Interaction Of H1 Research Articles

Co-operative interactions of oligonucleosomal DNA with the H1e histone variant and its poly(ADP-ribosyl)ated isoform.

H1 histone somatic variants from L929 mouse fibroblasts were purified by reverse-phase HPLC. We analysed the ability of each H1 histone variant to allow the H1-H1 interactions that are essential for the formation of the higher levels of chromatin structure, and we investigated the role played by the poly(ADP-ribosyl)ation process. Cross-linking analysis showed that H1e is the only somatic variant which, when bound to DNA, is able to produce H1-H1 polymers; the size of polymers was decreased when H1e was enriched in its poly(ADP-ribosyl)ated isoform. Measurement of the methyl-accepting ability in native nuclei compared with nuclei in which poly(ADP-ribosyl)ation was induced showed that the poly(ADP-ribosyl)ated H1 histone had not been removed from linker regions, in spite of its different interaction with DNA.

A preference of histone H1 for methylated DNA.

We have identified a clear preference of histone H1 for CpG-methylated DNA, irrespective of DNA sequence. The conditions under which this preference is observed allowed cooperative binding of H1; the H1-DNA complexes formed were shown earlier to be 'tramlines' of two DNA duplexes bridged by an array of H1 molecules, and multiples of these. The preference for methylated DNA is clear in sedimentation assays, which also show that the preference is greater with increased methylation level, and in gel retardation assays with an oligonucleotide containing a single methyl-CpG pair; it is shared by the globular domain which also binds cooperatively to DNA. A small intrinsic preference of H1 for methylated DNA is also apparent in Southwestern assays where the immobilized H1 presumably cannot bind cooperatively. Methylated DNA in H1-DNA complexes was partially protected (relative to unmethylated DNA) against digestion by MspI but not by enzymes whose cutting sites were not methylated, consistent with a direct interaction of H1 with methylated nucleotides; this was also true of GH1-DNA complexes. H1 variants (spH1 and H5) from transcriptionally repressed nuclei have a stronger preference than H1 for methylated DNA, suggesting that this may be relevant to the stabilization of chromatin higher order structure and transcriptional repression.

Binding of Histone H1 to DNA Is Indifferent to Methylation at CpG Sequences

The possibility that histone H1 binds preferentially to DNA containing 5-methylcytosine in the dinucleotide CpG is appealing, as it could help to explain the repressive effects of methylation on gene activity. In this study, the affinity of purified H1 for methylated and non-methylated DNA sequences has been tested using both naked DNA and chromatin. Based on a variety of assays (bandshifts, filter-binding assays, Southwestern blots, and nuclease sensitivity assays), we conclude that H1 has no significant preference for binding to naked methylated DNA. Similarly, H1 showed the same affinities for methylated and non-methylated DNA when assembled into chromatin in a Xenopus oocyte extract. Thus potential cooperative interaction of H1 with polynucleosomal complexes is not enhanced by the presence of DNA methylation.

Binding of histones H1 and H5 and their globular domains to four-way junction DNA.

We have compared chicken erythrocyte linker histones H1 and H5 binding to a synthetic four-way DNA junction. Each histone binds to form a single complex, with an affinity which permits competition against a large excess of linear duplex DNA. The affinity of H5 is higher than that of H1. The globular domain from either protein will also bind strongly, but in this case multiple binding occurs. Binding of intact H1 is inhibited by cations: Mg2+ and spermidine are very effective, Na+ much less so. This inhibition is not likely to be a general ion-competition effect, for Mg2+ is much less effective in inhibiting the binding of H1 to linear DNA. Instead, the inhibition of binding may be due to ion-dependent changes in the conformation of the four-way junction, which are known to occur under similar conditions. These results strongly suggest that the angle formed between the arms of the DNA junction could be a major determinant in the interaction of H1 with DNA crossovers.

Selectivity in the interaction of various DNA sequences with H1 histone.

