Ultraviolet light induced preferential cross-linking of histone H3 to deoxyribonucleic acid in chromatin and nuclei of chicken erythrocytes

Histones have been cross-linked to DNA in chicken erythrocyte nuclei and chromatin by using ultraviolet light irradiation at 254 nm. Following irradiation, cross-linked histone-DNA adducts were isolated and purified by hydroxylapatite chromatography, and the DNA component was subjected to acid hydrolysis. Of several hydrolysis techniques investigated, trichloroacetic hydrolysis of the DNA component of the adducts was found to be most effective. Histones isolated from hydrolyzed histone-DNA adducts were characterized by gel electrophoresis and fingerprint analysis. No histone-histone protein adducts were observed. All histone fractions have been shown to cross-link DNA in nuclei or chromatin by utilizing the technique employed, but with different propensities. The order of observed cross-linking, deduced from kinetic experiments, is H1 + H5, H3 greater than H4 greater than H2A much greater than H2B. The preferential binding of the core histone H3, as compared to the other core histones, is discussed in light of recent data concerning histone-DNA interactions and nucleosome structure. The use of the ultraviolet light technique as a conformational probe to study chromatin is also discussed.     

A model for chromatin structure.

A model for chromatin structure is presented. (a) Each of four histone species, H2A (IIbl or f2a2), H2B (IIb2 or f2b), H3 (III or f3) and H4 (IV or f2al) can form a parallel dimer. (b) These dimers can form two tetramers, (H2A)2(H2b)2 and (H3)2(H4)2. (C) These two tetramers bind a segment of DNA and condense it into a "C" segments. (d) The adjacent segments, termed extended or "E" segments, are bound by histone H1 (I or fl) for the major fraction of chromatin; the other "E" regions can be either bound by non-histone proteins or free of protein binding. (e) The binding of histones causes a structural distortion of the DNA which, depending upon the external conditions, may generate the formation of either an open structure with a heterogeneous and non-uniform supercoil or a compact structure with a string of beads. The model is supported by experimental data on histone-histone interaction, histone-DNA interaction and histone subunit-DNA interaction.     

Photochemical cross-linking of histones to DNA nucleosomes.

Ultraviolet (UV)-induced cross-linking was utilized in order to identify histone-DNA interacting regions in the chromatin repeating unit. Fractionated mononucleosomes which contained 185 base pairs of DNA and a full complement of the histones, including histone H1, were irradiated with light of lambda greater than 290nm in the presence of a photosensitizer. Equimolar amounts of histones H2A and H2B were found, by two independent labeling experiments, to be cross-linked to the DNA. Based on previous finding that the UV irradiation specifically cross-links residues which are in close proximity, irrespective of the nature of the amino acid side chain or the nucleotide involved, our results indicate that the four core histones are not positioned equivalently with respect to the DNA. This arrangement allows histones H2A and H2B to preferentially cross-link to the DNA. A water soluble covalent complex of DNA and histones was isolated. This complex was partially resistant to mild nuclease digestion, it exhibited a CD spectrum similar to that of chromatin, and was found to contain histone H1. These results are compatible with a model which suggests that histone H1, though anchored to the linker, is bound to the DNA at additional sites. By doing so it spans the whole length of the nucleosome and clamps together the DNA fold around the histone core.     

Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H1(0) linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.     

Arginine residues involved in strong histone--DNA interactions to fold DNA into the nucleosome core particle.

The numbers of the arginine residues involved in strong histone-DNA interactions to fold DNA into a nucleosome core particle were determined for each of the four core histones, by kinetic studies of chemical modification of the residues in the nucleosome core particle. It was suggested that the arginines in the globular region of H3 histone make major contributions to the strong binding of the octameric histones to the core DNA.     

