Xenopus laevis

The maintenance of the differentiated condition is supposed to be associated with the presence of a histone of the H1(0)/H5 subclass. If the H1(0)/H5 variant has an important role in differentiation distinct from that of H1, it should display differential expression in time and position during development. Here we report that this prediction is verified during Xenopus laevis development, in which tadpoles exhibit a very characteristic, developmentally regulated pattern of histone H1(0)/H5 expression that is different for the derivatives of each embryonic germ layer. However, the pattern of appearance of this variant during development does not reflect a simple correlation between its presence and the state of differentiation. Therefore, these results are pertinent to current ideas on differentiation and the involvement of lysine-rich histones in the repression of eukaryotic genes.     

The role of the central globular domain of histone H5 in chromatin structure

Histone H5 contains three tyrosines in the central, apolar region of the molecule. All three tyrosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain in essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result is a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higher-order chromatin structure.     

Chromatin on a membrane: accessibility of histone H5 for antibodies in the supernucleosomal structure

Histone H5 accessibility for the antibodies in chromatin was studied. Chromatin was immobilised on the nitrocellulose membrane in conditions which provide different levels of its compactization. Antiserum specific to the globular domain of histone H5 was used. It was shown, that for establishing real protection of histone H5 in the supernucleosomal structure it is necessary to use long fibers of chromatin. Their linking to the membrane must occur by a minimum quantity of points. It was established, that histone H5 is 5 times more accessive in the preparations of dispersed chromatin (low ionic strength) then in chromatin with the supernucleosomal organization (physiological ionic strength). We suppose that the small level of accessibility of histone H5 for the antibodies in the compact chromatin can be explained by some disruptions in the supernucleosomal organization. On the contrary, the long equable solenoid of nucleosomes provides complete protection of histone H5. In accordance with the results obtained, the model of ordered packaging of nucleosomes in the solenoid is discussed. In this model the point of entrance and exit of DNA on the nucleosomes, fixed by globular region of histone H5, is localized inside the solenoid.     

Restricted expression of an MHC alloantigen in cells of the erythroid series: A specific marker for erythroid differentiation

The spectrum of reactivity with various types of cells of a monoclonal antibody (CH-4) which detects a private MHC antigen of chickens was analysed. CH-4 agglutinates only RBCs that possess the B² (MHC) haplotype. A new rosetteforming cell (RFC) assay was devised to detect individual cells (excluding RBCs) that possess the CH-4 specificity on their cell surfaces. RBCs that have CH-4 chemically coupled to their surfaces attach to, and form rosettes with, B² antigen-bearing cells. Most non-RBC RFC were detected in active erythropoietic organs (adult bone marrow and embryonic spleen), and none were found in organs where erythropoiesis does not occur: adult thymus and bursa. Preincubation of bone marrow cells with CH-4 plus complement almost completely inhibits their capacity to form CFU-E without affecting their ability to form GM-CFU. In addition, CH-4 plus complement does not inhibit the capacity of B²/B² lymphocytes to induce a graft-versus-host reaction under conditions where anti-B² lymphocyte alloantisera are completely inhibitory. Our results strongly suggest that CH-4 monoclonal antibodies detect a private specificity on a gene product of the B-G locus whose expression is restricted to erythroid stem cells and erythrocytes.     

The N-terminally acetylated form of mammalian histone H1(o), but not that of avian histone H5, increases with age

We report here on the HPCE separation of two chicken H5 histones, which do not show the heterogeneity (Gln/Arg) at residue 15 first found by Greenaway and Murray [Greenaway and Murray (1971) Nat. New Biol. 229, 233-238]. The two subfractions obtained were identified using reversed-phase HPLC, hydrophilic interaction HPLC, Edman degradation, and MALDI-MS analysis. We found that the two H5 subcomponents differ only by an acetylated (designated H5a) and an unacetylated N-terminus (H5b). In contrast to the N-terminally acetylated form of rat kidney histone H1(o), which increased by about 40% with aging of the animal, the corresponding form of chicken H5 did not: the ratio N-terminally acetylated: unacetylated remained constant (30:70) when histone H5 was extracted from erythrocytes of newly hatched chickens and from adult chickens, respectively. The HPCE technique used in this investigation represents a quick and convenient method for analyzing N-terminally acetylated proteins in the presence of unacetylated forms.     

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.