Interaction of histone H1 from sea urchin sperm with superhelical and relaxed DNA

Complexes of histone H1 from sea urchin sperm (H1S) and calf thymus (H1T) with superhelical DNA I and relaxed circular DNA II have been analyzed by analytical sedimentation. Similar to H1T, the highly basic and relatively arginine-rich histone H1S preferentially interacts with DNA I compared to DNA II under competition conditions. However, H1S induces a stronger aggregation of bothforms of DNA than H1T. Below 0.05 M NaCl, the soluble complexes formed by both histones have similar properties, but aggregation proceeds in a different manner: H1S induces a stronger aggregation of DNA II as compared to DNA I, whereas H1T fails to aggregate DNA I.The results are explained on the basis of differences in amino acid sequence and structure of the two histones and related to the special chromatin condensing ability of histone H1S.     

Interaction of histone H1 with superhelical DNA conformational studies and influence of ionic strength

The interaction of histone H1 with superhelical SV40 DNA at low ionic strength (0.02 M NaCl) results in the formation of DNP double-fibers and bundle-and cablelike twisted side-by-side associates of several of these double-fibers. On the basis of simple cylindrical or ellipsoidal models the sedimentation properties of these structures can be calculated in accordance with the experiment allowing a direct assignment of electron microscopical and hydrodynamic results. Sedimentation measurements in dependence on the ionic strength indicate a redistribution of H1 resulting in the formation of associates at 0.04 M NaCl and of aggregates at higher salt concentration. Double-fibers are present up to physiological salt concentrations.     

Differential phosphorylation of histone H1 subfractions in vivo.

A study was made of the phosphorylation of chromatographically purified histone H1 subfractions from the liver of premetamorphic tadpoles ($\text{Rana}\text{catesbeiana}$). Two H1 subfractions were obtained which differed in terms of net incorporation of [³²P]phosphate $\text{in}\text{vivo}$. Analysis of N-bromosuccinimide cleavage products further revealed that the two subfractions also differed in the relative distribution of [³²P]phosphate in N- and C-terminal regions of the molecule. Incorporation of [³²P]phosphate into both regions of the molecule occurred virtually exclusively in serine residues.     

Salt-dependent co-operative interaction of histone H1 with linear DNA

The nature of the complexes formed between histone H1 and linear double-stranded DNA is dependent on ionic strength and on the H1 : DNA ratio. At an input ratio of less than about 60% (w/w) H1 : DNA, there is a sharp transition from non-co-operative to co-operative binding at a critical salt concentration that depends on the DNA size and is in the range 20 to 50 mM-NaCl. Above this critical ionic strength the H1 binds to only some of the DNA molecules leaving the rest free, as shown by sedimentation analysis. The ionic strength range over which this change in behaviour occurs is also that over which chromatin folding is induced. Above the salt concentration required for co-operative binding of H1 to DNA, but not below it, H1 molecules are in close proximity as shown by the formation of H1 polymers upon chemical cross-linking. The change in binding mode is not driven by the folding of the globular domain of H1, since this is already folded at low salt in the presence of DNA, as indicated by its resistance to tryptic digestion. The H1-DNA complexes at low salt, where H1 is bound distributively to all DNA molecules, contain thickened regions about 6 nm across interspersed with free DNA, as shown by electron microscopy. The complexes formed at higher salt through co-operative interactions are rods of relatively uniform width (11 to 15 nm) whose length is about 1.6 times shorter than that of the input DNA, or are circular if the DNA is long enough. They contain approximately 70% (w/w) H1 : DNA and several DNA molecules. These thick complexes can also be formed at low salt (15 mM-NaCl) when the H1 : DNA input ratio is sufficiently high (approximately 70%).     

A sedimentation study of the interaction of superhelical SV40 DNA with H1 histone.

By moving boundary sedimentation it is shown that the interaction of H1 histone with superhelical circular SV40 DNA results in the formation of giant heterogeneous aggregates. The size of these aggregates grows with increasing H1 concentration. s20,w values of some 10 000 S were measured. As compared with open relaxed circular DNA a preferential interaction of superhelical DNA with H1 histone is observed, irrespective of the sign of the superhelical turns which was reversed by the addition to DNA of ethidium bromide. The addition to the H1 complexed aggregates of ethidium bromide effects a progressive breakdown of the aggregates. Furthermore, the superhelicity of DNA is not changed by the addition of small amounts of H1 histone.     

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.     

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.     

Studies on the interaction of H1 histone with superhelical DNA: characterization of the recognition and binding regions of H1 histones.

The very lysine rich histone, H1, isolated from a variety of sources interacts preferentially with superhelical DNA compared to relaxed DNA duplexes. The nature of this specific interaction has been investigated by studying the ability of various purified fragments of H1 histone from calf thymus to recognize and bind superhelical DNA. The data suggest that the globular region of the H1 histone molecule (amino acid residues 72-106) is involved in the recognition of superhelical DNA. Thus, the H1 histone carboxy-terminal fragment, 72-212, resembles native H1 histone both quantitatively and qualitatively in its ability to discriminate between and bind to superhelical and relaxed DNA while the H1 histone carboxy-terminal fragment, residues 106-212, has lost this specificity, binding superhelical and relaxed DNA equally well. Furthermore, under conditions in which the globular region of the intact H1 histone has been unfolded, the molecule loses its ability to discriminate between superhelical and relaxed DNA, and binds both forms of DNA equally.     

