Identification of suberimidate cross-linking sites of four histone sequences in H1-depleted chromatin. Histone arrangement in nucleosome core

The arrangement of 8 histones in the nucleosome core has been investigated by identifying the sites of 4 histone sequences cross-linked with a bifunctional amino-group reagent, dimethyl suberimidate, selected from among 4 diimidoesters of various linker lengths examined. H1-depleted calf thymus chromatin was allowed to react with 14C-labeled suberimidate at pH 8.5 and 0 degrees C. The cross-linked chromatin was then digested exhaustively with trypsin. Almost all the histone fragments were released from the chromatin with 0.25 M HCl and chromatographed on several columns and on paper. Cross-linked peptides were detected by analyzing the content of radioactive suberimidoylbislysine after acid hydrolysis. The chromatographic procedure developed here showed that the whole histone fragments contained 29 mol% of the total linked reagent as suberimidoylbisylsine. The 5 finally purified cross-linked peptides were identified from the total and N-terminal amino acids of each pair of peptides separated by two-dimensional cellulose thin layer chromatography after cutting the linker by ammonolysis. Thus, intramolecular cross-linking was found between Lys-5 and Lys-9 of H2A, and Lys-34 and Lys-85 of H2B, while intermolecular cross-linking was found between Lys-24 (or 27) of H2B and Lys-74 of H2A, Lys-85 of H2B and Lys-91 of H4, and Lys-120 of H2B and Lys-115 of H3 and/or Lys-77 of H4. Most of these lysine residues are located in the DNA-binding segments of the 4 histone sequences identified previously [Kato, Y. & Iwai, K, (1977) J. Biochem. 81, 621--630]. All the 5 or 6 cross-links can be located in a heterotypic tetramer consisting of one molecule each of H2A, H2B, H3, and H4, and a model of the histone arrangement in the tetramer is proposed. Two such tetramers may compose to the histone octamer in the nucleosome core.     

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.     

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.     

H3 Cys-110 is in close proximity to the C-terminal regions of H2B and H4 in a nucleosome core with an altered internal arrangement of histones

A particle obtained by nuclease digestion of nucleohistone complexes prepared by direct mixing of histones with DNA in 0.15 M NaCl was indistinguishable by composition and physical properties from nucleosome cores prepared under the same conditions from nucleohistone preannealed in 0.6 M NaCl. We show here that different photo-cross-links form when these particles are prepared from H3 labeled with photoaffinity reagents on the unique histone H3 cysteine. H3-H3 histone dimers were dominant when the particles were prepared by dilution of the nucleohistone from 0.6 M NaCl while H3-H2B and H3-H4 histone dimers were prominent if the nucleohistone complex was prepared directly in 0.15 M NaCl. Peptide mapping of the novel H3-H4 and H3-H2B dimers showed that Cys-110 of histone H3 is cross-linked to the 18 amino acid C-terminal end of H4 or to the 66 amino acid C-terminal half of H2B.     

High mobility group protein 17 cross-links primarily to histone H2A in the reconstituted HMG 17-nucleosome core particle complex

The "neighbor relationship" of lamb thymus high mobility group (HMG) protein 17 to native HeLa nucleosome core particle histones in the reconstituted complex has been studied. 125I-Labeled HMG 17 was cross-linked to core histones using the protein-protein cross-linking reagent 2-iminothiolane. Specific cross-linked products were separated on a two-dimensional Triton-acid-urea/sodium dodecyl sulfate-gel system, located by autoradiography, excised, and quantified. Disulfide bonds in the cross-links were then cleaved, and the protein constituents were identified by sodium dodecyl sulfate-gel electrophoresis. HMG 17 cross-linked primarily to histone H2A while lower levels of cross-linking occurred between HMG 17 and the other histones. In contrast, cross-linking between 2 HMG 17 molecules bound on the same nucleosome core particle was relatively rare. We have concluded that H2A comprises part of the HMG 17 binding site. Less contact occurs between HMG 17 and the other core histones, and there is little contact possible between the 2 bound HMG 17 molecules. These results are in agreement with the current model for the structure of the nucleosome and the proposed binding sites for HMG 17.     