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

Intra- and inter-nucleosomal protein-DNA interactions of the core histone tail domains in a model system

The core histone tail domains are key regulators of eukaryotic chromatin structure and function and alterations in the tail-directed folding of chromatin fibers and higher order structures are the probable outcome of much of the post-translational modifications occurring in these domains. The functions of the tail domains are likely to involve complex intra- and inter-nucleosomal histone-DNA interactions, yet little is known about either the structures or interactions of these domains. Here we introduce a method for examining inter-nucleosome interactions of the tail domains in a model dinucleosome and determine the propensity of each of the four N-terminal tail domains to mediate such interactions in this system. Using a strong nucleosome "positioning" sequence, we reconstituted a nucleosome containing a single histone site specifically modified with a photoinducible cross-linker within the histone tail domain, and a second nucleosome containing a radiolabeled DNA template. These two nucleosomes were then ligated together and cross-linking induced by brief UV irradiation under various solution conditions. After cross-linking, the two templates were again separated so that cross-linking representing inter-nucleosomal histone-DNA interactions could be unambiguously distinguished from intra-nucleosomal cross-links. Our results show that the N-terminal tails of H2A and H2B, but not of H3 and H4, make internucleosomal histone-DNA interactions within the dinucleosome. The relative extent of intra- to inter-nucleosome interactions was not strongly dependent on ionic strength. Additionally, we find that binding of a linker histone to the dinucleosome increased the association of the H3 and H4 tails with the linker DNA region.     

Studies on erythrocruorin. V. Nuclear magnetic resonance quadrupole relaxation study of sodium, calcium and chloride binding.

Metabolically labeled non-histone chromosomal proteins of high specific activity were fractionated on the basis of their sequential extractability from Krebs II chromatin with urea/salt solutions according to Bekhor et al. (1974a). The binding of each of these NHCP² classes to protein-free DNA and histone-DNA complexes (nucleohistone) was measured and compared to the binding to DNA substituted with 5-bromo-2′-deoxyuridine. After reconstitution of the interacting components, the binding of NHCP and histones was measured according to Scatchard formalism by titration of fixed amounts of DNA with increasing inputs of protein ligands under stringent conditions of 0.25 ionic strength, pH 8.0. Histone binding to either native DNA or BrUrd-substituted DNA was found to be essentially the same. In the presence of histones, the binding for all NHCP classes, except for medium 3 NHCP, was enhanced by an order of magnitude over the binding values for NHCP to DNA in the absence of histones. The binding of NHCP to DNA was thus strongly influenced by histones bound to DNA. A general and significant decrease in histone content in the complexes relative to increased NHCP binding was also apparent, with medium 3 NHCP having the greatest activity to weaken histone interaction with DNA and medium 0 the least. Enhancement in NHCP binding to BrUd-substituted DNA in the presence of histones was decreased to about 50% of the binding to control DNA. The distribution and quantity of DNA binding and non-DNA binding NHCP was also estimated by photochemical attachment to 33% BrUrd-substituted DNA in tryptophan-labeled chromatin and by direct binding assays. We have obtained 30% crosslinking for either histones or NHCP to DNA in stringently formed complexes. In histone-NHCP-DNA complexes, histone crosslinking remained unchanged, while that of NHCP increased to 70%. This is further evidence for a modification in the binding of NHCP to DNA in the presence of histones. The percentage of NHCP crosslinked to DNA in native chromatin ranged from 24% for medium 0 NHCP to 50% for medium 1 and 3 NHCP with an average of 35% for total NHCP. These results plus the direct binding assays indicate that NHCP, in addition to high affinity DNA binding, also interacts non-specifically to DNA and to proteins in chromatin. A mechanism is also being proposed to account for the observed BrUrd effects in chromatin.     