An assay for the binding of H1 histone by DNA was developed based on extraction with phenol, which partitions free DNA into the aqueous layer and aggregates of H1 histone-DNA complexes into the phenol layer and interface. When this assay was performed on fragments of simian virus 40 (SV40) DNA, fragments containing the 21-bp repetitive element and a portion of the origin of replication were resistant to H1 binding. This result was corroborated when an endonuclease protection assay showed that the origin was poorly protected by H1 compared to other sites. DNase I protection mapping demonstrated that H1 "underprotected" sites immediately to either side of the AT element, which lies in the origin of replication. These sites were also hypersensitive to attack by hydroxyl radical in the absence of histone, probably indicative of some conformation aberration such as minor-groove distension. The same DNA sequences resistant to binding H1 histone resisted binding to H4 histone but showed much less selectivity, if any, in binding polylysine. These results clearly demonstrate that the interaction of DNA and H1 (and H4 histone) is more complicated than just charge neutralization and probably involves the conformation of the DNA.

Cell cycle-dependent suppressive effect of histone H1 on mitosis-specific H3 phosphorylation.

To analyze the mechanism by which histone H3 phosphorylation occurs specifically during mitosis, the effect of H1 on mitosis-specific H3 phosphorylation (Ser-10) was investigated in nucleosomes. H1 interaction with H1-depleted nucleosomes suppressed H3 phosphorylation including Ser-10 by approximately 50%. However, H1 interaction with DNA-free histone octamers failed to suppress H3 phosphorylation. The extent of suppression of H3 phosphorylation in nucleosomes with H1 prepared from synchronized HeLa cells was cell cycle-dependent. Binding with a highly phosphorylated mitotic H1 produced the least suppression of H3 phosphorylation, whereas binding with a lower H1 phosphorylation from G1 phase resulted in the greatest suppression. The results suggest that 1) mitotic H3 phosphorylation is suppressed with a lower level of H1 phosphorylation during interphase and 2) highly phosphorylated H1 during mitosis partially releases the suppression of mitotic H3 phosphorylation.

Effect of organic effectors on chromatin solubility, DNA-histone H1 interactions, DNA and histone H1 structures.

We have extended our previous investigations on the effect of organic osmolytes (glycine, proline, taurine, mannitol, sorbitol and trimethylammonium oxide (TMAO)) on chromatin solubility, to the study of their influence on DNA stability and DNA-histone interactions. Our aim was to understand the molecular origin of the protection effects observed. To this end, we determined the amount of histone H1 required to precipitate DNA or H1-depleted chromatin, at various salt concentrations, in the presence of the above mentioned organic compounds. We found a shift of the H1/DNA ratio required to reach 50% precipitation, towards higher values. Taurine was the most efficient compound followed by mannitol and glycine, then sorbitol and proline. On the contrary, TMAO favoured the precipitation process. We attempted to interpret these results on the basis of Manning's counterion condensation theory. Changes in histone H1 structure folding and in DNA melting temperature Tm were also analyzed. Glycine, taurine, sorbitol and TMAO increased the degree of secondary structure folding of the protein while mannitol and sorbitol had no effect. Taurine, glycine and proline decreased the Tm of DNA, TMAO largely destabilized DNA, but mannitol and sorbitol had no effect. Measurements of NaCl activity in the presence of organic osmolytes did not reveal sufficiently large changes to account for their protection effect against chromatin precipitation. The osmotic coefficient j of the organic effectors solutions increased in the order: taurine < glycine < sorbitol < mannitol < proline << TMAO. For the two latter compounds, the j values increased above 1 at high concentration. We consider that the organic compounds investigated may be classified into three categories: (i) class I (zwitterionic compounds: glycine, proline, taurine) would produce sodium ions release from the DNA surface; (ii) class II (the very polar molecule TMAO) would increase sodium counterions condensation on DNA together with histone H1 folding; (iii) class III compounds (mannitol and sorbitol) would possibly produce a modification of NaCl activity but no definite explanation could be found for the complex behavior of these compounds.

Structural studies on phenanthro[9,10-e]pyrene and its 9,10-dihydro-derivative: dependence of chemical shift on degree of planarity of and buttressing effect in the polycyclic aromatic compounds

The first example of a transannular reaction of a syn-[2,2]metacyclophane derivative via photochemical iodination has been employed in the preparation of 9,10-dihydrophenanthro[9,10-e]-pyrene (2). The structural studies of 2 and the phenanthropyrene 1 were carried out by 1D and 2D NMR spectroscopic analyses. Qualitative correlation between tilting of the fused moieties and the change in deshielding effect was observed by probing the chemical shifts of H3 and H1′(H6 and H8′). Buttressing effects resulting from the steric interaction of H3 and H1′(H6 and H8′), although differing in magnitudes for the two groups of protons (H3→H2→H1→H10 and H1′→H2′→H3′→H4′), seem to transmit evenly among those on the same moiety according to a simple additivity rule.

