Accessibility of the globular domain of histones H1 and H5 to antibodies upon folding of chromatin

Antibodies to the globular domain of histones H1 and H5 were purified by affinity chromatography and used to study the accessibility of this region of H1 and H5 in folded and unfolded rat liver and hen erythrocyte chromatin respectively. The different conformations of the chromatin filament were induced by varying the ionic strength from 1 mM to 80 mM NaCl and maintained by fixation with glutaraldehyde. Treatment with glutaraldehyde at a given salt concentration affected neither the orientation of nucleosomes relative to the fiber axis nor the compactness of chromatin. Solid-phase immunoassay and inhibition experiments showed no binding of the antibody against the globular domain of H1 to chromatin at the entire range of salt concentrations, while the antibody to the whole H1 molecule reacted with chromatin at low salt. On the other hand, the antibody to the globular region of H5 reacted with hen erythrocyte chromatin independently of the extent of chromatin condensation. These results indicate that the antigenic determinants of the globular domain of H5 are accessible to the antibody both in folded and unfolded chromatin, while those of the same region of H1 are masked, probably by interaction with DNA or proteins.     

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.     

The globular region of histone H5 is equally accessible to antibodies in relaxed and condensed chromatin

The accessibility of histone H5 in chromatin was examined with monoclonal antibodies recognizing several epitopes of the globular region (GH5) of the histone (Rózalski, M., Lafleur, L., and Ruiz-Carrillo, A. (1985) J. Biol. Chem. 260, 14379-14385). The stoichiometry of the chromatin-antibody complexes indicated that while 0-86% of the H5 molecules were able to react, depending on the particular epitope, the extent of antibody binding to relaxed chromatin (in 5 mM KCl) and condensed chromatin (in 100 mM KCl or 0.35 mM MgCl2) was virtually identical. This indicates that the topography of H5 does not change during the conformational transition of chromatin. The data suggest that H5 is not completely internalized in the 30-nm fiber or that the fiber is flexible enough to allow full exposure of the GH5 epitopes. Several control experiments, including monoclonal antibody binding, sedimentation analysis, DNase II digestion, and glutaraldehyde cross-linking, showed that epitope accessibility is not due to H5 exchange or to perturbation of the chromatin fiber. The accessibility of GH5 suggests ways in which inactive chromatin may be unfolded in vivo.     

The Role of the Central Globular Domain of Histone H5 in Chromatin Structure

Abstract

Histone H5 contains three tryosines in the central, a polar region of the molecule. All three tryosines 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 is 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 in 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 higherorder chromatin structure.     

Mesoscale Modeling of Nucleosome-Binding Antibody PL2-6: Mono- versus Bivalent Chromatin Complexes

Interactions of chromatin with bivalent immunoglobin nucleosome-binding antibodies and their monovalent (papain-derived) antigen-binding fragment analogs are useful probes for examining chromatin conformational states. To help interpret antibody-chromatin interactions and explore how antibodies might compete for interactions with chromatin components, we incorporate coarse-grained PL2-6 antibody modeling into our mesoscale chromatin model. We analyze interactions and fiber structures for the antibody-chromatin complexes in open and condensed chromatin, with and without H1 linker histone (LH). Despite minimal and transient interactions at physiological salt, we capture significant differences in antibody-chromatin complex configurations in open fibers, with more intense interactions between the bivalent antibody and chromatin compared to monovalent antigen-binding fragments. For these open chromatin fiber morphologies, antibody binding to histone tails is increased and compaction is greater for bivalent compared to monovalent and antibody-free systems. Differences between monovalent and bivalent binding result from antibody competition with internal chromatin fiber components (nucleosome core and linker DNA) for histone tail (H3, H4, H2A, H2B) interactions. This antibody competition for tail contacts reduces tail-core and tail-linker interactions and increases tail-antibody interactions. Such internal structural changes in open fibers resemble mechanisms of LH condensation, driven by charge screening and entropy changes. For condensed fibers at physiological salt, the three systems are much more similar overall, but some subtle tail interaction differences can be noted. Adding LH results in less-dramatic changes for all systems, except that the bivalent complex at physiological salt shows cooperative effects between LH and the antibodies in condensing chromatin fibers. Such dynamic interactions that depend on the internal structure and complex-stabilizing interactions within the chromatin fiber have implications for gene regulation and other chromatin complexes such as with LH, remodeling proteins, and small molecular chaperones that bind and modulate chromatin structure.     

Accessibility of histone H1° and its structural domains to antibody binding in extended and folded chromatin

The aim of this work was to study the accessibility of histone H1° and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1° which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1° which recognized epitopes located in the globular region of H1° and (3) anti-C-tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1° did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1° in chromatin was much less than that of histone H1.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - G-domain Globular domain - IgG Immunoglobulin G - SDS Sodium Dodecylsulphate     

The globular domain of histone H5 is internally located in the 30 nm chromatin fiber: an immunochemical study.