Isolation and characterisation of a 167 bp core particle isolated from stripped chicken erythrocyte chromatin

We have digested chicken erythrocyte soluble chromatin, both unstripped and stripped of histones H1 and H5 with either 0.6 M NaCl or DNA-cellulose, with micrococcal nuclease (MNase). Digestion of unstripped chromatin to monomeric particles initially paused at 188 bp DNA; continued digestion resulted in another pause at 177 before the 167 bp chromatosome and 146 bp core particle were obtained. Digestion of stripped chromatin to monomeric particles paused transiently at 177 bp; continued digestion resulted in marked pauses at 167 and 156 before the 146 bp core particle was obtained. These results suggested that 167 bp DNA representing two complete turns are bound to the histone octamer. Histone H1/H5 binds an additional two helical turns of DNA, thereby protecting up to 188 bp DNA against nuclease digestion. Monomeric particles containing 167 bp DNA were isolated from stripped chromatin and found by DNase I digestion to be a homogeneous population with a 10 bp DNA extension to either end relative to the 146 bp core particle. Thermal denaturation and circular dichroism spectroscopy showed stronger histone-DNA interactions and increased DNA winding as the length of DNA attached to the core histone octamer was decreased. Thermal denaturation also showed three classes of histone-DNA interaction: the core particle containing 167 bp DNA had tight binding of ten helical turns of DNA, intermediate binding of two helical turns and looser binding of four helical turns.     

The use of DNA-cellulose for analyzing histone-DNA interactions. Discovery of nucleosome-like histone binding to single-stranded DNA.

In this report, we introduce the use of DNA-cellulose chromatography for evaluating the strength of binding of histones to DNA under a variety of conditions. We have found that histones added directly to DNA-cellulose at physiological salt concentrations bind relatively weakly, with all histones eluting together at about 0.5 M NaCl when a salt gradient is applied. However, much tighter binding of the four nucleosomal histones to DNA-cellulose is obtained if gradual histone-DNA reconstitution conditions are used. In this case, the binding of histones H2A, H2B, H3, and H4 to DNA-cellulose closely resembles their binding to native chromatin. The nativeness of the binding is indicated both by the distinctive sodium chloride elution profile of these histones from DNA-cellulose and by their relative resistance to trypsin digestion when DNA-bound. The binding to DNA-cellulose of histones H2A, H2B, H3, and H4, which have had the first 20 to 30 amino acid residues removed from their NH2 termini, is indistinguishable from the binding to DNA-cellulose of the same intact histones, as judged by their salt elution profile. Thus, even though the NH2 termini contain 40 to 50% of the positively charged amino acid residues (thought to interact with the DNA backbone), a major contribution to the DNA binding comes from the remainder of the histone molecule. Finally, we have discovered that histones can form a "nucleosome-like" complex on single-stranded DNA. The same complex does not appear to form on RNA. Histones H3 and H4 play a predominant role in organizing this histone complex on single-stranded DNA, as they do on double-stranded DNA in normal nucleosomes. We suggest that, in the cell nucleus, nucleosomal structures may form transiently on single strands of DNA, as DNA and RNA polymerases traverse DNA packaged by histones.     

Binding of HMG14 non-histone protein to histones H2A, H2B, H1 and DNA in reconstituted chromatin

The interaction between calf thymus HMG14 and rat liver chromatin components has been studied via reconstitution and chemical cross-linking. Selective labeling of HMG14 with photoactivable reversible heterobifunctional reagents has allowed a clear identification of the histones interacting with it (histones H2A, H2B and H1). These results are not dependent on whether the chromatin samples used were bulk chromatin, mononucleosomes, or core particles (for H2A and H2B). In addition to histone proteins, DNA also seems to be involved in HMG14 attachment to nucleosome.     

Effect of the supranucleosomal chromatin organization on histone-DNA interrelations

It has been demonstrated by the method of competitive displacement of own chromatin histone by excess total histone that chromatin dispersity influence the strength of histone-DNA interactions in a medium of physiological ionic strength. Histone NI was removed from chromatin after the quantity of total histone added to chromatin was equivalent to that existing in chromatin. The proportion of histones H2A and H2B removed from chromatin was increased after mechanical of ultrasonic degradation of chromatin at 5-20-fold excess of total extra-histone. In some histone preparations, the removal of histones H2A and H2B was not detectable at even 200-fold excess of total histone. This may be explained by strengthening histone-DNA interactions in superhelical loops of chromatin.