Interaction of histones H1 and H1° with superhelical and linear DNA

By using direct competition experiments, the binding of histone H1AB (a mixture of H1A and H1B) and H1° to superhelical and linear DNA forms was studied. Mouse liver H1 isohistones and plasmid pαGD containing part of the 5′ flanking and part of the coding sequence of the mouse α-globin gene in pUC18 were used as partners in the binding reaction. The competition experiments were performed by direct mixing of the histone with labelled supercoiled DNA (at 125 mM NaCI and at a histone/DNA ratio of 1.0 and addition to the mixture of increasing amounts of cold competitor DNA, either supercoiled or linear. The radioactivity of the complex formed was determined by filter binding. The results show that both histones H1 and H1° possess a strong binding preference for supercoiled DNA forms. Thus, histone H1° resembles the regular somatic set of histone H1 and not the other differentiation-specific histone H5 studied thus far.

Histone-histone interaction mediates chromatin unfolding at physiological ionic strength.

High-resolution thermal denaturation data on chicken erythrocyte chromatin are reported over 4 orders of magnitude in NaCl concentration which includes the physiological region. A novel technique using critical-point polyacrylamide sols instead of ordinary solvents effectively stabilizes chromatin against precipitation at high salt concentrations. These sols are optically transparent from 260 to 320 nm and are thermally stable over the temperature ranges studied. At Na+ ion concentrations below 10 mM, the polyacrylamide slightly destabilizes chromatin at the nucleosome level, possibly through interactions of histones H1 and H5 with the carboxylic acid residues. At the same low salts, polyacrylamide stabilizes pure DNA against denaturation, presumably by mechanically stabilizing it against helix-distorting thermal fluctuations. In both cases, however, the polyacrylamide sols are entirely noninvasive at higher salts. Prominent low-temperature thermal transitions are observed in chromatin at and above 100 mM NaCl which evidently are associated with conformational changes in DNA. Our results are in accord with the idea that histone-histone interactions at physiological ionic strengths (approximately 100 mM Na+) may be comparable to histone-DNA interactions and hence may be sufficient to promote the destabilization of the DNA helix in chromatin under these conditions. The biological implications of this are discussed, and a possible model for the local decondensation of chromatin under physiological conditions is proposed.

Conformational effects of histones H1 on DNA structure: Comparative study between H1-1, H1 o, H5 and sperm holothuira φ o

Interactions of mammalian histones, H1-1 and H1°, Φ o from holothuria sperm and H5 with poly(dA-dT), poly(dG-dC) and poly(dG-me 5dC) were measured by a nitrocellulose filter binding assay and circular dichroism. All of the proteins bound to every one of the polymers, but differed in the extent of binding, which depended on the polynucleotide/protein ratios and ionic strength. The order of retention of all polymers was Φ o > H1-1 > H1°. The binding of H1° to poly(dG-me 5dC) was remarkably sensitive to ionic strength. The proteins caused changes in the spectral features of the polynucleotides, but differed in the type and extent of the change. Complexes prepared with H1-1 and H1° with all polymers showed a strongly negative ψ, spectrum. Complexes of poly(dA-dT) and Φ o, at a protein/polynucleotide ratio of 0.4, displayed a distinctive spectrum, giving the appearance of a Z-Iike DNA spectrum, at low ionic strength. At higher ionic strength the complexes showed a ψ spectrum. Complexes of poly(dG-me 5dC) in the Z or B conformation with φ o showed spectral features characteristic of a mixture of a Z-Iike and a ψ spectrum. In contrast, H5 reduced the Z-DNA spectral features in the presence of Mg, and produced an inversion of the B spectrum up to a polynucleotide/ protein ratio of 0.24. These findings demonstrate the ability of different proteins to produce changes in the conformation of DNA. This may reflect the ability of chromatin to undergo differential condensation, depending on both the base composition of DNA and the type of H1 histone bound to it.

Chromatin structure. Further evidence against the existence of a beaded subunit for the 30-nm fiber.