Interaction of histone H1 with superhelical DNA. Sedimentation and electron microscopical studies at low salt concentration

Complexes of histones H1 with superhelical SV40 DNA obtained by direct mixing were studied in 0.1 SSC buffer corresponding to 0.02 M Na+. Depending on the molar input ratio H1/DNA three classes of sedimenting species were observed: (1) a component sedimenting similar to superhelical DNA with a sedimentation coefficient s2o,w of 25 S observable up to 335 Mol H1/Mol DNA (w/w = 2); (2) a component with s2o,w = 120 S appearing at 135 Mol H1/Mol DNA and (3) growing amounts of heterogeneous aggregates greater than 1000 S. Electron micrographs revealed the 25 S component to consist of double-fibers formed from one DNA molecule and the 120 S component to consist of bundles of several such double-fibers. The aggregates represent cable-like structures. The addition of ethidium bromide to 25 S complexes induces the formation of bundles, if H1 is present in a quantity which alone is not sufficient to bring about this effect. This result indicates that ethidium bromide effects a redistribution of H1 molecules and that H1 is responsible for the bundle formation.     

Isolation and characterization of five subfractions of chicken erythrocyte histone H1

Chicken erythrocyte chromatin contains, besides the specific histone H5, a set of histone H1 subtypes. Five of them were isolated by ion-exchange chromatography and these very related proteins, called H1A, H1B, H1C, H1D and H1E, were characterized by their amino acid compositions.     

Asynchronous appearance of newly synthesized histone H1 subfractions in HeLa chromatin

Incorporation of radioactive alanine into chromatin-bound subfractions of H1 histone was studied in HeLa cells synchronized by the double thymidine block technique. The subfractions were resolved into three chromatographic peaks by Biorex-70. In the period 5-7 h after release from the thymidine block, peaks I and III showed twice as much incorporation as they did in the period 1-3 h after release, whereas peak II showed three times the incorporation at 5-7 h that it did at 1- 3 h. Thus, the H1-histone subfraction in peak II appears in chromatin somewhat later in S phase than do the subfractions in Peaks I and III.     

Mg-induced transition to strongly cooperative binding of histone H1 to linear DNA

By addition of Mg²⁺ ions to histone H1-DNA complexes formed at 20mM NaCl a transition to strongly cooperative binding of histone H1 to DNA is induced. In the analytical ultracentrifuge, above a critical Mg²⁺ concentration of about 0.05 mM, the single component representing the original H1-DNA complex is replaced by two components: a higher order H1-DNA complex type characterized by a much higher sedimentation coefficient, and a slow-sedimenting component consisting of essentially H1-free DNA above 0.1 mM Mg^2-. The fast complex diappears upon removal of Mg²⁺, showing that the process is reversible, and also upon addition of urea. Electron microscopy shows the cooperatively formed H1-DNA complexes to appear predominatly as loosely twisted cable rings in unfixed specimens, and as strongly condensed circular structures of different diameter, but approximately uniform thickness (of about 12nm) after fixation with glutaraldehyde. Besides these higher order structures, only single fibres indistinguishable from control DNA may be seen; individual double fibres which, in the absence of Mg²⁺, represent the predominant H1-DNA complexe structure at about 0.4–0.8 w/w H1/DNA are no longer visible. The transition to strongly cooperative binding of H1 occurs at approximately the same Mg²⁺ concentration which is known to induce the folding of the 10 nm nucleosome chain into the 30nm solenoid structure of chromatin.     

Linker histone binding to superhelical DNA: Electrophoretic and filter binding studies

It has been known for years that linker histones bind preferentially to supercoiled DNA. This preference has been demonstrated by a number of different techniques including deoxyfibonucleoprotein electrophoresis, sedimentation, and filter binding under non-competitive conditions. In an attempt to further study this issue, we used one- and two-dimensional electrophoretic gels and filter binding under competitive conditions, with all DNA forms of interest being simultaneously present in the incubation mixture. Comparison between results obtained by the two methods showed that whereas the preference for superhelical molecules was clearly seen in the electrophoretic gels, the filter binding assay failed to reveal this preference. These results reveal limitations to the filter binding technique, which must be borne in mind in studies involving superhelical DNA molecules.     

Purification of histone H10 and its subfractions under non-denaturing conditions

A quick and convenient method for a non-denaturing purification of histone H10 and its subfractions from different mammalian sources is presented. It is based on cation-exchange chromatography on a CM-Sephadex C-25 column developed by a linear gradient of NaCl. The procedure allows a satisfactory fractionation of H10 subfractions in a form suitable for further studies in assays requiring the native state of the protein.