SWI/SNF- and RSC-catalyzed nucleosome mobilization requires internal DNA loop translocation within nucleosomes

The multisubunit SWI/SNF and RSC complexes utilize energy derived from ATP hydrolysis to mobilize nucleosomes and render the DNA accessible for various nuclear processes. Here we test the idea that remodeling involves intermediates with mobile DNA bulges or loops within the nucleosome by cross-linking the H2A N- or C-terminal tails together to generate protein "loops" that constrict separation of the DNA from the histone surface. Analyses indicate that this intranucleosomal cross-linking causes little or no change in remodeling-dependent exposure of DNA sequences within the nucleosome to restriction enzymes. However, cross-linking inhibits nucleosome mobilization and blocks complete movement of nucleosomes to extreme end positions on the DNA fragments. These results are consistent with evidence that nucleosome remodeling involves intermediates with DNA loops on the nucleosome surface but indicate that such loops do not freely diffuse about the surface of the histone octamer. We propose a threading model for movement of DNA loops around the perimeter of the nucleosome core.     

Nucleosome remodeling by the human SWI/SNF complex requires transient global disruption of histone-DNA interactions

We utilized a site-specific cross-linking technique to investigate the mechanism of nucleosome remodeling by hSWI/SNF. We found that a single cross-link between H2B and DNA virtually eliminates the accumulation of stably remodeled species as measured by restriction enzyme accessibility assays. However, cross-linking the histone octamer to nucleosomal DNA does not inhibit remodeling as monitored by DNase I digestion assays. Importantly, we found that the restriction enzyme-accessible species can be efficiently cross-linked after remodeling and that the accessible state does not require continued ATP hydrolysis. These results imply that the generation of stable remodeled states requires at least transient disruption of histone-DNA interactions throughout the nucleosome, while hSWI/SNF-catalyzed disruption of just local histone-DNA interactions yields less-stable remodeled states that still display an altered DNase I cleavage pattern. The implications of these results for models of the mechanism of SWI/SNF-catalyzed nucleosome remodeling are discussed.     

Tissue transglutaminase serves as an inhibitor of apoptosis by cross-linking caspase 3 in thapsigargin-treated cells

Thapsigargin (THG) is an inhibitor of the endoplasmic reticulum Ca2+-ATPase that induces caspase 3 activation and apoptosis in HCT116 cells through a Bax-dependent pathway. In Bax-deficient HCT116 cells, however, THG specifically generates two additional species of caspase 3, termed p40 and p64, with molecular masses of approximately 40 and 64 kDa, respectively, through unknown mechanisms. Here, we report that the Ca2+-dependent protein cross-linking enzyme tissue transglutaminase (tTGase) is involved in THG-induced p40 and p64 formation by catalyzing caspase 3 cross-linking reactions, thereby inactivating caspase 3 and apoptosis in Bax-deficient cells. Overexpression of tTGase increases p40 and p64 in THG-treated cells, and purified tTGase catalyzes procaspase 3 cross-linking in vitro. Inhibition of tTGase activity by either the tTGase inhibitor monodansylcadaverine or short-hairpin RNA reduces the cross-linked species p40 and p64 and restores caspase 3 activation in response to THG treatment. Moreover, prolonged exposure to THG results in a decrease in protein levels of XIAP and cIAP-1, which is subsequently followed by an increase in tTGase protein expression and activity. Expression of cytosolic Smac sensitizes Bax-deficient cells to THG-induced apoptosis; however, this effect is diminished by coexpression of tTGase. Taken together, these results suggest a novel role for tTGase as a new type of caspase 3 inhibitor in THG-mediated apoptosis.     

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.     

Chemical acetylation, trypsinolysis and stability of nucleosomes

Gel electrophoretic analysis of the histone chemical acetylation in the nucleosome core particles with acetic andydride revealed availability of about 14 lysine residues of histone H2A, 15-21 of H2B, 8-11--H3 and 6-9--H4. Moderately lysine-rich histones H2A and H2B were found to be more susceptible to acetylation than arginine-rich H3 and H4. Chemical acetylation enhanced the rate of trypsin digestion in acetylated nucleosomes as evidenced by gel electrophoresis of histone fragments. A more pronounced trypsin digestion was evident at acetylation of only 3-5 histone amino groups per nucleosome. However, even heavily acetylated nucleosomes yielded in familiar trypsin limit digest pattern of histone fragments thus indicating persistence of histone octamer. Nucleosomes which were trace acetylated (up to 3-5 histone amino groups neutralized per nucleosome) and treated with trypsin to remove highly charged terminal histone regions revealed remarkable unfolding and partial dissociation when analyzed by gel electrophoresis. The same trace acetylated nucleosomes did not show such destabilization prior to trypsin digestion.     