The size distribution of chromatin fragments released by micrococcal nuclease digestion of liver chromatin at various ionic strengths was examined. Below 20 mM ionic strength, gradient profiles with a peak centered at 6 nucleosomes are generated, whereas between 20 and 50 mM the peak is always centered on 12 nucleosomes, and above 50 mM ionic strength the 30-nm fiber becomes less accessible to the nuclease and there is a corresponding increase in the size distribution of fragments in the gradients. However, extensive digestions always give profiles with a peak of 12 nucleosomes as nuclease-resistant dodecamers accumulate. All of these observations are consistent with the winding of the 10-nm polynucleosome chain into a helical coil commencing at about 20 mM ionic strength. The helical turns are stabilized by histone H1 interactions between 20 and 50 mM ionic strength producing stable dodecamers. Above 50 mM ionic strength the coil condenses longitudinally and the profiles are consistent with a random attack of this fiber by the nuclease. Consequently it is not necessary to invoke the existence of a subunit bead to explain the profiles. We further define the conditions at which specific structural transitions take place and provide methodology for the preparation of chromatin at various levels of condensation.

Formation and characterization of soluble complexes of histone H1 with supercoiled DNA

We have analyzed the interaction of rat liver histone H1 with superhelical DNA. Depending on the ratio of H1 to DNA and the concentration of salt, two different types of complexes were found. Above a critical ratio of H1 to DNA, called the aggregation point, large aggregates are formed, which have a cable-like appearance in the electron microscope. Below the aggregation point, individual soluble complexes are formed, which are the subject of this study. With increasing ionic strength, the aggregation point is shifted towards lower ratios of H1 to DNA. In the soluble complexes, H1 appears to bind along superhelically intertwined DNA strands, forming a polymer. Partial digestion of the complexes with protease suggests protection of the N-terminal tail and the globular domain of H1. Similar soluble complexes were observed with various H1 fragments but not with the core histones. In the soluble complexes, similar regions of the H1 molecule are considered to be protected from cleavage by protease, as in chromatin. Therefore, these complexes appear to be a valuable model for the interaction of H1 in chromatin fibers.

Classical scaling functions for moments of the order parameter profile in critical adsorption of a binary liquid mixture

Because knowledge of the moments of the order parameter profile is required to understand reflected light studies of critical adsorption of a liquid mixture, the scaling functions in classical Landau theory for the two lowest order moments have been calculated. A contact substrate-liquid interaction of strength h1 is presumed. The first moment is found to saturate at large scaled h1. Finally, the connection with a recent experiment is discussed.

Interactions of H1 and H5 histones with polynucleotides of B- and Z-DNA conformations.

Interactions of chicken H1 and H5 histones with poly(dA-dT), poly(dG-dC), and the Z-DNA structure brominated poly(dG-dC) were measured by a nitrocellulose filter binding assay and circular dichroism. At low protein:DNA ratios, both H1 and H5 bound more Z-DNA than B-DNA, and binding of Z-DNA was less sensitive to interference by an increase in ionic strength (to 600 mM NaCl). H5 histone bound a higher percentage of all three polynucleotides than did H1 and caused more profound CD spectral changes as well. For spectral studies, histones and DNA were mixed in 2.0 M NaCl and dialyzed stepwise to low ionic strength. Prepared in this way or by direct mixing in 150 mM NaCl, complexes made with right-handed poly(dG-dC) showed a deeply negative psi spectrum (deeper with H5 than with H1). Complexes of histone and Br-poly(dG-dC) showed a reduction in the characteristic Z-DNA spectral features, with H5 again having a greater effect. Complexes of poly(dA-dT) and H5, prepared by mixing them at a protein:DNA ratio of 0.5, displayed a distinctive spectrum that was not achieved with H1 even at higher protein:DNA ratios. It included a new negative band at 287 nm and a large positive band at 255 nm, giving the appearance of an inverted spectrum relative to spectra of various forms of B-DNA. These findings may reflect an ability of the different lysine-rich histones to cause varying conformational changes in the condensation of chromatin in DNA regions of highly biased base sequence.

Involvement of the globular domain of histone H1 in the higher order structures of chromatin

We have attacked H1-containing soluble chromatin by α-chymotrypsin under conditions where chromatin adopts different structures. Soluble rat liver chromatin fragments depleted of non-histone components were digested with α-chymotrypsin in NaCl concentrations between 0 m m and 500 m m. at pH 7, or at pH 10, or at pH 7 in the presence of 4 m-urea. α-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by α-chymotrypsin can be easily monitored. The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: (1) under conditions of H1 dissociation at 400 m m and 500 m m-NaCl (pH 7 and 10); (2) at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; (3) at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; (4) in the presence of 4 m-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 m m and 300 m m-NaCl. Under these conditions H1 is degraded by α-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin. Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for α-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers. Based on the present and earlier observations we propose a model for H1 in which the globular domains eventually together with the N-terminal tails form a backbone in the fiber axis, and the nucleosomes are mainly attached to this polymer by the C-terminal tails.

