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.     

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.     

Competition between histone H1 and HMGN proteins for chromatin binding sites

The ability of regulatory factors to access their nucleosomal targets is modulated by nuclear proteins such as histone H1 and HMGN (previously named HMG-14/-17 family) that bind to nucleosomes and either stabilize or destabilize the higher-order chromatin structure. We tested whether HMGN proteins affect the interaction of histone H1 with chromatin. Using microinjection into living cells expressing H1–GFP and photobleaching techniques, we found that wild-type HMGN, but not HMGN point mutants that do not bind to nucleosomes, inhibits the binding of H1 to nucleosomes. HMGN proteins compete with H1 for nucleosome sites but do not displace statically bound H1 from chromatin. Our results provide evidence for in vivo competition among chromosomal proteins for binding sites on chromatin and suggest that the local structure of the chromatin fiber is modulated by a dynamic interplay between nucleosomal binding proteins.     

Copper effective binding with 32-62 and 94-125 peptide fragments of histone H2B

In an attempt to investigate the role of histone H2B in Cu(II) induced toxicity and carcinogenesis, we synthesized the terminally blocked peptides H2B_32-62 (SRKESYSVYVYKVLKQVH₄₈PDTGISSKAMGIM) and Η2Β_94-125 (IQTAVRLLLPGELAKH₁₁₀AVSEGTKAVTKYTSS), mimicking the N-terminal histone-fold domain and C-terminal tail of histone H2B, respectively and studied their interaction with Cu(II) ions by means of potentiometric titrations and spectroscopic techniques (UV-visible, CD and EPR). Both peptides, H2B_32-62 and H2B_94-125, interacted efficiently with Cu(II) ions, forming several species from pH 4 to 11, with His₄₈ and His₁₁₀ serving as anchors for metal binding. In H2B_32-62, the effective Cu(II) binding is emphasized by the formation of a soluble Cu(II)-H2B_32-62 complex, unlike the unbound peptide that precipitated over pH 7.9. At physiological pH, both peptides form tetragonal 3N species with a {N_Im, 2N⁻} coordination mode. At this pH, H2B_32-62 presented the formation of coordination isomers, differentiated by the presence in one of them, of an axial coordination of the carboxylate group of Asp₅₀. Copper binding with both H2B_32-62 and H2B_94-125 may induce a conformational change in the peptides' original structure. At physiological conditions, this effect may interfere with nucleosome's structure and dynamics, including the ubiquitination of Lys₁₂₀ which is linked to gene silencing.     

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.     

Exposure of histone antigenic determinants in chromatin.

The exposure of antigenic determinants of histones present in "native" chromatin was studied by: (1) testing their ability to elicit anti-histone antibodies and (2) measuring their ability to interact with anti-histone sera. To this end, antisera specific to purified histone fractions and to purified rat liver chromatin were elicited in rabbits. The anti-chromatin sera did not react with pure histone fractions and pure histone fractions F2b, F3, F2a1, and F2a2 failed to inhibit the complement fixation resulting from the binding of anti-chromatin to chromatin. These results suggest that in native chromatin, determinants in these histones are not immunogenic. Histone F1, however, inhibited the reaction between chromatin and anti-chromatin. Antisera elicited by histone fractions reacted weakly with "native" chromatin. The maximal complement fixations (obtained with 5-10 mug of chromatin DNA) were as follows: 60% with anti-F2b, 20% with anti-F1 and anti-F3, and less than 5% with either anti-F2a1 or anti-F2a2. Studies of the interaction between anti-histone antibodies and chromatin in which chromatin was used as an immunoadsorbent indicated that antibodies against different histones were adsorbed to a different degree by the same amount of chromatin. Differences in the immunoadsorbing capacity between sonicated and nonsonicated chromatin were found. Quantitative adsorbtion studies revealed that in the "native" chromatin structure, antigenic determinants of F1 and F2b were more available to interact with homologous antibody than those of F3 and F2a1 and that determinants in F2a2 were the least available. It could be calculated that the "equivalent antigenicity" of the histones in chromatin was 9.6% for F1, 3.2% for F2b, and 0.90% for F3 and F2a1. Upon sonication these values did not change for F1 but increased two-, three-, and fourfold for F2b, F3, and F2a1, respectively. Digestion of chromatin with trypsin totally abolished the ability of chromatin to adsorb anti-histone antibodies.     