New findings on interactions among the yeast oligosaccharyl transferase subunits using a chemical cross-linker

At present, there is very limited knowledge about the structural organization of the yeast oligosaccharyl transferase (OT) complex and the function of each of its nine subunits. Because of the failure of the yeast two-hybrid system to reveal interactions between luminal domains of these subunits, we utilized a membrane permeable, thiocleavable cross-linking reagent dithiobis-succinimidyl propionate to biochemically study the interactions of various OT subunits. Four essential gene products, Ost1p, Wbp1p, Swp1p, and Stt3p were shown to be cross-linked to each other in a pairwise fashion. In addition, Ost1p was found to be cross-linked to all other eight OT subunits individually. This led us to propose that Ost1p may reside in the core of the OT complex and could play an important role in its assembly. Ost4p and Ost5p were found to only interact with specific components of the OT complex and may function as an additional anchor for optimal stability of Stt3p and Ost1p in the membrane, respectively. Interestingly, Ost3p and Ost6p subunits exhibited a surprisingly identical pattern of cross-linking to other subunits, which is consistent with their proposed redundant function. Based on these findings, we analyzed the distribution of the lysine residues that are likely to be involved in cross-linking of OT subunits and propose that the OT subunits interact with each other through either their transmembrane domains and/or a region proximal to it, rather than through their luminal or cytoplasmic domains.     

Nonrandom assembly of chromatin during hydroxyurea inhibition of DNA synthesis

Incubation of MSB-1 chicken lymphoblastoid cells with hydroxyurea leads to a rapid 25-fold decrease in the incorporation of [3H]thymidine into DNA and a 5-fold decrease [3H]lysine into the nucleosome core histones. I have investigated whether the distortion in the normal proportion of histone-DNA synthesis results in alterations in the nucleosome assembly process and find that neither the stoichiometry of new histone synthesis nor the deposition is appreciably changed during hydroxyurea incubation. Protein cross-linking and micrococcal nuclease digestion show that the histones synthesized during hydroxyurea treatment form octamer structures and are assembled into typical nucleosome particles. Minor nucleosome subpopulations are found which exhibit altered sensitivity to nuclease digestion and which are depleted in new histones H3 and H4. When MSB-1 cells incubated in hydroxyurea are pulsed briefly with density-labeled amino acids and [3H]lysine, the radiolabeled core histone octamers formed are as dense as individual monomer histones. These results suggest that the newly synthesized histone octamers are uniformly dense and do not contain mixtures of new and old histones. Thus, histones synthesized during hydroxyurea incubation are deposited nonrandomly and do not exchange with preexisting histones.     

Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure—a FRET study

Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor–acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry–exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.     

Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus

The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.     

Histones H1 and H5: one or two molecules per nucleosome?

We have determined histone stoichiometries in nuclei from several sources by a direct chemical method, with the particular aim of quantitating histone H1 and, in chicken erythrocytes, H5, and of distinguishing between one and two molecules per nucleosome. The four histones H3, H4, H2A and H2B are found in equimolar amounts, as expected for the core histone octamer. The molar ratio of H1 in lymphocyte and glial nuclei is 1.0 per octamer, and in liver nuclei from three species 0.8 per octamer. These results suggest that each nucleosome has one H1 molecule; nucleosomes could acquire two molecules of H1 only at the expense of others containing none. The stoichiometry of H5 in chicken erythrocyte nuclei is similar to that of H1 in other nuclei, being about 0.9 molecules per nucleosome; the H1 also present in these nuclei amounts to 0.4 molecules per nucleosome.     

Chemical cross-linking of H1 histone to the nucleosomal histones.

When whole steer kidney nuclei were treated with dimethyl-3,3'-dithiobisproprionimidate, N,N'-bis(2-carboxyimidomethyl) tartaramide dimethyl ester, or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under approximately physiological ionic conditions, H1 histone was cross-linked to each of the four histones in the nucleosome core. The carbodiimide reagent, which introduces no atoms between the amino acid side chains being joined, seemed to give the same result as did the longer di-imidate cross-linking reagents. When conditions were optimized for the production of of H1-containing dimers, the total yield of H1-core histone heterodimers was nearly equal to the yield of H1 homodimers. Naturally occurring H1 dimers and cross-linked heterodimers of high mobility group proteins 14 and 17 with H1 and core histones were also observed.     

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.