Poly(dA-dT) complexes with histone H1 and pancreatic ribonuclease: specific base recognition evidenced by ultraviolet resonance raman spectroscopy.

AbstractThe conformational changes of poly(dA‐dT) from random coil to ordered structure with stacked bases produce important changes in the Raman line intensities (hypochromism) when the polymer is excited under the preresonance Raman conditions (λ excitation = 300 nm). Poly(dA‐dT)–RNase and poly(dA‐dT)–histone H1 interactions have been studied as models of mechanisms of destabilization and stabilization by proteins of the DNA secondary structure, respectively, following this intense preresonance Raman hypochromism. In addition, the specific variation of the intensity of the 1582‐cm−1 line of adenine is interpreted in terms of the interaction of the amino group with the RNase (thus involving the large groove). In the poly(dA‐dT)–H1 complex, the intensity of the 1665‐cm−1 line of thymine increases. This increase appears to involve the C2O group of thymine, located in the narrow groove.

Nucleosome spacing in rat liver cluomatin. A study with exonuclease III

Exonuclease III was used to uniformly trim DNA ends of micrococcal nuclease-prepared chromatin fragments down to the first major impediment encountered by the enzyme, which arises from the interaction of H1 with the nucleosome. This trimming, when performed on nucleosome dimers, allowed one to quantitatively determine the center-to-center distance of nucleosomes. This distance, of mean 198 base pairs, was found to essentially vary between about 180 and 215 base pairs, with extremes of 165 and 230 base pairs. Trimming of trimers further revealed that the overall arrangement of nucleosome center-to-center distances along the chromatin fiber is that expected on a statistical basis.

Histone H1 from prophase aggregates DNA better than histone H1 from S phase

Published data from Physarum polycephalum plasmodia show a correlation between chromosome condensation before metaphase in the cell cycle and a six-fold increase in phosphate content of histone H1. Histone H1 was isolated from naturally synchronous plasmodia of Physarum either in S phase or in early prophase. The interaction of H1 with DNA was studied using a turbidity assay and the results showed that H1 from early prophase was more effective at causing turbidity with DNA than was H1 from S phase, consistent with the proposed role of H1 phosphorylation in chromosome condensation.

Influence of chew on swallow: Robert J. NelsonJ.A.M.A. 241:4, Jan. 19, 1979

Lysyl oxidases (LOXs) are enzymes that permit the covalent crosslinking of the component chains of collagen and elastin. These enzymes are present inside the nuclei of certain mammalian cells. Previous studies have proposed LOX binding to histone H1 in vitro, and histone H1 is known to control global chromatin compaction and mitotic chromosome architecture. Therefore, in the present study, we analyzed chromatin supraorganizational changes, mitotic abnormalities, mitotic indices and cell death ratios in COS-7 and NRK-49F cells with high and low lox expression levels, respectively. The objective was to support biochemical data of LOX–H1 interaction, by providing evidence of chromatin remodeling in vivo, under different lox expressions. Chromatin decondensation assessed by image analysis was observed in COS-7 cells with increased lox expression. This decondensation is suggested to be promoted by LOX actions on histone H1, which loosens the DNA–H1 complex. In NRK-49F cells transfected with antisense lox or subjected to treatment with beta-aminopropionitrile (BAPN), chromatin condensation and nuclear phenotypic variability were found, which may be due to reduced LOX–H1 interaction. When lox expression was increased in COS-7 cells, the frequency of irregular chromosome plates was not affected, but cell proliferation decreased and “cell death preceded by multinucleation” increased. In NRK-49F cells there was accelerated proliferation induced by transfection with the antisense lox, and confirmed when cells were treated with BAPN. Apoptosis increased in NRK-49F cells only with BAPN treatment whereas cell death preceded by multinucleation increased only after antisense lox transfection. The data presented herein regarding chromatin remodeling indirectly support the hypothesis that LOX binds to histone H1 in vivo. Cell proliferation in COS-7 and NRK-49F cells and cell death at least in COS-7 cells agree with predicted effects of LOX interference in these processes.