H1 histone exchange is limited to particular regions of chromatin that differ in aggregation properties

Chromatin fragments produced by mild nuclease digestion were chromatographed on Bio-Gel A-50m to give fractions ranging in size from 0.4 to 30 kilobase pair-DNA. The fragments that were larger than about 8-10 nucleosomes accounted for 80% of the chromatin, and the H1/core histone ratio was constant throughout these fractions. When adjusted to 150 mM NaCl, aggregates precipitated in each fraction, the largest fragments yielding 60% and the smallest 25%. In all of these fractions, after aggregation was induced by NaCl, the H1/core histone ratio in the aggregation-resistant chromatin (S) was 0.7 that in the aggregated chromatin (P). To show that the H1 deficiency and aggregation resistance were not produced by transfer of H1 from little fragments to bigger one, big aggregation-resistant fragments were incubated with little aggregation-prone fragments in 75 mM NaCl for 2 h, and readjusted to 150 mM. The little aggregation-prone fragments retained their aggregatibility after exposure to big aggregation-resistant fragments. By mixing [3H]P with [14C]S and vice versa, incubating at 75 mM NaCl for 2 h, and separating P from S with 150 mM NaCl, it was demonstrated that H1 histone did not equilibrate between S and P. Similarly, mixing combinations of radioactive and unlabeled, big and little, S and P fractions, and fractionating by size after 2 h or more incubation at 75 mM NaCl, it was shown that H1 equilibrates between different S fragments, and between different P fragments, but not between S and P. The distribution of H1 variants between S and P fractions was not correlated with the affinity of the variants for DNA. The order of binding affinities was H10 greater than H1ab = H1c, but the deficits of H1's in the aggregation-resistant S fractions were ranked H1ab greater than H1c greater than H10. It is suggested that chromatin is a mosaic of aggregation-resistant and aggregation-prone regions which differ in H1 content quantitatively and qualitatively.     

The structural role of histone H1: properties of reconstituted chromatin with various H1 subfractions (H1-1, H1-2, and H1o).

A previous study on the distribution of histone H1 subfractions in chromatin suggested that these proteins differ in the protection they confer to DNA. To elucidate further this suggestion, reconstitution experiments were carried out with purified H1 subfractions (H1-1, H1-2, H1o) and H1-depleted chromatin. We have studied the structural properties of H1o as compared to those of other H1 fractions by electrophoretic analysis of DNA and mononucleosomes obtained after micrococcal nuclease digestion, thermal denaturation, and electron microscopy. The three fractions studied reassociate to H1-depleted chromatin. However, differences in the extent of DNA protection are observed between H1o and the other fractions: H1o induces a more rapid degradation of long oligomers into mononucleosomes; these mononucleosomes bearing H1o only, have a greater electrophoretic mobility; furthermore, thermal denaturation shows that a small fraction of DNA is less efficiently protected by H1o than by the other fractions. Electron microscopy, on the other hand, shows that these differences are not due to areas of chromatin devoid of H1o in the reconstitute and that the reconstituted samples are able, under proper ionic conditions, to refold in a higher-order structure.     

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.     

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.     

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.     

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.     

HMG-D and histone H1 alter the local accessibility of nucleosomal DNA

There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.     

Changes in the accessibility of chromatin antigenic determinants induced by different influences

The accessibility of protein antigenic determinants of rat thymocyte chromatin was studied in a reaction of complement fixation, using antisera from animals immunized with chromatin or non-histone proteins and control sera containing natural antichromatin IgG. It was shown that the bulk of the antigenic determinants of intact chromatin are inaccessible for antibodies. The reactivity of chromatin in the complement fixation assay increases after ultrasonication or irradiation in vitro as well as the enzymatic cleavage of chromatin down to nucleosomes and their oligomers in dying thymocytes in vivo. This effect can mainly be due to changes of chromatin compactization.