The location of the globular domain of histone H5 relative to the axis of the 30 nm chromatin fiber was investigated by following the accessibility of this region of the molecule in chicken erythrocyte chromatin to specific antibodies as a function of chromatin structure. Antibodies to the globular domain of H5 as well as their Fab fragments were found to react with chromatin at ionic strengths ranging from 1-80 mM NaCl, the reaction gradually decreasing upon increase of salt concentration. If, however, Fab fragments were conjugated to ferritin, no reaction of the complex with chromatin was observed at salt concentrations higher than 20 mM. The accessibility of the globular part of H5 in unfolded chromatin to the Fab-ferritin complex was also demonstrated with trypsin-digested chromatin. The experiments were carried out by both solid-phase immunoassay and inhibition experiments. The data obtained are consistent with a structure in which the globular domain of H5 is internally located in the 30 nm chromatin fiber.     

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.     

Inaccessibility of theEuplotes telomere binding protein

The telomere binding protein (TP) from the macronucleus of the ciliateEuplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequences. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augumented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.     

Nucleosome packing in interphase chromatin.

Higher-order chromatin fibers (200--300 A in diameter) are reproducibly released from nuclei after lysis in the absence of formalin and/or detergent. Electron microscope analysis of these fibers shows that they are composed of a continuous array of closely apposed nucleosomes which display several distinct packing patterns. Analysis of the organization of nucleosomes within these arrays and their distribution along long stretches of chromatin suggest that the basic 100-A chromatin fiber is not packed into discrete superbeads and is not folded into a uniform solenoid within the native 250-A fiber. Furthermore, because similar higher-order fibers have been visualized in metaphase chromosomes, the existence of this fiber class appears to be independent of the degree of in vivo chromatin condensation.     

Monoclonal antibodies against histone H5. Epitope mapping and binding to chromatin

Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed.     

Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure

We have used light microscopy and serial thin-section electron microscopy to visualize intermediates of chromosome decondensation during G1 progression in synchronized CHO cells. In early G1, tightly coiled 100-130-nm "chromonema" fibers are visualized within partially decondensed chromatin masses. Progression from early to middle G1 is accompanied by a progressive uncoiling and straightening of these chromonema fibers. Further decondensation in later G1 and early S phase results in predominantly 60-80-nm chromonema fibers that can be traced up to 2-3 microns in length as discrete fibers. Abrupt transitions in diameter from 100-130 to 60-80 nm along individual fibers are suggestive of coiling of the 60-80-nm chromonema fibers to form the thicker 100-130-nm chromonema fiber. Local unfolding of these chromonema fibers, corresponding to DNA regions tens to hundreds of kilobases in length, reveal more loosely folded and extended 30-nm chromatin fibers. Kinks and supercoils appear as prominent features at all observed levels of folding. These results are inconsistent with prevailing models of chromosome structure and, instead, suggest a folded chromonema model of chromosome structure.     

Chromatin fiber folding: requirement for the histone H4 N-terminal tail

We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53S when maximally folded in 1mM or 100mM MgCl(2). The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.     

The alternatively folded state of the antibody C(H)3 domain

The C(H)3 domain of antibodies is characterized by two antiparallel beta-sheets forming a disulfide-linked sandwich-like structure. At acidic pH values and low ionic strength, C(H)3 becomes completely unfolded. The addition of salt transforms the acid-unfolded protein into an alternatively folded state exhibiting a characteristic secondary structure. The transition from native to alternatively folded C(H)3 is a fast reaction. Interestingly, this reaction involves the formation of a defined oligomer consisting of 12-14 subunits. Association is completely reversible and the native dimer is quantitatively reformed at neutral pH. This alternatively folded protein is remarkably stable against thermal and chemical denaturation and the unfolding transitions are highly cooperative. With a t(m) of 80 degrees C, the stability of the alternatively folded state is comparable to that of the native state of C(H)3. The defined oligomeric structure of C(H)3 at pH 2 seems to be a prerequisite for the cooperative unfolding transitions.     

Distinct properties of the two putative "globular domains" of the yeast linker histone, Hho1p

The putative linker histone in Saccharomyces cerevisiae, Hho1p, has two regions of sequence (GI and GII) that are homologous to the single globular domains of linker histones H1 and H5 in higher eukaryotes. However, the two Hho1p "domains" differ with respect to the conservation of basic residues corresponding to the two putative DNA-binding sites (sites I and II) on opposite faces of the H5 globular domain. We find that GI can protect chromatosome-length DNA, like the globular domains of H1 and H5 (GH1 and GH5), but GII does not protect. However, GII, like GH1 and GH5, binds preferentially (and with higher affinity than GI) to four-way DNA junctions in the presence of excess linear DNA competitor, and binds more tightly than GI to linker-histone-depleted chromatin. Surprisingly, in 10 mM sodium phosphate (pH 7.0), GII is largely unfolded, whereas GI, like GH1 and GH5, is structured, with a high alpha-helical content. However, in the presence of high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate) GII is also folded; the anions presumably mimic DNA in screening the positive charge. This raises the possibility that chromatin-bound Hho1p may be bifunctional, with two folded nucleosome-binding domains.     

Two homologous domains of similar structure but different stability in the yeast linker histone, Hho1p

The Saccharomyces cerevisiae homologue of the linker histone H1, Hho1p, has two domains that are similar in sequence to the globular domain of H1 (and variants such as H5). It is an open question whether both domains are functional and whether they play similar structural roles. Preliminary structural studies showed that the two isolated domains, GI and GII, differ significantly in stability. In 10 mM sodium phosphate (pH 7), the GI domain, like the globular domains of H1 and H5, GH1 and GH5, was stably folded, whereas GII was largely unstructured. However, at high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate), which might mimic the charge-screening effects of DNA phosphate groups, GII was folded. In view of the potential significance of these observations in relation to the role of Hho1p, we have now determined the structures of its GI and GII domains by NMR spectroscopy under conditions in which GII (like GI) is folded. The backbone r.m.s.d. over the ordered residues is 0.43 A for GI and 0.97 A for GII. Both structures show the "winged-helix" fold typical of GH1 and GH5 and are very similar to each other, with an r.m.s.d. over the structured regions of 1.3 A, although there are distinct differences. The potential for GII to adopt a structure similar to that of GI when Hho1p is bound to chromatin in vivo suggests that both globular domains might be functional. Whether Hho1p performs a structural role by bridging two nucleosomes remains to be determined.     

The transition of the higher-order structure of rat liver chromatin takes place at about 85% neutralization of DNA charges

The salt dependence of the stoichiometry in the binding of linker histone to rat liver chromatin was analysed. It was found that the transition of the higher-order structure of chromatin (between folded and unfolded) takes place at the point of approximately 85% neutralization of DNA charges in chromatin, as in a related process in the B-Z transition of double-helical DNA.     

Diffusion-enhanced energy transfer investigation of histone H5 in chromatin with a fluorescently-labelled antibody fragment Fab'.

The location of chicken erythrocyte H5 histone relative to the axis the 30 nm chromatin fibre axis has been investigated by diffusion-enhanced energy transfer. In this investigation, a neutral lanthanide chelate as donor and a fluorescent probe specific to H5 as acceptor have been used. The acceptor probe consists of H5 antibody Fab' fragment, which has been labeled with 5-iodoacetamidofluorescein (5-IAF). Using H5 fragments we have shown by ELISA that the antibodies recognized the N- and C-terminal ends of this histone. A neutral chelate of terbium (TbHED3A) was chosen as a suitable donor for energy transfer with IAF-labelled Fab' (Fab'-IAF) bound to H5 in various chromatin structures. The ionic strength dependence of the energy transfer from TbHED3A to chromatin-bound Fab'-IAF was used to estimate the accessibility and the location of the Fab' in chromatin. The rate constants for energy transfer, obtained from the lifetimes of the TbHED3A excited state in presence and absence of acceptor, indicated a decrease in transfer efficiency upon increase of salt concentration from 5 to 80 mM NaCl. This can be correlated with the chromatin folding occurring in this ionic strength range and is consistent with the location of at least some of the N and C-termini of H5 within the condensed chromatin structure.     

The arrangement of H5 molecules in extended and condensed chicken erythrocyte chromatin.

Chemical cross-linking with dithiobis(succinimidyl propionate) has been used to investigate the relative disposition of neighbouring H5 (H1) molecules in chicken erythrocyte chromatin in the extended (nucleosome filament) and condensed (300 A filament) states; in this chromatin H5 and H1 are interspersed along the nucleosome filament, rather than segregated into blocks, as shown by the nature of the cross-linked dimers and their relative amounts. Detailed analysis of the cross-linked H5 homopolymers from extended chromatin and condensed nuclear chromatin indicates which domains of H5 are in contact (or close proximity) in the two states. Two results suggest a polar, head-to-tail arrangement of H5 molecules along the nucleosome filament. This arrangement persists when chromatin adopts higher-order structure but in the folded state neighbouring basic C-terminal domains, in particular, are more closely juxtaposed than they are in extended chromatin.